ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2013

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 871

Aspects of Regulation of GFRand Tubular Function in theDiabetic Kidney

Roles of Adenosine, Nitric Oxide and OxidativeStress

PATRIK PERSSON

ISSN 1651-6206ISBN 978-91-554-8610-5urn:nbn:se:uu:diva-195956

Dissertation presented at Uppsala University to be publicly examined in A1:107a, BMC,Husargatan 3, Uppsala, Friday, April 19, 2013 at 09:15 for the degree of Doctor of Philosophy(Faculty of Medicine). The examination will be conducted in Swedish.

AbstractPersson, P. 2013. Aspects of Regulation of GFR and Tubular Function in the Diabetic Kidney:Roles of Adenosine, Nitric Oxide and Oxidative Stress. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine871. 54 pp. Uppsala. ISBN 978-91-554-8610-5.

Diabetic nephropathy is the main cause for initiation of renal replacement therapy and earlysymptoms in patients include increased glomerular filtration rate (GFR), decreased oxygentension and albuminuria, followed by a progressive decline in GFR and loss of kidney function.Experimental models of diabetes display increased GFR, decreased tissue oxygenation andnitric oxide bioavailability. These findings are likely to be intertwined in a mechanisticpathway to kidney damage and this thesis investigated their roles in the development ofdiabetic nephropathy. In vivo, diabetes-induced oxidative stress stimulates renal tubular Na+

transport and in vitro, proximal tubular cells from diabetic rats display increased transport-dependent oxygen consumption, demonstrating mechanisms contributing to decreased kidneyoxygenation. In control animals, endogenous adenosine reduces vascular resistance of theefferent arteriole via adenosine A2-receptors resulting in reduced filtration fraction. However,in diabetes, adenosine A2-signalling is dysfunctional resulting in increased GFR via increasedfiltration fraction. This is caused by reduced adenosine A2a receptor-mediated vasodilationof efferent arterioles. The lack of adenosine-signaling in diabetes is likely due to reducedlocal adenosine concentration since adenosine A2a receptor activation reduced GFR only indiabetic animals by efferent arteriolar vasodilation. Furthermore, sub-optimal insulin treatmentalso alleviates increased filtration pressure in diabetes. However, this does not affect GFRdue to a simultaneously induction of renal-blood flow dependent regulation of GFR byincreasing the filtration coefficient. In diabetes, there is decreased bioavailability of nitric oxide,resulting in alterations that may contribute to diabetes-induced hyperfiltration and decreasedoxygenation. Interestingly, increased plasma concentration of l-arginine, the substrate fornitric oxide production, prevents the development of increased GFR and proteinuria, but notincreased oxygen consumption leading to sustained intra-renal hypoxia in diabetes. This thesisconcludes that antioxidant treatment directed towards the NADPH oxidase as well maneuversto promote nitric oxide production is beneficial in diabetic kidneys but is targeting differentpathways i.e. transport-dependent oxygen consumption in the proximal tubule by NADPHoxidase inhibition and intra-renal hemodynamics after increased plasma l-arginine. Also, theinvolvement and importance of efferent arteriolar resistance in the development of diabetes-induced hyperfiltration via reduced adenosine A2a signaling is highlighted.

Keywords: diabetes, diabetic nephropathy, glomerular filtration rate, renal blood flow, insulin,renal hemodynamics, micropuncture, oxygen, NADPH-oxidase, apocynin, streptozotocin, l-arginine, CGS21680, rats, mice

Patrik Persson, Uppsala University, Department of Medical Cell Biology, IntegrativePhysiology, Box 571, SE-751 23 Uppsala, Sweden.

© Patrik Persson 2013

ISSN 1651-6206ISBN 978-91-554-8610-5urn:nbn:se:uu:diva-195956 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-195956)

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Persson, P., Hansell, P., Palm, F. (2012) NADPH oxidase inhi-

bition reduces tubular sodium transport and improves kidney oxygenation in diabetes. Am J Physiol Regul Integr Comp Phys-iol, 15(302):1443–9.

II Pihl, L*., Persson, P*., Fasching, A., Hansell, P., DiBona, G.F., Palm, F. (2012) Insulin induces the correlation between renal blood flow and glomerular filtration rate: implications for mechanisms causing hyperfiltration. Am J Physiol Regul Integr Comp Physiol, 1(303):39–47.

III Persson, P., Hansell, P., Palm, F. (2013) Adenosine A2 recep-

tor-mediated regulation of renal hemodynamics and glomerular filtration rate is abolished in diabetes. Adv Exp Med Biol, 765:225–30.

IV Persson, P., Hansell, P., Palm, F. (2013) Reduced adenosine A2a receptor-mediated efferent arteriolar vasodilation causes di-abetes-induced glomerular hyperfiltration. Submitted.

V Persson, P., Fasching, A., Teerlink, T., Hansell, P., Palm, F. (2013) L-citrulline, but not L-arginine, prevents diabetes-induced glomerular hyperfiltration and proteinuria in rat. Sub-mitted.

Reprints were made with kind permission from the American Physiological Society for study I and II and Springer Science+Business Media for study III.

*Equal contribution

Contents

Introduction ..................................................................................................... 9 The Kidney ................................................................................................. 9 Regulation of GFR ................................................................................... 11

Determinants of GFR ........................................................................... 11 Adenosine in the kidney ...................................................................... 13

Diabetic nephropathy ............................................................................... 14 Diabetes-induced hyperfiltration ......................................................... 14 Reactive oxygen species ...................................................................... 16 Nitric oxide .......................................................................................... 17

Aims .............................................................................................................. 18

Materials and Methods .................................................................................. 19 Animals and chemicals ............................................................................. 19

Animal procedures ............................................................................... 19 Experimental protocols ............................................................................ 20 In vivo kidney function in rat (Study I, II, IV and V) and mouse (Study III) ............................................................................................................ 21

Surgery................................................................................................. 21 GFR (Study I, II, III, IV and V) ........................................................... 21 Renal oxygen tension (PO2) (Study I and V) ....................................... 21 In vivo oxygen consumption (QO2) (Study V) ..................................... 22 Electrolyte excretion and Li+ clearance (Study I and III) .................... 22 Thiobarbituric acid reactive substances (Study I and V) ..................... 22 Micropuncture (Study II and IV) ......................................................... 22

In vitro oxygen consumption (QO2)(Study I) ........................................... 23 Isolation of proximal tubular cells and QO2 measurement .................. 23

Statistical analysis .................................................................................... 23

Results ........................................................................................................... 25 NAPDH oxidase a determinant of kidney oxygenation and proximal tubular Na+ transport in diabetes (Study I) .......................................... 25 Insulin affects filtration hemodynamics and the mechanism causing diabetes-induced hyperfiltration (Study II).......................................... 27 Adenosine A2-signaling reduces filtration fraction in controls but not in diabetics (Study III) ................................................................... 29

Reduced adenosine A2a signaling mediates increased filtration fraction in diabetes (Study IV) ............................................................ 30 L-citrulline improves plasma L-arginine in diabetes and prevents diabetes-induced glomerular hyperfiltration and proteinuria (Study V) ......................................................................................................... 32

Discussion ..................................................................................................... 35

Summary and conclusions ............................................................................ 40

Sammanfattning på svenska .......................................................................... 41

Acknowledgements ....................................................................................... 43

References ..................................................................................................... 45

Abbreviations

πGC colloid osmotic pressure in glomerular capillaries A1AR adenosine A1 receptors A2AR adenosine A2 receptors ADH antidiuretic hormone Ang II angiotensin II ASL argininosuccinate lyase ASS argininosuccinate synthase BH4 tetrahydrobiopterin CAT1 cationic amino acid transporter 1 DMA dimethylamiloride FF filtration fraction GFR glomerular filtration rate Kf filtration coefficient L-NAME N-ω-Nitro-L-arginine methyl ester hydrochloride MAP mean arterial pressure MD macula densa mTAL medullary thick ascending limb NADPH nicotinamide adenine dinucleotide phosphate NHE sodium/hydrogen-exchanger NKCC sodium-potassium-2-chloride transporter NO nitric oxide NOS nitric oxide synthase O2

.- superoxide anion O2ct oxygen content ONOO.- peroxynitrite PBow hydrostatic pressure in Bowman’s space PFF tubular free flow pressure PGC hydrostatic pressure in glomerular capillaries PNET net filtration pressure PSF tubular stop flow pressure PTC proximal tubular cells QO2 oxygen consumption RAAS renin-angiotensin-aldosterone system RBF renal blood flow ROS reactive oxygen species SGLT sodium/glucose-linked transporter

SOD superoxide dismutase TBARS thiobarbituric acid reactive substances TGF tubuloglomerular feedback

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Introduction

The Kidney The kidneys maintain a stable internal environment by regulating plasma volume, adjusting blood pH and excreting metabolic waste products. To efficiently fulfill these functions the kidneys receive a high blood flow equaling around 25% of cardiac output during rest. This results in a high glomerular filtration rate (GFR), around 125 ml/min in the human kidneys resulting in a production of 180 L of primary urine each day. The primary urine is basically plasma, except large plasma proteins, that is handled along the nephron by active and passive reabsorption of electrolytes, valuable mol-ecules like glucose, amino acids and small proteins and water but also active and passive secretion of molecules that needs to be excreted in the final urine. This processing results in a production of a final urine of 1-2 L each day with varying osmolality depending on salt and fluid intake. Considering a blood volume of around 5 L in the human and a production of 180 L pri-mary urine each day it is easy to imagine that only a very small mismatch in this system will either result in a complete depletion of body fluid and drop in blood pressure or an extreme accumulation in plasma volume and increase in blood pressure. Several systems cooperate to achieve a perfect match so that blood pressure is maintained, both locally in the kidney and systemic, including the renin-angiotensin-aldosterone system (RAAS), central nervous system innervation, neuro-endocrine regulation and endocrine hormones.

Anatomically the basic structure of the kidney is the nephron, which is derived from the Greek word for just kidney. Each nephron is a functional unit that works to a great extent independently of each other and the anatomy and function is conserved between mammals. The human kidney consists of around 0.8 to 1.5 million nephrons whereas the rat kidney consist of around 30 000 nephrons. Each kidney is supplied by blood by one renal artery that is further divided to segmental arteries, interlobar arteries, arcuate arteries, interlobular arteries and finally afferent arterioles. The afferent arteriole sup-plies the renal glomeruli with blood in which the filtration occurs. The glo-meruli consists of a tuft of fenestrated capillaries surrounded by mesangial cells which is a modified smooth muscle cell with the ability to regulate contraction and thereby hydraulic conductivity and effective surface area for filtration which will be discussed in forthcoming sections. Mesangial cells are under influence of several hormones including angiotensin II (Ang II)

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and nitric oxide (NO). A specialized epithelial cell, the podocyte, covers the glomerular capillaries; possessing processes that cover the capillaries. The processes are kept separated at a distance of 25 nm forming filtration slits. The glomerular capillary tuft including the podocytes are completely sur-rounded by a second epithelial layer forming a urinary space known as Bowman´s capsule, which is in direct connection to the tubular system. Glomerular capillaries are united into one efferent arteriole leaving the glo-meruli, which tonus, and thereby pressure in glomerular capillaries is under influence of several factors and in this thesis are the influence of adenosine and NO investigated. A second capillary network of either peritubular capil-laries or vasa recta is formed after the efferent arteriole, supplying the renal cells with oxygen (O2), before the blood enters a venous system ending in the renal vein.

The tubular system consists of several different parts that are both ana-tomically and functionally different. The first part of the tubular system is proximal tubule, performing a major part of the active reabsorption in the kidney, 2/3rds of the filtered electrolytes are reabsorbed and the same amount of water is passively diffused by the osmotic gradient. Filtered glu-cose and amino acids are fully reabsorbed in the proximal tubule as long as maximal transport capacity is not exceeded as will happen in uncontrolled diabetes. The driving force for reabsorption is the basolateral Na+/K+-ATPase creating a Na+ gradient that is used for secondary active transport by luminal transporters. Important transporters that are discussed in this thesis are the sodium/hydrogen-exchanger (NHE) isoform 3 and sodium/glucose-linked transporter (SGLT). The nephron proceeds into the medullary region in a U-shaped structure, the loop of Henle, that can be further divided into a thick descending, thin descending, thin ascending and finally a thick ascend-ing part (mTAL). The renal medulla has high interstitial osmolality that ena-bles reabsorption of water and concentration of the urine. The high osmolali-ty is maintained by medullary countercurrent exchange of urea in vasa recta together with a low blood flow to allow for diffusion. The low medullary blood flow results in low oxygenation of this part of the kidney as will be discussed in forthcoming sections. The descending part of loop of Henle is water permeable leading to reabsorption of water as the filtrate passes, whereas the ascending part is water impermeable but has abundant expres-sion of the furosemide-sensitive sodium-potassium-2-chloride (NKCC) transporter, which contributes to approximately 10-15% of total electrolyte reabsorption. The nephron returns to cortex and forms the distal convoluted tubules, with high expression of thiazide-sensitive Na+-Cl- co-transporters which consist of two cell types, principal cells and intercalated cells where fine-tuning of Na+ and H+ reabsorption occurs to maintain blood pressure and acid-base balance, respectively. The last part of the nephron is the corti-cal and medullary collecting duct where the tubular system one more time will go through the hyperosmotic medulla to be able to reabsorb water. The

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water reabsorption is regulated by plasma osmolality and therefore hydration status via osmo-sensitive receptors in hypothalamus. High plasma osmolality that occurs during dehydration will result in secretion of antidiuretic hor-mone (vasopressin, ADH) from the posterior pituitary gland. ADH will sig-nal via G-protein coupled V2 receptors in the distal tubule and collecting duct, stimulating insertion of aquaporins in the apical membrane promoting water reabsorption.

Regulation of GFR GFR is under strict autoregulation maintaining GFR during fluctuations in blood pressure, matching GFR with the capacity for tubular reabsorption. Normal regulation to match oxygen supply with demand aims to increase organ blood flow and oxygen delivery during increased metabolism. How-ever, this regulation is significantly more complicated in the kidney since renal blood flow (RBF) and therefore O2 supply is correlated to GFR and subsequently O2 demand, if maintaining Na+ balance. Two independent mechanisms, the myogenic mechanism and tubuloglomerular feedback (TGF), cooperate to regulate diameter of the afferent arteriole and thereby the driving forces for filtration in form of RBF and hydrostatic pressure in glomerular capillaries (PGC). The myogenic mechanism constricts the affer-ent arteriole in response to increased arterial blood pressure whereas TGF works from the tubular side by specialized cells in macula densa (MD) in the early distal nephron, a region in each nephron where the distal tubule makes contact with its own glomeruli. MD cells sense the tubular flow of electro-lytes by NKCC, specifically Cl-, and interpret electrolyte flow as a function of GFR. Increased reabsorption by MD NKCC results in a depolarization of the cell which triggers release of ATP which is further enzymatically de-graded to adenosine in the interstitium in close proximity to the afferent arte-riole where it elicit a vasoconstriction by binding to adenosine A1 receptors, reducing RBF and PGC and subsequently GFR. Decreased GFR will reduce electrolyte flow passing MD and therefore cease the ATP release (1).

Determinants of GFR As mentioned in the previous section the driving force for filtration is PGC. This driving force is opposed by a hydrostatic pressure in Bowmans´space (PBow) and colloid osmotic force in glomerular capillaries (πGC). PGC is natu-rally derived from the arterial pressure together with the vascular resistance of the afferent arteriole, but also by post-glomerular resistance in the efferent arteriole. In contrast to the afferent arteriole, the efferent arteriole can regu-late GFR with minimal impact on total RBF. A constriction of the afferent arteriole will reduce PGC and RBF, but a constriction of the efferent arteriole

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will increase PGC and also GFR without influencing RBF. This leads to an increased filtration fraction (FF), a common observation in diabetes that can pose a problem when it comes to match O2 supply with demand, but this will be discussed in detail in paper III and IV. Normal values for PGC are in the range of 44-50 mmHg (2-4). PBow is mainly derived from flow resistance in the tubular system and can be modulated by the degree of proximal tubular reabsorption. A normal range is between 12-15 mmHg (2; 3; 5-9). The filtra-tion barrier is normally impermeable for proteins leaving them in the glo-merular capillaries where they will exert an osmotic pressure opposing filtra-tion. πGC will thereby increase along the capillaries as filtration occurs which will reduce net filtration pressure (PNET), and eventually even stop filtration. However, considering a mean πGC around 25 mmHg (10; 11), leads to the conclusion that PNET is between 4-13 mmHg. Certain studies suggest that πGC will rise to a level that will oppose PGC and stop filtration before the end of the glomerular capillaries, a concept named filtration equilibrium, which appears to occur in a special rat strain named the Munich-Wistar rat, due to a very low PGC. This phenomenon would result in regulation of GFR solely dependent on the afferent arteriole and RBF and completely rule out the importance of the efferent arteriole in regulation of GFR. However, in other rat strains studied and also other species PGC is higher and filtration equilib-rium does not occur, opening for an important role for the efferent arteriole as discussed in paper III and paper IV. The filtration barrier consists of fe-nestrated capillary endothelial cells, a glomerular basement membrane and podocytes, which is permeable for ions, small organic molecules and water, but rather impermeable for proteins. However, a debate exists to which ex-tent small proteins are filtered and subsequently reabsorbed in the proximal tubule, and it was recently demonstrated in vivo that Ang II directly regulates permeability of the filtration barrier for macromolecules, by inducing the number of large pores (12). The collective permeability of the filtration bar-rier is usually summarized into a filtration coefficient (Kf), defined as vol-ume filtered per mmHg of PNET, yielding the formula that determines GFR.

𝐺𝐹𝑅 = 𝐾𝑓 (𝑃𝐺𝐶 − 𝑃𝐵𝑜𝑤) − (𝜋𝐺𝐶 − 𝜋𝐵𝑜𝑤) (Equation 1) In the above Equation 1 forces determining net filtration pressure and there-by GFR are displayed. Glomerular capillary pressure (PGC) originating from the interplay between diameter of the afferent and efferent arteriole is the driving force for filtration. Factors opposing filtration are pressure in Bow-man’s space (PBow) originating from tubular reabsorption and hydraulic re-sistance of the nephron, and plasma colloid osmotic pressure πGC determined by plasma protein concentration and filtration fraction. Filtration of proteins is usually very low and therefore is colloid osmotic pressure in Bowman’s space (πBow) considered to be zero. Filtration coefficient (Kf) is determined by the permeability and surface area of the filtration barrier.

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Adenosine in the kidney Factors regulating afferent and efferent arteriole diameter, determining PNET, includes Ang II, adenosine, endothelin-1, prostaglandins, NO and other fac-tors. This thesis however, will focus on adenosine and mainly its effects via the adenosine A2 receptors. Adenosine mediates its effects by activating P1 purinoceptors that are G protein-coupled. Four subtypes are identified, aden-osine A1, A2a, A2b and A3 receptor. Adenosine A1 activation inhibits adenyl-ate cyclase and increases intracellular [Ca2+] in afferent arterioles (13), lead-ing to vasoconstriction (14), which is a component of the renal autoregula-tion of GFR through the TGF-mechanism (15; 16). Hence, infusion of aden-osine leads to vasoconstriction of the afferent arteriole of superficial nephrons, resulting in a quick reduction in RBF (17). This is opposite to the effect in most other organs where adenosine elicits a vasodilation to increase blood flow at increased metabolic demands (18). In addition to expression on the afferent arteriole, adenosine A1 receptors are found in mesangial cells, vasa recta and throughout the tubular system (19-21). Functionally, the main effects of adenosine in regulation of tubular reabsorption are in the proximal tubule and medullary thick ascending limb (mTAL). Adenosine A1 receptor stimulation increases proximal tubule reabsorption of Na+, HCO3

- and fluid (22). More specifically, activation of the adenosine A1 receptor in isolated renal cells regulates NHE3 in biphasic manner, where low concentration of adenosine stimulates and high concentration inhibits NHE3 (23). In mTAL however, adenosine A1 activation inhibits reabsorption (24). The renal me-dulla is capable of adenosine release that increases secondary to hypoxia (25). Physiologically, these disparate effects of adenosine A1 receptor activa-tion in regulation of tubular transport will shift site of tubular reabsorption to the well-oxygenated renal cortex during increased workload. Adenosine A2a and A2b receptor activation increases adenylate cyclase activity and causes vasodilation (26; 27) and adenosine has higher affinity to A2a compared to A2b receptors (28). Adenosine A2b is expressed on pre-glomerular vessels (29) in rat but found on both afferent and efferent arteriole in mouse (30). The efferent arteriole appears to express both A2a and A2b receptor. A2a is also found in outer medullary descending vasa recta. Indeed, medullary blood flow increases after adenosine infusion whereas cortical blood flow decreases (31; 32).

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Figure 1. Simplified graph of beginning of a nephron with the factors determining GFR. See also Equation 1.

Diabetic nephropathy Diabetes mellitus is associated with several complications and organs that possesses insulin-independent glucose uptake are extra susceptible. This includes the kidneys, which reabsorb glucose from the tubular lumen via SGLT leading to high intracellular concentrations of glucose. Indeed, diabe-tes is the main cause to end-stage renal disease requiring renal replacement therapy affecting up to 30% of all type-1 diabetes and increasing numbers of type-2 diabetes patients (33). The earliest sign of altered kidney function in diabetes is increased GFR in some patients, although this is not defined as renal disease. Clinically the degree of renal disease is based on presence of albumin in the urine, categorized in microalbuminuria (30-300 mg/24 h) and macroalbuminuria (>300 mg/24 h) and the actual GFR divided into 5 inter-vals where a GFR of less than 15 ml/min/1.73 m2 is classified as chronic kidney disease stage 5 or end stage renal disease (34).

Diabetes-induced hyperfiltration The mechanisms behind the diabetes-induced hyperfiltration are under de-bate, and also whether presence of hyperfiltration predicts development of renal disease is not fully clarified. Recent studies suggest that there is no correlation between hyperfiltration and progression to proteinuria but it is associated with a faster decline in GFR in later stages of the disease (35; 36). Increased GFR seems to be initiated by an increased proximal tubular reab-sorption driven by the hyperglycemia. Glucose is freely filtered in the kidney

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leading to high proximal tubular glucose concentration. Glucose is normally completely reabsorbed in the proximal tubule by secondary active transport by the SGLT, driven by the low intracellular Na+ concentration caused by the basolateral Na+/K+-ATPase. Two isoforms of SGLT are expressed in proximal tubule, SGLT2 in the early S1 segment of proximal tubule which posses’ low affinity but high capacity for glucose reabsorption and transports glucose and Na+ in a 1:1 ratio (37), mediating reabsorption of the bulk of tubular glucose (38), whereas high affinity low capacity SGLT1 in the later S3 segment of proximal tubule transports glucose and Na+ in a 1:2 ratio, cleaning up any remaining glucose (39; 40). Together they are able to reab-sorb all filtered glucose up to a blood glucose concentration of 15 mM. In-deed, progressive increase in tubular glucose load results in net Na+ and fluid reabsorption (41). With more severe hyperglycemia, glucosuria will occur causing an osmotic diuresis.

Increased proximal tubular reabsorption will have two consequences for GFR. First, it will initiate an error signal to MD leading to a reduced TGF signal. Luminal Na+, Cl- and K+ concentration is reduced in the early distal nephron (4). This will be interpreted by MD as a low GFR, reducing adeno-sine release and subsequently A1AR signaling, increasing RBF and GFR. However, A1AR knock-out mice lacking a functional TGF mechanism (15; 16) still display diabetes-induced hyperfiltration (42; 43) and increased RBF is not a requirement for diabetes-induced glomerular hyperfiltration (9; 44; 45), although it is observed in some studies (46-48). Second, proximal tubu-lar pressure is reduced in diabetes increasing PNET, secondary to increased proximal tubular reabsorption through SGLT but also tubular hypertrophy reducing flow resistance in the distal nephron, buffering the increased tubu-lar flow rate due to osmotic diuresis (49). Inhibition of SGLT increases PBow and reduces GFR exclusively in diabetes, and knockout of SGLT2 in mice attenuates glomerular hyperfiltration but not tubular hypertrophy (50). PGC in diabetic animals can be increased, decreased or unchanged compared to con-trol animals (7; 47; 51). Increased PGC can be mediated by an afferent arteri-ole dilation, which would result in a concomitant increase in RBF. As al-ready mentioned, RBF can be increased or unchanged in the hyperfiltration phase of diabetes but this will be discussed in detail in the discussion of pa-per II. Increased PGC with unchanged RBF that is also observed in experi-mental diabetes is compatible with a constriction of the efferent arteriole, partly mediated by increased renal Ang II concentration, binding to efferent arteriolar AT1 receptors. Indeed, AT1 receptor inhibition decreases FF and low dose Ang II infusion increases GFR and decreases RBF resulting in increased FF in early type 1 diabetic patients (52). Furthermore, ACE-inhibition reduce, although not normalize GFR exclusively in hyperfiltering type 1 diabetics (53) as well as in animal models of type 1 diabetes (51). More recently, attention has been directed toward afferent and efferent arte-riolar adenosine A2 receptors (A2AR) and its involvement in renal autoregu-

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lation (54), and the aim of paper III and IV was to investigate A2AR signal-ing in the involvement of diabetes-induced hyperfiltration.

Reactive oxygen species Diabetes is associated with increased load of oxidative stress, defined as an imbalance between production of reactive oxygen species (ROS) and antiox-idant defense resulting in oxidative damage. In health, when ROS producing systems and antioxidant defense is in balance, the basal ROS production is important for normal redox signaling, for instance oxidation of cysteine resi-dues, known to regulate activity of several enzymes (55). Also a massive but well regulated oxidative burst in neutrophils is important for the immune system to be able to kill pathogens (56). However, a generalized increased ROS production, as occur in diabetes, is an important component in the dis-ease progression. However, important to mention though is that clinical trials with antioxidant treatment have shown on increased mortality (57; 58), stressing the fact that ROS are involved in normal physiological processes and a disruption might be harmful. Sources of increased ROS in diabetes include the mitochondria (59), uncoupled nitric oxide synthase (NOS) (60), xanthine oxidase (61; 62) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (63).

The most common ROS molecules are the superoxide anion (O2.-), formed

by one electron donation to molecular oxygen, the hydroxyl radical (OH.-), hydrogen peroxide (H2O2) and peroxynitrite (ONOO.-). ONOO.- is the reac-tion product between O2

.- and NO thereby linking increased ROS production to reduced bioavailability of NO. Redox status is balanced by enzymatic antioxidant defense systems degrading, and molecular antioxidants scaveng-ing ROS. A key system is the superoxide dismutases (SOD) catalyzing the reaction from O2

.- to H2O2. Three isoforms are present in the human: SOD1 in the cytoplasm, SOD2 in the mitochondria and SOD3 in the extracellular space. H2O2 is further catalyzed to water and O2 by catalase (64). Reduced SOD1 activity accelerates diabetic nephropathy in mice (65) and total anti-oxidant capacity is decreased in diabetic patients (66), and correlated to the degree of complications (67), linking the development of diabetic nephropa-thy to both increased ROS production but also reduced antioxidant defense. Of special interest for this thesis is the NADPH oxidase, the only enzyme with the sole purpose of producing O2

.-. Different isoforms of the NADPH oxidase is expressed in phagocytes, smooth muscle cells, endothelial cells and renal tubules to create huge amounts of O2

.- in phagocytes during oxida-tive burst and a low basal O2

.- production in the other tissues contributing to regulation of vascular tone, and thereby blood pressure, and tubular reab-sorption, respectively (68-70). The originally discovered phagocytic NADPH oxidase consists of membrane bound enzymatic component, gp91phox or NOX-2, and regulatory cytosolic subunits, p47phox, p67phox,

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p40phox and Rac 1 (71). Today however, at least seven different homologues has been discovered with different tissue expression where Nox-1, Nox-2 and Nox-4 is detected in the microvasculature and tubule of the kidney (72). Diabetes results in up regulation of this enzyme system at different levels observed as increased phosphorylation, indicating activation, of the regulato-ry subunits p47phox and p67phox as well as increased protein expression of Nox-2 and Nox-4 (73-76). Therefore was the outcome of acute NADPH oxidase inhibition in diabetes investigated in study I.

Nitric oxide NO determines vascular tone and thereby RBF (77) and tubular electrolyte transport, partly via direct interference with tubular transporters (78) as well as inhibition of mitochondria respiration (79). Thereby, NO is involved in determining renal oxygenation (80; 81). NO is produced by three different NOS, endothelial, inducible and neuronal, from the substrate L-arginine and several co-factors, including tetrahydrobiopterin (BH4), NADPH and O2. Production is regulated by availability of both substrate in form of L-arginine (82) and co-factors, mainly BH4 (83) but also directly regulated depending on NO needs. Most important are phosphorylation at serine 1177, increasing NOS activity, mediated by Akt/protein kinase B, protein kinase A, 5´-AMP-activated protein kinase (AMPK) and calmodulin-dependent kinase II, and phosphorylation at threonine 495, decreasing NOS activity, mediated by protein kinase C (84). NO bioavailability is reduced in diabetes and is linked to intra-renal hypoxia (80). Uptake of L-arginine is mediated by the amino acid transport system y+ or system y+L where y+ is Na+-independent and transports cationic amino acids and y+L is Na+-dependent and transports both neutral and cationic amino acids (85). System y+ is rep-resented in the kidney by cationic amino acid transporter 1 (CAT1) (82). This result in a direct competition between several amino acids for the same transporter and the individual concentration of each will determine its transport. Importantly, NO production rate is limited by L-arginine transport by CAT1 and L-arginine concentration is reduced in diabetes and therefore an interesting intervention that is discussed in paper V.

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Aims

The overall aim of this thesis was to investigate changes occurring in the kidney very early in the disease progression, usually before the classical clinical sign of diabetic nephropathy. Focus was directed towards events initiating the diabetes-induced increase in GFR but also changes in the tubu-lar system, with emphasis on electrolyte reabsorption and oxygen metabo-lism.

Specific aims for the papers included in this thesis:

Paper I To determine the role of NADPH oxidase derived ROS on tubular Na+

transport in vivo and its relation to kidney oxygenation. In isolated proximal tubular cells localize exaggerated ROS-dependent tubular reabsorption in diabetes to renal cortex. Paper II

To elucidate the role of insulin in regulation of factors determining GFR in diabetes. Paper III

To determine the role of adenosine A2 receptors in regulation of GFR in diabetes. Paper IV

To determine if reduced adenosine A2a receptor signaling in diabetes is contributing to increased FF and GFR. Paper V

To determine if restored plasma L-arginine concentration in diabetes pre-vents diabetes-induced kidney dysfunction.

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Materials and Methods

Animals and chemicals All chemicals were from Sigma-Aldrich (St. Louis, MO, USA) if not other-wise stated. Male Sprague-Dawley rats (Charles River, Sulzfeldt, Germany) were used in study I, II, IV and V. Male C57/BL6 mice (Charles River, Sulzfeldt, Germany) were used in study III. Animals had free access to tap water and standard rat (Ewos, Södertälje, Sweden) or mouse chow (LAB-FOR Lantmännen, Sweden). Animals were housed in groups in a tempera-ture and light controlled environment and received daily care.

Animals were divided into the following groups:

Study I Control and diabetes.

Study II Control, diabetes and diabetes+insulin.

Study III Control and diabetes.

Study IV Control and diabetes.

Study V Control and diabetes with and without chronic L-arginine or L-citrulline supplementation in the drinking water.

Animal procedures All animal procedures were performed in accordance with national guide-lines of animal care and use and approved by the Uppsala animal ethics committee.

Induction of diabetes (Study I, II, III, IV and V) Diabetes was induced by a single injection of either 55 mg/kg bw strepto-zotocin dissolved in 0.2 ml saline in the tail vein (Study I, II, IV and V) or 70 mg/kg bw alloxan dissolved in 0.2 ml saline in the tail vein (Study III). Animals were considered diabetic if blood glucose concentration was in-creased >15 mmol/L within 48 hours and remained elevated. Blood glucose concentration was determined with test reagent strips (FreeStyle, Abbott

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Laboratories, Abbott Park, IL, USA) on blood samples obtained from the cut tip of the tail. Diabetes duration was 14-28 days.

Insulin treatment (Study II) and supplementation of L-arginine and L-citrulline (Study V) Insulin treatment (10 IU/kg, subcutaneous) was given once a day and started 24 hours after diabetes was induced and carried out throughout the course of diabetes. L-arginine and L-citrulline supplementation was given in the drink-ing water and treatment started the same day as diabetes induction.

Experimental protocols In study I untreated control (n=11) and diabetic (n=13) rats were used with a total diabetes-duration of 14±4 days until acute experiments. Additional control (n=7) and diabetic (n=8) rats with similar diabetes-duration were allocated for isolation of proximal tubular cells (PTC).

In study II untreated control (n=17), untreated diabetic (n=16) and insulin-treated diabetic (n=18) rats were used with a total diabetes-duration of 14±2 days until acute experiments. Additional untreated control (n=4), untreated diabetic (n=4) and insulin-treated diabetic (n=4) rats were allocated to mi-cropuncture studies. Insulin-treated diabetic rats were given one daily subcu-taneous injection of insulin (9 IU/kg/day) (Lantus, Sanofi Aventis, Frankfurt am Main, Germany).

In study III untreated control (n=11) and diabetic mice (n=10) were used with a total diabetes-duration 25±4 days until acute experiments.

In study IV untreated control (n=8) and diabetic (n=11) rats were used with a diabetes-duration of 14±4 days until acute experiments for investigation of whole kidney function. Additional control (n=5) and diabetic (n=8) rats with similar diabetes-duration were used for micropuncture.

In study V untreated control (n=12) and diabetic rats (n=9), L-arginine treat-ed control (n=10) and diabetic (n=10) rats and L-citrulline treated control (n=11) and diabetic (n=10) rats were used with a total diabetes-duration of 21±5 days until acute experiments. L-arginine (1.25% to controls and 0.35% to diabetics) or L-citrulline (1.20% to controls and 0.20% to diabetics) treatment was administered in the drinking water throughout the course of diabetes.

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In vivo kidney function in rat (Study I, II, IV and V) and mouse (Study III) Surgery Rats were anesthetized with thiobutabarbital (Inactin, 120 mg/kg bw for non-diabetic and 80 mg/kg bw for diabetic animals, intraperitoneal injection) and placed on a servo-controlled heating pad to maintain body temperature at 37.5°C. A tracheotomy was performed to assure unobstructed spontaneous breathing. A polyethylene catheter was placed in a femoral artery for moni-toring of blood pressure and blood sampling and in a femoral vein for infu-sion of saline (5 ml/kg bw/h to non-diabetic animals and 10 ml/kg bw/h to diabetic animals). The bladder was catheterized for urinary drainage fol-lowed by a subcostal flank incision exposing the left kidney that then was immobilized in a plastic cup. The left ureter was catheterized for timed urine collections. An ultrasound flow probe (Transonic Systems Inc., Ithaca, NY, USA) was placed around the left renal artery to measure RBF. After surgery, all animals were allowed to recover for 40 minutes before the experiments were commenced.

Mice were anesthetized with isoflurane 1.5-2.0% in 100% O2 (Abbott), placed on a servo-controlled heating pad maintaining body temperature at 37.5°C. Catheters were placed in carotid artery and jugular vein for determi-nation of blood pressure, blood sampling and infusion of saline (0.35 ml/h to non-diabetic and 0.7 ml/h to diabetic mice). The bladder was catheterized for urine collection and the left kidney was exposed through a sub-costal flank incision. An ultrasound flow-probe was placed around the renal artery.

GFR (Study I, II, III, IV and V) GFR was estimated by inulin-clearance. 3H-inulin (American Radiolabeled Chemicals, St. Louis, MO, USA) was administered as a bolus dose (185 kBq/kg) followed by a continuous infusion in Ringer solution (185 kBq/kg bw/h). 3H-inulin activity in urine from timed collections and in plasma was measured with standard liquid scintillation technique. Urine flow was meas-ured gravimetrically and GFR calculated according to clearance equation, GFR=[inulin]urine*urine flow/[inulin]plasma.

Renal oxygen tension (PO2) (Study I and V) Renal tissue oxygen tension was determined using modified Clark-type mi-croelectrodes with an outer diameter of the tip of 10 µm (Unisense, Aarhus, Denmark). Electrodes were two-point calibrated in water saturated with ei-ther Na2S2O5 to set zero or air to set 147 mmHg PO2. Microelectrodes were adjusted by a micromanipulator to measure PO2 at either 1 mm depth from

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kidney surface for cortical PO2 or at 5 mm depth from kidney surface for determining medullary PO2.

In vivo oxygen consumption (QO2) (Study V) In vivo QO2 was determined from the arterio-venous difference in oxygen content (O2ct) multiplied by the RBF according to the formula: O2ct=(([hemoglobin]*oxygen saturation*1.34)+(oxygen tension*0.003).

Electrolyte excretion and Li+ clearance (Study I and III) Na+ and Li+ concentrations in urine and plasma samples were determined by flame spectrophotometry (model IL543, Instrumentation Lab, Milan, Italy). Li+ clearance, as a marker of proximal tubular reabsorption, was measured in study I. Li+ was administered as a 4 mg intraperitoneal injection of LiCl at the end of surgery followed by a continuous infusion of 2.1 mg/h. This re-sulted in plasma Li+ concentration of 0.5-1.0 mM.

Thiobarbituric acid reactive substances (Study I and V) Thiobarbituric acid reactive substances (TBARS) to assess oxidative stress level were measured in urine samples and determined fluorometrically. 100 µl of diluted urine sample were mixed with 125 µl thiobarbituric acid (Merck, Darmstadt, Germany) and heated to 97°C for 60 minutes. Standards were prepared from malondialdehyde-bis-(diethylacetate) (Merck-Schuchart, Schuchart, Germany). Samples were cooled on ice and 150 µl of 1 M NaOH and methanol (91:9) were added. Samples were centrifuged (3000 rpm for five minutes) and fluorescence was measured in the supernatant (ex. 532 nm, em. 553 nm, Safire 2, TECAN, Männedorf, Switzerland).

Micropuncture (Study II and IV) Intratubular pressure was measured with a servo-controlled pressure system (World Precision Instruments, New Haven, CT, USA). Proximal tubular free flow pressure (PFF) and stop flow pressure (PSF) were measured. PFF in the unobstructed nephron of an early loop of a proximal tubule whereas PSF is the pressure recorded after which injecting mineral oil distal of the pressure pipette stops the tubular flow. PSF is a surrogate marker for PGC minus the oncotic pressure. Accordingly, the driving pressure for filtration, PNET was calculated from PSF-PFF.

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In vitro oxygen consumption (QO2)(Study I) Isolation of proximal tubular cells and QO2 measurement PTCs were isolated from control and diabetic rats. Rats were anesthetized with thiobutabarbital and both kidneys were immediately removed and placed in ice-cold buffer. Renal cortex pooled from both kidneys were minced through a metallic mesh strainer and incubated with buffer contain-ing collagenase (0.05% wt/vol) at 37°C for 90 minutes. Incubation was con-stantly bubbled with a gas-mixture of 95% O2 and 5% CO2. Suspension was then cooled on ice for 10 minutes followed by filtration through graded fil-ters with pore sizes of 180, 75, 53 and 38 µm. Cells were centrifuged (200 g, 2 min) and the pellet was suspended in collagenase-free buffer. This process was repeated three times and cells were kept on ice until QO2 was measured. Isolation procedure and subsequent QO2 measurements were conducted in a buffer solution containing in mM, 113.0 NaCl, 4.0 KCl, 27.2 NaHCO3, 1.0 KH2PO4, 1.2 MgCl2, 1.0 CaCl2, 10.0 HEPES, 0.5 Ca-lactate, 2.0 glutamine and 50 U/ml streptomycin. Osmolality and pH was adjusted to 300 mOsm/kg and 7.4, respectively. Glucose concentration in the medium was 5.8 mM for cells from normoglycemic control rats and 23.2 mM for cells from diabetic rats.

QO2 was measured in an Oxygraph 2k (OROBOROS Instruments, Inns-brück, Austria). PTCs were incubated with vehicle, dimethylamiloride (DMA; 1 mM), apocynin (1 mM), ouabain (2 mM) and apocynin in combi-nation with either DMA or ouabain for 10 minutes at 37°C. DMA was used to inhibit NHE3, apocynin to inhibit the NADPH oxidase and ouabain to inhibit the Na+K+-ATPase. After incubation 50 µl of PTC suspension was injected into the oxygraph, and the rate of O2 disappearance was recorded. At the end of each recording, 1 ml was removed to determine protein con-centration. To avoid interference with the protein assay samples were centri-fuged (15,000 g, 10 minutes) and resuspended in 200 µl dH2O. Protein con-centration was determined according to the Lowry method with DC Protein Assay (Bio-Rad, Hercules, CA, USA) and QO2 was adjusted for protein concentration.

Statistical analysis All statistical analysis were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) or SAS (SAS Institute, Cary, NC, USA) and for all analysis a P<0.05 was considered significant. Descriptive statis-tics are presented as means ± SEM.

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In study I all data were analyzed by 2-way repeated measure ANOVA fol-lowed by Bonferroni´s multiple comparisons test. In addition, data presented in Figure 5 was analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test.

In study II correlation analysis were performed using least-squares linear regression to test whether regression slopes were significantly different from zero. All other analysis used a mixed model approach to account for the study design.

In study III and IV all data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test.

In study V in vivo data were grouped in untreated, L-arginine treated and L-citrulline treated and analyzed with two-way ANOVA followed by Bonfer-roni’s multiple comparisons test. Plasma concentration of amino acids and related compounds were analyzed with one-way ANOVA followed by Fish-er’s Least Significant Difference (LSD) test.

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Results

NAPDH oxidase a determinant of kidney oxygenation and proximal tubular Na+ transport in diabetes (Study I) In vivo kidney function Diabetic rats displayed increased GFR, but similar RBF, resulting in in-creased calculated FF compared to normoglycemic controls. NADPH oxi-dase inhibition did not affect any of these parameters but increased both absolute and fractional excretion of Na+ (Figure 2), as well as fractional ex-cretion of Li+ (Figure 3). Cortical and medullary PO2 were reduced in diabet-ics and PO2 was increased by NADPH oxidase inhibition only in diabetics (Figure 4). Urinary excretion of TBARS was increased in diabetics, reflect-ing increased oxidative stress, and acute NAPDH oxidase inhibition reduced it, confirming the effect of apocynin.

Figure 2. Absolute (A) and fractional (B) excretion of Na+ in control and diabetic rats during baseline and after NADPH oxidase inhibition with apocynin.

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Figure 3. Fractional excretion of Li+ in control and diabetic rats during baseline and after NADPH oxidase inhibition using apocynin.

Figure 4. Cortical (A) and medullary (B) oxygen tension in control and diabetic rats during baseline and after NADPH oxidase inhibition using apocynin.

In vitro QO2

QO2 in isolated PTC was increased in cells isolated from diabetic rats com-pared to cells from normoglycemic control rats. QO2 was decreased in both groups after inhibition of the NADPH oxidase with apocynin, NHE3 by DMA, and the Na+/K+-ATPase by ouabain. Combination of apocynin with either DMA or ouabain decreased QO2 as well, but did not result in additive decrease in QO2 compared to incubation with DMA or ouabain alone (Figure 5).

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Figure 5. Oxygen consumption in PTC isolated from control and diabetic rats during baseline and after incubation with apocynin to inhibit the NADPH oxidase, DMA to inhibit NHE3 and ouabain to inhibit the Na+/K+-ATPase or apocynin in combination with either DMA or ouabain. * denotes P<0.05 compared to baseline within the same group. # denotes P<0.05 compared to corresponding control

Insulin affects filtration hemodynamics and the mechanism causing diabetes-induced hyperfiltration (Study II) GFR was increased in both untreated and insulin-treated diabetics compared to controls (Figure 6). Increased GFR was accompanied by increased RBF (Figure 7A) in diabetes+insulin and increased FF (Figure 7B) in untreated diabetes. A correlation between RBF and GFR was observed in diabe-tes+insulin, and the regression line was significantly different from both controls and untreated diabetes (Figure 8). PNET was increased in untreated diabetes, but not significantly different from control in diabetes+insulin (Figure 9).

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Figure 6. Glomerular filtration rate in control, untreated diabetes and diabe-tes+insulin treated rats during baseline and after unselective nitric oxide synthase inhibition using L-NAME. * denotes P<0.05 compared to corresponding control.

Figure 7. Renal blood flow (A) and filtration fraction (B) in control, untreated diabe-tes and diabetes+insulin treated rats during baseline and after unselective nitric ox-ide synthase inhibition using L-NAME. * denotes P<0.05 compared to correspond-ing control.

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Figure 8. Correlation between glomerular filtration rate and renal blood flow in control, untreated diabetes and diabetes+insulin treated rats. The slope of the regres-sion line is significantly different between diabetes+insulin compared to both other groups.

Figure 9. Free flow (left), stop flow (middle) and calculated net filtration pressure (right) in control, untreated diabetes and diabetes+insulin treated rats.

Adenosine A2-signaling reduces filtration fraction in controls but not in diabetics (Study III) The adenosine A2 antagonist DMPX increased GFR but decreased RBF in normoglycemic control but was without effect in diabetics (Figures 10 and 11), resulting in increased FF in controls.

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Figure 10. Glomerular filtration rate in control and diabetic mice during baseline and after acute administration of the adenosine A2 receptor antagonist DMPX.

Figure 11. Renal blood flow in control and diabetic mice during baseline and after acute administration of the adenosine A2 receptor antagonist DMPX.

Reduced adenosine A2a signaling mediates increased filtration fraction in diabetes (Study IV) Mean arterial pressure (MAP) was lower in diabetics during baseline but both groups decreased MAP in response to adenosine A2a activation (Figure 12). RBF was not significantly different between the groups and unaffected by adenosine A2a activation in controls and in the low dose in diabetics, but decreased during the high dose (Figure 12). GFR was higher in diabetics and adenosine A2a activation decreased it, but had no effect in controls (Figure 13), resulting in reduced calculated FF only in diabetics (Figure 13).

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Figure 12. Mean arterial pressure (left) and renal blood flow (right) in controls and diabetics during baseline and after infusion of two doses of the adenosine A2a-agonist CGS21680 into the renal artery. * denotes P<0.05 compared to baseline analyzed by Bonferroni´s multiple comparisons test. Result from 2-way ANOVA in left panel: interaction: ns, diabetes: P<0.05, CGS21680: P<0.05 and right panel: interaction: P<0.05, diabetes: ns, CGS21680: ns.

Figure 13. Glomerular filtration rate (left) and filtration fraction (right) in controls and diabetics during baseline and after infusion of two doses of the adenosine A2a-agonist CGS21680 into the renal artery. * denotes P<0.05 compared to baseline analyzed by Bonferroni´s multiple comparisons test. Result from 2-way ANOVA left panel: interaction: P<0.05, diabetes: P<0.05, CGS21680: P<0.05 and right panel: interaction: P<0.05, diabetes: ns, CGS21680: ns

Micropuncture PFF was lower in diabetics compared to controls and not affected by adeno-sine A2a activation (Figure 14). PSF was not significantly different between the groups during baseline, but was decreased by adenosine A2a activation in diabetics (Figure 14). Accordingly, calculated PNET was decreased by adeno-sine A2a activation only in diabetics (Figure 15).

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Figure 14. Free flow pressure (left) and stop flow pressure (right) in controls and diabetics during baseline and after infusion of adenosine A2a-agonist CGS21680 into the renal artery. * denotes P<0.05 compared to baseline analyzed by Bonferroni´s multiple comparisons test. Result from 2-way ANOVA. Left panel: interaction: ns, diabetes: P<0.05, CGS21680: ns. Right panel: interaction: P<0.05, diabetes: P<0.05, CGS21680: P<0.05.

Figure 15. Net filtration pressure in controls and diabetics during baseline and after infusion of the adenosine A2a-agonist CGS21680 into the renal artery. Calculated from PSF-PFF. * denotes P<0.05 compared to baseline analyzed by Bonferroni´s multiple comparisons test. Result from 2-way ANOVA: interaction: P<0.05, diabe-tes: ns, CGS21680: P<0.05.

L-citrulline improves plasma L-arginine in diabetes and prevents diabetes-induced glomerular hyperfiltration and proteinuria (Study V) Baseline GFR was higher in untreated diabetics and diabetics treated with L-arginine compared to corresponding controls, whereas GFR in diabetics treated with L-citrulline was not (Figure 16). RBF was higher in untreated diabetics, but similar to corresponding controls in diabetics treated with ei-ther L-arginine or L-citrulline. Indeed, FF was increased in untreated diabet-ics but not significantly different from corresponding controls in L-arginine and L-citrulline treated diabetics (Figure 17).Untreated and L-arginine treat-

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ed diabetics had elevated urinary protein excretion but L-citrulline treatment prevented this increase (Figure 18).

Figure 16. Glomerular filtration rate in untreated controls and diabetics (A), L-arginine treated controls and diabetics (B) and L-citrulline treated controls and dia-betics (C), during baseline and after unselective nitric oxide synthase inhibition using L-NAME. Result from 2-way ANOVA (A) type: P<0.05 treatment: ns interac-tion: ns (B) type: P<0.05 treatment: ns interaction: ns (C) type: ns treatment: ns interaction: ns.

Figure 17. Filtration fraction in untreated controls and diabetics (A), L-arginine treated controls and diabetics (B) and L-citrulline treated controls and diabetics (C), during baseline and after unselective nitric oxide synthase inhibition using L-NAME. Result from 2-way ANOVA (A) type: P<0.05 treatment: P<0.05 interac-tion: ns (B) type: ns treatment: P<0.05 interaction: ns (C) type: ns treatment: P<0.05 interaction: ns.

Figure 18. Urinary protein excretion in untreated controls and diabetics (A), L-arginine treated controls and diabetics (B) and L-citrulline treated controls and dia-betics (C), during baseline and after unselective nitric oxide synthase inhibition using L-NAME. Result from 2-way ANOVA (A) type: P<0.05 treatment: ns interac-tion: ns (B) type: P<0.05 treatment: ns interaction: ns (C) type: P<0.05 treatment: P<0.05 interaction: P<0.05

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Plasma concentration of L-arginine was decreased in untreated diabetics and not significantly increased by L-arginine supplementation. However, L-citrulline treatment increased plasma L-arginine in both controls and diabet-ics (Figure 19). L-arginine transport by CAT1 is competitively inhibited by L-ornithine and L-lysine. Calculating a ratio between L-arginine and L-ornithine together with L-lysine reveals that this ratio is 1:9 and 1:13 in un-treated controls and diabetics, respectively. This is improved to 1:5 in diabet-ics treated with L-arginine and further improved to 1:2 controls and diabetics treated with L-citrulline (Figure 19).

Figure 19. Plasma concentration of L-arginine (A) and ratio (L-Lys+L-Orn/L-Arg) (B) in untreated controls and diabetics, L-arginine treated controls and diabetics and L-citrulline treated controls and diabetics. * denotes P<0.05 compared to corresponding control, † denotes P<0.05 compared to untreated control and ‡ denotes P<0.05 compared to untreated diabetes.

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Discussion

The main findings from the studies presented in this thesis are that the diabe-tes-induced increase in NADPH oxidase activity increases transport-dependent QO2 in the proximal tubule and contributes to intra-renal hypoxia, but does not affect diabetes-induced hyperfiltration. Insulin on the other hand affects intra-renal hemodynamics, inducing a RBF-dependent regula-tion of GFR and alleviates the diabetes-induced increased PNET and subse-quently FF. Adenosine A2 receptor inhibition increase GFR, despite de-creased RBF in control animals but lacks effect in diabetics. Indeed, adeno-sine A2a activation reduces GFR with maintained RBF, decreasing FF in diabetics by a preferential dilatation of the efferent arteriole decreasing PNET. Furthermore, L-citrulline supplementation in diabetes increases plasma L-arginine concentration, in contrary to L-arginine supplementation, and pre-vents diabetes-induced GFR and proteinuria.

In study I was the correlation between NADPH oxidase activity and tubu-lar reabsorption of Na+ and kidney oxygenation investigated. O2

.- stimulates tubular reabsorption of electrolytes in isolated mTAL by activating the transporters NKCC and NHE3 (70; 86; 87). NADPH oxidase activity is in-creased in diabetes resulting in increased O2

.- production (73; 74; 76) and diabetes-induced transport-dependent QO2 in cells isolated from mTAL has been correlated to NADPH oxidase activity. However, in study I it was shown that diabetes-induced NADPH oxidase activity stimulates Na+ reab-sorption in vivo. Absolute and fractional Na+ excretion was increased sec-ondary to NADPH oxidase inhibition only in diabetics and some parts of this increased reabsorption was localized to the proximal tubule using clearance of Li+ as a marker (88). Though it has been shown that smaller amounts of Li+ can be reabsorbed in mTAL, and under situations of Na+ restriction in cortical collecting duct, increased transport-dependent QO2 from the proxi-mal tubule was confirmed using isolated PTC. Inhibition of either NADPH oxidase or Na+/K+-ATPase reduced QO2 in isolated PTC but their effects were not additive suggesting that NADPH oxidase inhibition directly inter-feres with transport-dependent QO2 in the proximal tubule. NHE3 has previ-ously been shown to be stimulated by O2

.- in isolated mTAL (86), and corti-cal NHE3 activity is increased in diabetes (89), probably via up regulation of Ang II AT1 receptors by the increased oxidative stress, leading to an over activation of NHE3 (90). Since the vast majority of filtered Na+ is reabsorbed by NHE3 it is likely that diabetes-induced increase in NADPH oxidase activ-

36

ity can stimulate proximal tubular transport by this mechanism. Indeed, it was no additive effect on QO2 by NADPH oxidase inhibition when NHE3 was inhibited, providing further support for its involvement. Tissue PO2 was decreased in diabetics in study I confirming previous reports from both ani-mal models and patients (91-93). Decreased PO2 in diabetes could be sec-ondary to increased GFR increasing tubular load of electrolytes that needs to be reabsorbed to maintain Na+ balance. However, this is not likely since NADPH oxidase inhibition increased both cortical and medullary PO2 with-out affecting GFR. Furthermore, chronic treatment with the antioxidant DL-alpha-tocopherol prevented the diabetes-induced decrease in PO2 but not glomerular hyperfiltration (91). Instead oxidative stress seems to reduce tubular transport efficiency and thereby increase basal QO2. This is support-ed by the higher QO2 in PTC isolated from diabetics compared to controls after Na+/K+-ATPase inhibition. This is mechanistically explained by in-creased mitochondria uncoupling in diabetes and its sensitivity to redox sta-tus (94-96).

In study II was the influence of sub-optimal insulin treatment on regula-tion of GFR investigated. Diabetes-induced glomerular hyperfiltration is observed in both animal models of diabetes and in patients (4; 45; 97; 98). Increased GFR in diabetes has been suggested to be either RBF-dependent or pressure-driven. Hyperglycemia results in increased proximal tubular Na+ reabsorption mediated by SGLT, resulting in decreased Na+ delivery to the early distal nephron (4; 99). This will exert an error signal to MD resulting in TGF-inactivation and afferent arteriole dilation increasing RBF and GFR. However, mice lacking a functional TGF-mechanism still develop glomeru-lar hyperfiltration (42; 43), but inhibition of SGLT or knockout of SGLT2 in diabetics normalizes and prevents the diabetes-induced increase in GFR, respectively (4; 50; 99). This is proof for proximal tubular reabsorption as crucial for development of glomerular hyperfiltration. Indeed, PBow is de-creased in diabetes, contributing to increase PNET, and is directly influenced by proximal tubular reabsorption (4; 5; 99) and a correlation between frac-tional Na+ reabsorption and GFR exists in hyperfiltering type 1 diabetic pa-tients (100). Indeed, increased GFR can be accompanied by increased RBF (46-48; 101), but it is not a pre-requisite for development of glomerular hy-perfiltration (9; 45; 97; 102-104) suggesting two different mechanisms that are able to maintain elevated GFR during early diabetes; insulin seems to be an important factor regulating this. Insulin stimulates NO production and induces vasodilation of both afferent and efferent arteriole (105-107). Sub-optimal insulin treatment induced a correlation between RBF and GFR in diabetes, an observation not present in either controls or untreated diabetics. Furthermore, insulin induced a correlation between blood glucose and RBF, and although sub-optimal insulin treatment did not normalize blood glucose it was significantly lower compared to untreated diabetics indicating that both RBF and GFR should be considerably higher in insulin treated diabetics

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if blood glucose levels were comparable. Micropuncture data revealed that untreated diabetics had lower PFF compared to both controls and insulin treated diabetics. Although PSF was not significantly different between the three groups calculated PNET was increased in untreated diabetics but not significantly different from controls in insulin treated diabetics. This is in line with previous a study where acute administration of insulin reduced PNET with maintained glomerular hyperfiltration in diabetics (108). This clearly demonstrates that untreated diabetics have a pressure driven glomerular hy-perfiltration and insulin treated diabetics, presenting normal PNET, have a RBF-dependent glomerular hyperfiltration. Interestingly, RBF was similar between the two diabetic groups suggesting that insulin mechanistically af-fects Kf, since GFR was maintained despite reduced PNET. Intra-renal Ang II is increased in diabetes and contributes to increased FF by constriction of the efferent arteriole (51; 109; 110), which can be off-set by NO-mediated vaso-dilation (111), eventually induced by insulin, explaining the normal PNET. Vasoactive hormones, including Ang II also modulates mesangial cell tone and subsequently GFR, and local renal inhibition of NOS reduces Kf impli-cating a direct tonic control of Kf by NO (112-114). NOS3 and insulin recep-tors are expressed in glomeruli mediating glomerular NO production (115-117). Insulin treatment could therefore have resulted in increased glomerular NO production inducing mesangial cell relaxation, thereby increasing Kf. Higher Kf will drive filtration dynamics towards filtration equilibrium known to result in RBF-dependent regulation of GFR (11; 118) whereas during a higher Kf PNET will be more important (11; 119; 120).

In study III and IV was the influence of adenosine A2a receptor signaling in the glomerular hyperfiltration investigated. Adenosine A2 inhibition in-creased GFR and reduced RBF in controls but was without effect in diabet-ics. As mentioned in previous sections renal autoregulation through the TGF-mechanism is mediated by adenosine A1 receptors, but the response is dampened by simultaneous activation of adenosine A2 receptors (54; 121) and expression studies reveal that adenosine A2 receptors are expressed on both the afferent and efferent arteriole (30; 122). However, the main physio-logical effect of endogenous adenosine signaling is a vasodilation of the efferent arteriole to reduce FF. The physiological effect after blocking ambi-ent adenosine A2 signaling can be explained by differences in sub-type dis-tribution where low-affinity adenosine A2b mediates afferent arteriole dila-tion (122) and efferent arteriole dilation is mediated by combined adenosine A2a and A2b activation (30). Adenosine A1 and A2 receptors are upregulated in diabetes, especially adenosine A2a receptors in renal cortex (123) and dia-betics have increased vascular reactivity to infusion of adenosine (124). This would implicate that lack of effect in diabetics of adenosine A2 inhibition is due to reduced endogenous ligand-activation. Therefore the hypothesis was tested that reduced adenosine A2a signaling in diabetes mediates glomerular hyperfiltration by increasing FF. Indeed, infusion of the adenosine A2a-

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agonist CGS21680 was able to reduce FF and GFR in diabetics but was without effect in controls. This would suggest that adenosine A2a receptors are able to reduce FF and that diabetes is associated with reduced ligand activation and not deranged down-stream signaling. NO bioavailability is reduced in diabetes (80) and adenosine A2a receptor activation increases eNOS activity stimulating NO production (125). Furthermore, eNOS expres-sion is inversely correlated to GFR and antioxidant treatment can prevent diabetes-induced glomerular hyperfiltration (95), indicating that reduced NO bioavailability is initiating increased GFR, eventually due to reduced adeno-sine A2a activation.

Figure 20. Mechanisms investigated in this thesis contributing to increased GFR in diabetes. Increased GFR is caused by increased filtration fraction mediated by re-duced adenosine A2a receptor activation, as shown in study III and IV, most likely due to reduced concentration of adenosine locally around the arterioles leading to a vasoconstriction preferentially of the efferent arteriole. Decreased pressure in Bow-man’s space, increasing net filtration pressure was observed in study II and IV, also known to be able to affect filtration fraction.

In study V was the outcome of normalized plasma concentration of L-arginine to improve NO production in diabetes investigated. L-citrulline but not L-arginine supplementation was effective in improving plasma concen-tration of L-arginine. This prevented the glomerular hyperfiltration and in-creased urinary protein excretion but not diabetes-induced QO2. Plasma L-arginine is reduced in diabetes (126), and this might be crucial for disease progression. Despite high intracellular concentration above the Km-value for NOS (127), NO production is highly dependent on extracellular L-arginine (128), and subsequently L-arginine transport across the plasma membrane via CAT1 which is a rate-limiting step in the NO production due to a caveo-lar complex between CAT1 and eNOS (129-131). This suggests a direct

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presentation of L-arginine by CAT1. The superior effect of L-citrulline com-pared to L-arginine in improving plasma L-arginine concentration is ex-plained by induction of de novo synthesis of L-arginine by L-citrulline. L-citrulline is normally synthesized from L-glutamine in enterocytes and re-leased to the venous blood (132), filtered by the kidneys and reabsorbed in the proximal tubule. Proximal tubules express argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), and are therefore able to synthe-size L-arginine from L-citrulline (133-135). Furthermore, ASL is an adaptor protein binding CAT1 and NOS together keeping de novo synthesis of L-arginine in close proximity to the NOS (136). It has previously been shown that NO regulates kidney QO2 and PO2 (80; 81), but the outcome of study V with normalized GFR and proteinuria after increased plasma L-arginine con-centration, but not QO2 and PO2, indicates that the potential NO-mediated effect occurred primarily in the vasculature and not in the proximal tubule.

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Summary and conclusions

The main findings of this thesis are that increased NADPH oxidase activity increases transport-dependent QO2 in the proximal tubule and contributes to intra-renal hypoxia in diabetes, but does not affect the hyperfiltration. Insulin on the other hand affect the mechanisms causing diabetes-induced hyperfil-tration by altering intra-renal hemodynamics, inducing a RBF-dependent regulation of GFR but reduces PNET and subsequently FF. Adenosine A2 receptor inhibition increase GFR, despite decreased RBF in control animals but lacks effect in diabetics. Indeed, adenosine A2a activation reduces GFR with maintained RBF, decreasing FF in diabetics by a preferential dilatation of the efferent arteriole decreasing PNET. Furthermore, L-citrulline supple-mentation in diabetes increases plasma L-arginine concentration, in opposite to L-arginine supplementation, which subsequently prevents diabetes-induced hyperfiltration and proteinuria.

• NADPH oxidase activity determines transport-dependent QO2 and intra-renal PO2 in diabetes.

• Insulin induces RBF-dependent regulation of GFR but alleviates in-creased PNET in diabetes.

• Adenosine A2 receptor activity decreases FF in controls but not in dia-betics.

• Reduced adenosine A2a receptor activation causes diabetes-induced hy-perfiltration by increasing FF.

• L-citrulline increases plasma L-arginine in diabetics and prevents diabe-tes-induced hyperfiltration and proteinuria.

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Sammanfattning på svenska

Syftet med undersökningarna som ingår i denna avhandling har varit att klargöra vilka mekanismer som bidrar till en förändrad filtrationshastighet och syrgasmetabolism i njuren vid diabetes. Bakgrunden till genomförandet bygger på att diabetes är den vanligaste orsaken till en sviktande njurfunkt-ion, så kallad diabetesnefropati, som kräver dialysbehandling eller organ-transplantation. Den tidigaste förändringen i njuren vid diabetes är att glome-rulära filtrationshastigheten (GFR) stiger, dock ej hos alla individer, vilket har lett till att undersökningar initierats med mål att utvärdera om ett ökat GFR i det tidiga förloppet kan sammankopplas med en mer progressiv för-sämring av njurfunktionen. Kliniskt definieras graden av njursjukdom efter utsöndringen av albumin i urinen, graderat som normoalbuminuri <30 mg/24 h, mikroalbuminuri 30-300 mg/24 h och slutligen makroalbuminuri >300 mg/24 h. Med ökad albuminuri avtar GFR succesivt tills uremi uppstår och dialysbehandling krävs. I nyligen publicerade meta-analyser har diabetisk hyperfiltration inte kunnat kopplas samman med utveckligen av albuminuri men med ett GFR som sjunker snabbare. Tidiga förändringar, observerade framförallt i djurmodeller, som föregår kliniska tecken på njursjukdom som albuminuri och reducerat GFR inkluderar ökad produktion av fria syrgasra-dikaler och minskad syrgastension i vävnaden. Diabetes via hyperglykemin stimulerar radikalproduktion från flera källor, bland annat från mitokondrien, överaktiverat NADPH-oxidas och frikopplat kväveoxidsyntas. I studie I visas att fria syrgasradikaler producerade från ett överaktiverat NADPH-oxidas stimulerar transportberoende syrgaskonsumtion i isolerade proximala tubulusceller och Na+-transport in vivo, då både absolut och fraktionell ut-söndring av Na+ ökade efter akut inhibition av NADPH-oxidaset i diabetiska råttor men ej i friska kontroller. Detta resulterade också i en förbättrad syr-gastension i både cortex och medulla. I studie II undersöktes mekanismerna bakom diabetesinducerad hyperfiltration och effekterna av insulin. Tre grup-per studerades, kontroller, diabetiker och diabetiker med suboptimal insulin-behandling. Huvudfynden visar att obehandlade diabetiker har ett ökat netto-filtrationstryck och således en tryckberoende hyperfiltration medan subopti-mal insulinbehandling normaliserar nettofiltrationstrycket men orsakar en blodflödesmedierad hyperfiltration. I studie III utvärderades om en förändrad adenosin A2-receptormedierad signalering vid diabetes påverkade GFR. Två grupper studerades, kontroll och diabetes, dels basalt, dels efter akut adeno-sin A2-inhibition. Huvudfynden visar att kontroller har en endogen A2-

42

signalering vilken primärt dilaterar post-glomerulära kärl, vilket minskar GFR genom att minska filtrationsfraktionen. Denna signalering är frånva-rande i diabetiker då adenosin A2-inhibition är utan effekt, vilket genererar hypotesen att minskad endogen adeonsin A2-signalering medierar hyperfilt-rationen. Detta testades i studie IV där adeonsin A2a-agonisten CGS21680 infunderades i njurartären till kontroller och diabetiker. Huvudfynden visar att exogen adenosin A2a-aktivering minskar GFR utan att påverka blodflödet i diabetiker men ej i kontroller där behandligen är utan effekt. I mikropunkt-ionsexperiment verifieras att adenosin A2a-aktivering minskar nettofiltrat-ionstrycket genom att minska trycket i glomerulära kapillärer i diabetiker. I studie V var målet att normalisera plasmakoncentrationen av L-arginin för att inducera ökad kväveoxidproduktion. Huvudfynden visar att behandling med L-citrullin, till skillnad från L-arginin, effektivt ökar plasmakoncentrat-ionen av L-arginin. Denna behandling förhindrar glomerulär hyperfiltration och även ökad proteinuri vid diabetes.

Sammanfattningsvis visar denna avhandling att glomerulär hyperfiltration vid diabetes orsakas av minskad adenosinsignalering via A2a-receptorn och minskad plasmakoncentration av L-arginin, vilket även bidrar till utvecklan-det av proteinuri. Vidare, fria syrgasradikaler från NADPH-oxidaset ökar transportberoende syrgaskonsumtion i njuren vilket samtidigt bidrar till minskad renal syrgastension.

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Acknowledgements

This thesis was carried out at the Department of Medical Cell Biology, Divi-sion of Integrative Physiology, Uppsala University, Sweden.

I would like to thank everyone involved in one way or another in the prepa-ration of this thesis. Especially I would like to thank my excellent supervisor Fredrik Palm for good discussions, advices and support and for giving great input when necessary but perhaps more importantly for allowing me to work independently and test my own ideas. I think it is hard to find a better super-visor. Also, big thanks to my co-supervisor Peter Hansell for always having the time to explain and making any kind of administrative issue to quickly disappear. Angelica Fasching for always keeping the lab organized and en-during through the never-ending l-arginine, l-citrulline, alpha-tocopherol, nitrate/nitrite free diets and insulin treatments. And last but not least, every-body else in the kidney group, Ebba, Lina, Liselotte, Micke, Per2, Sara and Stephanie for contributing to a great research group.

My co-authors for fruitful collaborations.

The administrative staff at the department for making everyday life easy, especially Gunno, Antoine, Björn, Lina, Camilla, Marianne and Shumin for making teaching assignments and administration running smooth.

Seniors and fellow PhD-students, especially in the physiology corridor for creating a good atmosphere. Örjan Källskog for spending time to teach me the renal micropuncture technique (at least a fraction of it) during the first year, and even if the initial micropuncture plans failed, it became useful and was applied in study II and IV.

Thanks to Drs. Christopher Wilcox and William Welch at the Division of Nephrology and Hypertension, Georgetown University for the time I spent at your lab during spring 2011.

My parents Lena and PerOla for always believing in me. Karin, Anita, Leif and Emy for all support during the years. Also my parents-in-law, Irene and Pelle, it is always fun to come to Hjälsta.

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Finally, Malou for all love, support and understanding.

The work presented was supported by grants from the Swedish Research Council, the Swedish Diabetes Foundation, the Wallenberg Foundations, the Ernfors Foundation, the Åke Wiberg Foundation, NIH/NIDDK K99/R00 and the Swedish Society for Medical Research.

Uppsala, March 2013

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References

1. Bell PD, Komlosi P, Zhang ZR: ATP as a mediator of macula densa cell signalling. Purinergic signalling 2009;5:461-471

2. Leyssac PP, Karlsen FM, Skott O: Dynamics of intrarenal pressures and glomerular filtration rate after acetazolamide. Am J Physiol 1991;261:F169-178

3. Hostetter TH, Troy JL, Brenner BM: Glomerular hemodynamics in experimental diabetes mellitus. Kidney Int 1981;19:410-415

4. Vallon V, Richter K, Blantz RC, Thomson S, Osswald H: Glomerular hyperfiltration in experimental diabetes mellitus: potential role of tubular reabsorption. J Am Soc Nephrol 1999;10:2569-2576

5. Leyssac PP, Karlsen FM, Holstein-Rathlou NH, Skott O: On determinants of glomerular filtration rate after inhibition of proximal tubular reabsorption. Am J Physiol 1994;266:R1544-1550

6. Leyssac PP, Holstein-Rathlou NH, Skott O: Renal blood flow, early distal sodium, and plasma renin concentrations during osmotic diuresis. Am J Physiol Regul Integr Comp Physiol 2000;279:R1268-1276

7. Thorup C, Ollerstam A, Persson AE, Torffvit O: Increased tubuloglomerular feedback reactivity is associated with increased NO production in the streptozotocin-diabetic rat. J Diabetes Complications 2000;14:46-52

8. Sorensen CM, Leyssac PP, Salomonsson M, Skott O, Holstein-Rathlou NH: ANG II-induced downregulation of RBF after a prolonged reduction of renal perfusion pressure is due to pre- and postglomerular constriction. Am J Physiol Regul Integr Comp Physiol 2004;286:R865-873

9. Nordquist L, Brown R, Fasching A, Persson P, Palm F: Proinsulin C-peptide reduces diabetes-induced glomerular hyperfiltration via efferent arteriole dilation and inhibition of tubular sodium reabsorption. Am J Physiol Renal Physiol 2009;297:F1265-1272

10. Brenner BM, Troy JL, Daugharty TM: The dynamics of glomerular ultrafiltration in the rat. J Clin Invest 1971;50:1776-1780

11. Arendshorst WJ, Gottschalk CW: Glomerular ultrafiltration dynamics: euvolemic and plasma volume-expanded rats. Am J Physiol 1980;239:F171-186

12. Axelsson J, Rippe A, Oberg CM, Rippe B: Rapid, dynamic changes in glomerular permeability to macromolecules during systemic angiotensin II (ANG II) infusion in rats. Am J Physiol Renal Physiol 2012;303:F790-799

13. Gutierrez AM, Kornfeld M, Persson AE: Calcium response to adenosine and ATP in rabbit afferent arterioles. Acta Physiol Scand 1999;166:175-181

46

14. Weihprecht H, Lorenz JN, Briggs JP, Schnermann J: Vasomotor effects of purinergic agonists in isolated rabbit afferent arterioles. Am J Physiol 1992;263:F1026-1033

15. Brown R, Ollerstam A, Johansson B, Skott O, Gebre-Medhin S, Fredholm B, Persson AE: Abolished tubuloglomerular feedback and increased plasma renin in adenosine A1 receptor-deficient mice. Am J Physiol Regul Integr Comp Physiol 2001;281:R1362-1367

16. Sun D, Samuelson LC, Yang T, Huang Y, Paliege A, Saunders T, Briggs J, Schnermann J: Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors. Proc Natl Acad Sci U S A 2001;98:9983-9988

17. Osswald H, Spielman WS, Knox FG: Mechanism of adenosine-mediated decreases in glomerular filtration rate in dogs. Circ Res 1978;43:465-469

18. Deussen A, Ohanyan V, Jannasch A, Yin L, Chilian W: Mechanisms of metabolic coronary flow regulation. J Mol Cell Cardiol 2012;52:794-801

19. Vitzthum H, Weiss B, Bachleitner W, Kramer BK, Kurtz A: Gene expression of adenosine receptors along the nephron. Kidney Int 2004;65:1180-1190

20. Smith JA, Sivaprasadarao A, Munsey TS, Bowmer CJ, Yates MS: Immunolocalisation of adenosine A(1) receptors in the rat kidney. Biochemical pharmacology 2001;61:237-244

21. Yamaguchi S, Umemura S, Tamura K, Iwamoto T, Nyui N, Ishigami T, Ishii M: Adenosine A1 receptor mRNA in microdissected rat nephron segments. Hypertension 1995;26:1181-1185

22. Takeda M, Yoshitomi K, Imai M: Regulation of Na(+)-3HCO3- cotransport in rabbit proximal convoluted tubule via adenosine A1 receptor. Am J Physiol 1993;265:F511-519

23. Di Sole F, Cerull R, Petzke S, Casavola V, Burckhardt G, Helmle-Kolb C: Bimodal acute effects of A1 adenosine receptor activation on Na+/H+ exchanger 3 in opossum kidney cells. J Am Soc Nephrol 2003;14:1720-1730

24. Beach RE, Good DW: Effects of adenosine on ion transport in rat medullary thick ascending limb. Am J Physiol 1992;263:F482-487

25. Beach RE, Watts BA, 3rd, Good DW, Benedict CR, DuBose TD, Jr.: Effects of graded oxygen tension on adenosine release by renal medullary and thick ascending limb suspensions. Kidney Int 1991;39:836-842

26. Freissmuth M, Nanoff C, Tuisl E, Schuetz W: Stimulation of adenylate cyclase activity via A2-adenosine receptors in isolated tubules of the rabbit renal cortex. Eur J Pharmacol 1987;138:137-140

27. Nakamoto H, Ogasawara Y, Kajiya F: Visualisation of the effects of dilazep on rat afferent and efferent arterioles in vivo. Hypertens Res 2008;31:315-324

28. Fredholm BB, Irenius E, Kull B, Schulte G: Comparison of the potency of adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cells. Biochemical pharmacology 2001;61:443-448

29. Jackson EK, Zhu C, Tofovic SP: Expression of adenosine receptors in the preglomerular microcirculation. Am J Physiol Renal Physiol 2002;283:F41-51

30. Al-Mashhadi RH, Skott O, Vanhoutte PM, Hansen PB: Activation of A(2) adenosine receptors dilates cortical efferent arterioles in mouse. Kidney Int 2009;75:793-799

47

31. Agmon Y, Dinour D, Brezis M: Disparate effects of adenosine A1- and A2-receptor agonists on intrarenal blood flow. Am J Physiol 1993;265:F802-806

32. Zou AP, Nithipatikom K, Li PL, Cowley AW, Jr.: Role of renal medullary adenosine in the control of blood flow and sodium excretion. Am J Physiol 1999;276:R790-798

33. Hovind P, Tarnow L, Rossing P, Jensen BR, Graae M, Torp I, Binder C, Parving HH: Predictors for the development of microalbuminuria and macroalbuminuria in patients with type 1 diabetes: inception cohort study. BMJ 2004;328:1105

34. Levey AS, Coresh J: Chronic kidney disease. Lancet 2012;379:165-180 35. Moriya T, Tsuchiya A, Okizaki S, Hayashi A, Tanaka K, Shichiri M:

Glomerular hyperfiltration and increased glomerular filtration surface are associated with renal function decline in normo- and microalbuminuric type 2 diabetes. Kidney Int 2012;81:486-493

36. Ruggenenti P, Porrini EL, Gaspari F, Motterlini N, Cannata A, Carrara F, Cella C, Ferrari S, Stucchi N, Parvanova A, Iliev I, Dodesini AR, Trevisan R, Bossi A, Zaletel J, Remuzzi G, Investigators GFRS: Glomerular hyperfiltration and renal disease progression in type 2 diabetes. Diabetes care 2012;35:2061-2068

37. Kanai Y, Lee WS, You G, Brown D, Hediger MA: The human kidney low affinity Na+/glucose cotransporter SGLT2. Delineation of the major renal reabsorptive mechanism for D-glucose. J Clin Invest 1994;93:397-404

38. Vallon V, Platt KA, Cunard R, Schroth J, Whaley J, Thomson SC, Koepsell H, Rieg T: SGLT2 mediates glucose reabsorption in the early proximal tubule. J Am Soc Nephrol 2011;22:104-112

39. Balen D, Ljubojevic M, Breljak D, Brzica H, Zlender V, Koepsell H, Sabolic I: Revised immunolocalization of the Na+-D-glucose cotransporter SGLT1 in rat organs with an improved antibody. Am J Physiol Cell Physiol 2008;295:C475-489

40. Lee WS, Kanai Y, Wells RG, Hediger MA: The high affinity Na+/glucose cotransporter. Re-evaluation of function and distribution of expression. J Biol Chem 1994;269:12032-12039

41. Bank N, Aynedjian HS: Progressive increases in luminal glucose stimulate proximal sodium absorption in normal and diabetic rats. J Clin Invest 1990;86:309-316

42. Sallstrom J, Carlsson PO, Fredholm BB, Larsson E, Persson AE, Palm F: Diabetes-induced hyperfiltration in adenosine A(1)-receptor deficient mice lacking the tubuloglomerular feedback mechanism. Acta Physiol (Oxf) 2007;190:253-259

43. Faulhaber-Walter R, Chen L, Oppermann M, Kim SM, Huang Y, Hiramatsu N, Mizel D, Kajiyama H, Zerfas P, Briggs JP, Kopp JB, Schnermann J: Lack of A1 adenosine receptors augments diabetic hyperfiltration and glomerular injury. J Am Soc Nephrol 2008;19:722-730

44. Persson P, Hansell P, Palm F: NADPH oxidase inhibition reduces tubular sodium transport and improves kidney oxygenation in diabetes. Am J Physiol Regul Integr Comp Physiol 2012;302:R1443-1449

48

45. Palm F, Fasching A, Hansell P, Kallskog O: Nitric oxide originating from NOS1 controls oxygen utilization and electrolyte transport efficiency in the diabetic kidney. Am J Physiol Renal Physiol 2010;298:F416-420

46. Bell TD, DiBona GF, Biemiller R, Brands MW: Continuously measured renal blood flow does not increase in diabetes if nitric oxide synthesis is blocked. Am J Physiol Renal Physiol 2008;295:F1449-1456

47. Jensen PK, Christiansen JS, Steven K, Parving HH: Renal function in streptozotocin-diabetic rats. Diabetologia 1981;21:409-414

48. Veelken R, Hilgers KF, Hartner A, Haas A, Bohmer KP, Sterzel RB: Nitric oxide synthase isoforms and glomerular hyperfiltration in early diabetic nephropathy. J Am Soc Nephrol 2000;11:71-79

49. Seyer-Hansen K, Hansen J, Gundersen HJ: Renal hypertrophy in experimental diabetes. A morphometric study. Diabetologia 1980;18:501-505

50. Vallon V, Rose M, Gerasimova M, Satriano J, Platt KA, Koepsell H, Cunard R, Sharma K, Thomson SC, Rieg T: Knockout of Na-glucose transporter SGLT2 attenuates hyperglycemia and glomerular hyperfiltration but not kidney growth or injury in diabetes mellitus. Am J Physiol Renal Physiol 2012;

51. Zatz R, Dunn BR, Meyer TW, Anderson S, Rennke HG, Brenner BM: Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. J Clin Invest 1986;77:1925-1930

52. Miller JA: Impact of hyperglycemia on the renin angiotensin system in early human type 1 diabetes mellitus. J Am Soc Nephrol 1999;10:1778-1785

53. Sochett EB, Cherney DZ, Curtis JR, Dekker MG, Scholey JW, Miller JA: Impact of renin angiotensin system modulation on the hyperfiltration state in type 1 diabetes. J Am Soc Nephrol 2006;17:1703-1709

54. Carlstrom M, Wilcox CS, Welch WJ: Adenosine A(2) receptors modulate tubuloglomerular feedback. Am J Physiol Renal Physiol 2010;299:F412-417

55. Thannickal VJ, Fanburg BL: Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 2000;279:L1005-1028

56. El-Benna J, Dang PM, Gougerot-Pocidalo MA: Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane. Seminars in immunopathology 2008;30:279-289

57. Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C: Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases. Cochrane database of systematic reviews 2008:CD007176

58. Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, Keogh JP, Meyskens FL, Valanis B, Williams JH, Barnhart S, Hammar S: Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. The New England journal of medicine 1996;334:1150-1155

59. Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino I, Brownlee M: Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000;404:787-790

49

60. Youn JY, Gao L, Cai H: The p47phox- and NADPH oxidase organiser 1 (NOXO1)-dependent activation of NADPH oxidase 1 (NOX1) mediates endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction in a streptozotocin-induced murine model of diabetes. Diabetologia 2012;55:2069-2079

61. Desco MC, Asensi M, Marquez R, Martinez-Valls J, Vento M, Pallardo FV, Sastre J, Vina J: Xanthine oxidase is involved in free radical production in type 1 diabetes: protection by allopurinol. Diabetes 2002;51:1118-1124

62. Matsumoto S, Koshiishi I, Inoguchi T, Nawata H, Utsumi H: Confirmation of superoxide generation via xanthine oxidase in streptozotocin-induced diabetic mice. Free radical research 2003;37:767-772

63. Sonta T, Inoguchi T, Tsubouchi H, Sekiguchi N, Kobayashi K, Matsumoto S, Utsumi H, Nawata H: Evidence for contribution of vascular NAD(P)H oxidase to increased oxidative stress in animal models of diabetes and obesity. Free Radic Biol Med 2004;37:115-123

64. Fukai T, Ushio-Fukai M: Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal 2011;15:1583-1606

65. Fujita H, Fujishima H, Takahashi K, Sato T, Shimizu T, Morii T, Shimizu T, Shirasawa T, Qi Z, Breyer MD, Harris RC, Yamada Y, Takahashi T: SOD1, but not SOD3, deficiency accelerates diabetic renal injury in C57BL/6-Ins2(Akita) diabetic mice. Metabolism: clinical and experimental 2012;61:1714-1724

66. Marra G, Cotroneo P, Pitocco D, Manto A, Di Leo MA, Ruotolo V, Caputo S, Giardina B, Ghirlanda G, Santini SA: Early increase of oxidative stress and reduced antioxidant defenses in patients with uncomplicated type 1 diabetes: a case for gender difference. Diabetes care 2002;25:370-375

67. Opara EC, Abdel-Rahman E, Soliman S, Kamel WA, Souka S, Lowe JE, Abdel-Aleem S: Depletion of total antioxidant capacity in type 2 diabetes. Metabolism: clinical and experimental 1999;48:1414-1417

68. Modlinger P, Chabrashvili T, Gill PS, Mendonca M, Harrison DG, Griendling KK, Li M, Raggio J, Wellstein A, Chen Y, Welch WJ, Wilcox CS: RNA silencing in vivo reveals role of p22phox in rat angiotensin slow pressor response. Hypertension 2006;47:238-244

69. Lassegue B, Clempus RE: Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol 2003;285:R277-297

70. Ortiz PA, Garvin JL: Superoxide stimulates NaCl absorption by the thick ascending limb. Am J Physiol Renal Physiol 2002;283:F957-962

71. Selemidis S, Sobey CG, Wingler K, Schmidt HH, Drummond GR: NADPH oxidases in the vasculature: molecular features, roles in disease and pharmacological inhibition. Pharmacol Ther 2008;120:254-291

72. Gill PS, Wilcox CS: NADPH oxidases in the kidney. Antioxid Redox Signal 2006;8:1597-1607

73. Edlund J, Fasching A, Liss P, Hansell P, Palm F: The roles of NADPH-oxidase and nNOS for the increased oxidative stress and the oxygen consumption in the diabetic kidney. Diabetes Metab Res Rev 2010;26:349-356

50

74. Asaba K, Tojo A, Onozato ML, Goto A, Quinn MT, Fujita T, Wilcox CS: Effects of NADPH oxidase inhibitor in diabetic nephropathy. Kidney Int 2005;67:1890-1898

75. Etoh T, Inoguchi T, Kakimoto M, Sonoda N, Kobayashi K, Kuroda J, Sumimoto H, Nawata H: Increased expression of NAD(P)H oxidase subunits, NOX4 and p22phox, in the kidney of streptozotocin-induced diabetic rats and its reversibity by interventive insulin treatment. Diabetologia 2003;46:1428-1437

76. Yang J, Lane PH, Pollock JS, Carmines PK: Protein kinase C-dependent NAD(P)H oxidase activation induced by type 1 diabetes in renal medullary thick ascending limb. Hypertension 2010;55:468-473

77. Welch WJ, Wilcox CS, Thomson SC: Nitric oxide and tubuloglomerular feedback. Semin Nephrol 1999;19:251-262

78. Ortiz PA, Garvin JL: Role of nitric oxide in the regulation of nephron transport. Am J Physiol Renal Physiol 2002;282:F777-784

79. Koivisto A, Matthias A, Bronnikov G, Nedergaard J: Kinetics of the inhibition of mitochondrial respiration by NO. FEBS Lett 1997;417:75-80

80. Palm F, Buerk DG, Carlsson PO, Hansell P, Liss P: Reduced nitric oxide concentration in the renal cortex of streptozotocin-induced diabetic rats: effects on renal oxygenation and microcirculation. Diabetes 2005;54:3282-3287

81. Laycock SK, Vogel T, Forfia PR, Tuzman J, Xu X, Ochoa M, Thompson CI, Nasjletti A, Hintze TH: Role of nitric oxide in the control of renal oxygen consumption and the regulation of chemical work in the kidney. Circ Res 1998;82:1263-1271

82. Kakoki M, Kim HS, Arendshorst WJ, Mattson DL: L-Arginine uptake affects nitric oxide production and blood flow in the renal medulla. Am J Physiol Regul Integr Comp Physiol 2004;287:R1478-1485

83. Werner ER, Gorren AC, Heller R, Werner-Felmayer G, Mayer B: Tetrahydrobiopterin and nitric oxide: mechanistic and pharmacological aspects. Exp Biol Med (Maywood) 2003;228:1291-1302

84. Fleming I, Busse R: Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 2003;284:R1-12

85. Deves R, Boyd CA: Transporters for cationic amino acids in animal cells: discovery, structure, and function. Physiol Rev 1998;78:487-545

86. Juncos R, Hong NJ, Garvin JL: Differential effects of superoxide on luminal and basolateral Na+/H+ exchange in the thick ascending limb. Am J Physiol Regul Integr Comp Physiol 2006;290:R79-83

87. Juncos R, Garvin JL: Superoxide enhances Na-K-2Cl cotransporter activity in the thick ascending limb. Am J Physiol Renal Physiol 2005;288:F982-987

88. Koomans HA, Boer WH, Dorhout Mees EJ: Evaluation of lithium clearance as a marker of proximal tubule sodium handling. Kidney Int 1989;36:2-12

89. Klisic J, Nief V, Reyes L, Ambuhl PM: Acute and chronic regulation of the renal Na/H+ exchanger NHE3 in rats with STZ-induced diabetes mellitus. Nephron Physiol 2006;102:p27-35

90. Banday AA, Lokhandwala MF: Oxidative stress causes renal angiotensin II type 1 receptor upregulation, Na+/H+ exchanger 3 overstimulation, and hypertension. Hypertension 2011;57:452-459

51

91. Palm F, Cederberg J, Hansell P, Liss P, Carlsson PO: Reactive oxygen species cause diabetes-induced decrease in renal oxygen tension. Diabetologia 2003;46:1153-1160

92. Edlund J, Hansell P, Fasching A, Liss P, Weis J, Glickson JD, Palm F: Reduced oxygenation in diabetic rat kidneys measured by T2* weighted magnetic resonance micro-imaging. Adv Exp Med Biol 2009;645:199-204

93. Yin WJ, Liu F, Li XM, Yang L, Zhao S, Huang ZX, Huang YQ, Liu RB: Noninvasive evaluation of renal oxygenation in diabetic nephropathy by BOLD-MRI. European journal of radiology 2012;81:1426-1431

94. Friederich M, Fasching A, Hansell P, Nordquist L, Palm F: Diabetes-induced up-regulation of uncoupling protein-2 results in increased mitochondrial uncoupling in kidney proximal tubular cells. Biochim Biophys Acta 2008;1777:935-940

95. Persson MF, Franzen S, Catrina SB, Dallner G, Hansell P, Brismar K, Palm F: Coenzyme Q10 prevents GDP-sensitive mitochondrial uncoupling, glomerular hyperfiltration and proteinuria in kidneys from db/db mice as a model of type 2 diabetes. Diabetologia 2012;55:1535-1543

96. Echtay KS, Roussel D, St-Pierre J, Jekabsons MB, Cadenas S, Stuart JA, Harper JA, Roebuck SJ, Morrison A, Pickering S, Clapham JC, Brand MD: Superoxide activates mitochondrial uncoupling proteins. Nature 2002;415:96-99

97. Berg UB, Torbjornsdotter TB, Jaremko G, Thalme B: Kidney morphological changes in relation to long-term renal function and metabolic control in adolescents with IDDM. Diabetologia 1998;41:1047-1056

98. O'Donnell MP, Kasiske BL, Keane WF: Glomerular hemodynamic and structural alterations in experimental diabetes mellitus. FASEB J 1988;2:2339-2347

99. Pollock CA, Lawrence JR, Field MJ: Tubular sodium handling and tubuloglomerular feedback in experimental diabetes mellitus. Am J Physiol 1991;260:F946-952

100. Vervoort G, Veldman B, Berden JH, Smits P, Wetzels JF: Glomerular hyperfiltration in type 1 diabetes mellitus results from primary changes in proximal tubular sodium handling without changes in volume expansion. European journal of clinical investigation 2005;35:330-336

101. Laborde K, Levy-Marchal C, Kindermans C, Dechaux M, Czernichow P, Sachs C: Glomerular function and microalbuminuria in children with insulin-dependent diabetes. Pediatr Nephrol 1990;4:39-43

102. Berg UB, Thalme B: Early renal functional changes in children with insulin-dependent diabetes mellitus--their relation to metabolic control. Int J Pediatr Nephrol 1984;5:16-21

103. Ditzel J, Junker K: Abnormal glomerular filtration rate, renal plasma flow, and renal protein excretion in recent and short-term diabetics. Br Med J 1972;2:13-19

104. Mogensen CE: Glomerular filtration rate and renal plasma flow in short-term and long-term juvenile diabetes mellitus. Scand J Clin Lab Invest 1971;28:91-100

52

105. Hayashi K, Fujiwara K, Oka K, Nagahama T, Matsuda H, Saruta T: Effects of insulin on rat renal microvessels: studies in the isolated perfused hydronephrotic kidney. Kidney Int 1997;51:1507-1513

106. Hayashi K, Matsuda H, Nagahama T, Fujiwara K, Ozawa Y, Kubota E, Honda M, Tokuyama H, Saruta T: Impaired nitric oxide-independent dilation of renal afferent arterioles in spontaneously hypertensive rats. Hypertens Res 1999;22:31-37

107. Trovati M, Massucco P, Mattiello L, Cavalot F, Mularoni E, Hahn A, Anfossi G: Insulin increases cyclic nucleotide content in human vascular smooth muscle cells: a mechanism potentially involved in insulin-induced modulation of vascular tone. Diabetologia 1995;38:936-941

108. Scholey JW, Meyer TW: Control of glomerular hypertension by insulin administration in diabetic rats. J Clin Invest 1989;83:1384-1389

109. Hsieh TJ, Zhang SL, Filep JG, Tang SS, Ingelfinger JR, Chan JS: High glucose stimulates angiotensinogen gene expression via reactive oxygen species generation in rat kidney proximal tubular cells. Endocrinology 2002;143:2975-2985

110. Ohashi N, Urushihara M, Satou R, Kobori H: Glomerular angiotensinogen is induced in mesangial cells in diabetic rats via reactive oxygen species--ERK/JNK pathways. Hypertens Res 2010;33:1174-1181

111. De Nicola L, Blantz RC, Gabbai FB: Nitric oxide and angiotensin II. Glomerular and tubular interaction in the rat. J Clin Invest 1992;89:1248-1256

112. Blantz RC, Konnen KS, Tucker BJ: Angiotensin II effects upon the glomerular microcirculation and ultrafiltration coefficient of the rat. J Clin Invest 1976;57:419-434

113. Ichikawa I, Miele JF, Brenner BM: Reversal of renal cortical actions of angiotensin II by verapamil and manganese. Kidney Int 1979;16:137-147

114. Deng A, Baylis C: Locally produced EDRF controls preglomerular resistance and ultrafiltration coefficient. Am J Physiol 1993;264:F212-215

115. Shultz PJ, Tayeh MA, Marletta MA, Raij L: Synthesis and action of nitric oxide in rat glomerular mesangial cells. Am J Physiol 1991;261:F600-606

116. Kurokawa K, Silverblatt FJ, Klein KL, Wang MS, Lerner RL: Binding of 125I-insulin to the isolated glomeruli of rat kidney. J Clin Invest 1979;64:1357-1364

117. Potenza MA, Addabbo F, Montagnani M: Vascular actions of insulin with implications for endothelial dysfunction. Am J Physiol Endocrinol Metab 2009;297:E568-577

118. Brenner BM, Troy JL, Daugharty TM, Deen WM, Robertson CR: Dynamics of glomerular ultrafiltration in the rat. II. Plasma-flow dependence of GFR. Am J Physiol 1972;223:1184-1190

119. Kallskog O, Lindbom LO, Ulfendahl HR, Wolgast M: Kinetics of the glomerular ultrafiltration in the rat kidney. An experimental study. Acta Physiol Scand 1975;95:293-300

120. Kallskog O, Lindbom LO, Ulfendahl HR, Wolgast M: Kinetics of the glomerular ultrafiltration in the rat kidney. A theoretical study. Acta Physiol Scand 1975;95:191-200

53

121. Carlstrom M, Wilcox CS, Welch WJ: Adenosine A2A receptor activation attenuates tubuloglomerular feedback responses by stimulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol 2011;300:F457-464

122. Feng MG, Navar LG: Afferent arteriolar vasodilator effect of adenosine predominantly involves adenosine A2B receptor activation. Am J Physiol Renal Physiol 2010;299:F310-315

123. Pawelczyk T, Grden M, Rzepko R, Sakowicz M, Szutowicz A: Region-specific alterations of adenosine receptors expression level in kidney of diabetic rat. Am J Pathol 2005;167:315-325

124. Pflueger AC, Schenk F, Osswald H: Increased sensitivity of the renal vasculature to adenosine in streptozotocin-induced diabetes mellitus rats. Am J Physiol 1995;269:F529-535

125. Hansen PB, Hashimoto S, Oppermann M, Huang Y, Briggs JP, Schnermann J: Vasoconstrictor and vasodilator effects of adenosine in the mouse kidney due to preferential activation of A1 or A2 adenosine receptors. J Pharmacol Exp Ther 2005;315:1150-1157

126. Palm F, Friederich M, Carlsson PO, Hansell P, Teerlink T, Liss P: Reduced nitric oxide in diabetic kidneys due to increased hepatic arginine metabolism: implications for renomedullary oxygen availability. Am J Physiol Renal Physiol 2008;294:F30-37

127. Palmer RM, Ashton DS, Moncada S: Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 1988;333:664-666

128. Vukosavljevic N, Jaron D, Barbee KA, Buerk DG: Quantifying the L-arginine paradox in vivo. Microvasc Res 2006;71:48-54

129. Li C, Huang W, Harris MB, Goolsby JM, Venema RC: Interaction of the endothelial nitric oxide synthase with the CAT-1 arginine transporter enhances NO release by a mechanism not involving arginine transport. Biochem J 2005;386:567-574

130. McDonald KK, Zharikov S, Block ER, Kilberg MS: A caveolar complex between the cationic amino acid transporter 1 and endothelial nitric-oxide synthase may explain the "arginine paradox". J Biol Chem 1997;272:31213-31216

131. Zani BG, Bohlen HG: Transport of extracellular l-arginine via cationic amino acid transporter is required during in vivo endothelial nitric oxide production. Am J Physiol Heart Circ Physiol 2005;289:H1381-1390

132. Windmueller HG, Spaeth AE: Uptake and metabolism of plasma glutamine by the small intestine. J Biol Chem 1974;249:5070-5079

133. Dhanakoti SN, Brosnan JT, Herzberg GR, Brosnan ME: Renal arginine synthesis: studies in vitro and in vivo. Am J Physiol 1990;259:E437-442

134. Aperia A, Broberger O, Larsson A, Snellman K: Studies of renal urea cycle enzymes. I. Renal concentrating ability and urea cycle enzymes in the rat during protein deprivation. Scand J Clin Lab Invest 1979;39:329-336

135. Goutal I, Fairand A, Husson A: Expression of the genes of arginine-synthesizing enzymes in the rat kidney during development. Biol Neonate 1999;76:253-260

136. Erez A, Nagamani SC, Shchelochkov OA, Premkumar MH, Campeau PM, Chen Y, Garg HK, Li L, Mian A, Bertin TK, Black JO, Zeng H, Tang Y,

54

Reddy AK, Summar M, O'Brien WE, Harrison DG, Mitch WE, Marini JC, Aschner JL, Bryan NS, Lee B: Requirement of argininosuccinate lyase for systemic nitric oxide production. Nat Med 2011;17:1619-1626

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 871

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