Novel Approaches to Treatment and Prevention of Diabetic...

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2007

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 290

Novel Approaches to Treatmentand Prevention of DiabeticNephropathy

LINA NORDQUIST

ISSN 1651-6206ISBN 978-91-554-7021-0urn:nbn:se:uu:diva-8308

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List of original papers

This thesis is based on the following original papers, referred to in the text by their Roman numerals:

I. The C-peptide fragment EVARQ reduces glomerular hy-

perfiltration in streptozotocin-induced diabetic rats. Lina Nordquist, Erika Moe, and Mats Sjöquist. Diabetes Metab Res Rev 2007, 23(5): 400-404

II. Proinsulin C-peptide constricts glomerular afferent arteri-

oles in diabetic mice. A potential reno-protective mecha-nism. Lina Nordquist, En Yin Lai, Mats Sjöquist, Andreas Pat-zak, and A. Erik G. Persson. Submitted.

III. Proinsulin C-peptide reduces diabetes-induced glomerular

hyperfiltration via efferent arteriole dilation and inhibition of proximal tubular reabsorption in streptozotocin-induced diabetic rats. Lina Nordquist, Russell Brown, Angelica Fasching, Mats Sjöquist, and Fredrik Palm. Submitted.

IV. Novel microneedle patches for active insulin delivery are

efficient in maintaining glycaemic control: an initial com-parison with subcutaneous administration. Lina Nordquist, Niclas Roxhed, Patrick Griss, and Göran Stemme. Pharm Res 2007, 24(7): 1381-1388

Reprints of papers and figures were made by permission of the publishers.

Contents

Introduction...................................................................................................11 Diabetes mellitus ......................................................................................11 Renal anatomy and physiology ................................................................11 Development of diabetic nephropathy .....................................................13

Alterations in glomerular filtration rate ...............................................13 Prevention and treatment of diabetic nephropathy...................................14 Prevention by proinsulin C-peptide..........................................................15

Mechanisms for C-peptide in diabetic nephropathy ............................16 Reducing diabetic glomerular hyperfiltration......................................17

The importance of glycemic control ........................................................19 Intradermal delivery of insulin ............................................................19

Aims of the investigation ..............................................................................23

Methods ........................................................................................................24 Animals and induction of diabetes ...........................................................24

Rats (Studies I, III, and IV) .................................................................24 Mice (Study II) ....................................................................................25

Experimental protocols, treatment regimens, drugs and preparations......25 Preparations and measurements ...............................................................26

Anesthesia (Study I, III, and IV) and euthanasia .................................26 Surgical procedure (Study I, III and IV)..............................................26 Blood pressure (Studies I, III and IV)..................................................27 Tissue isolation, dissection and perfusion (Study II)...........................27 Measurements of oxygen consumption (Study III) .............................28 Blood flow estimations (Study III) ......................................................29 Measurements of tubular pressures (Study III)....................................29 Intradermal infusion of insulin (Study IV) ..........................................30

Analyses and calculations ........................................................................30 Estimation of glomerular filtration rate (Study I and III) ....................30 Electrolyte analysis (Study I and III)...................................................31 Measurements of arteriole contraction (Study II)................................31 Calculations in Study III......................................................................31 Insulin analysis (Study IV) ..................................................................31

Statistical method and presentation of data ..............................................32

Results...........................................................................................................33 Study I ......................................................................................................33

Glomerular filtration rate.....................................................................33 Blood glucose concentrations ..............................................................34 Urinary sodium excretion ....................................................................34

Study II.....................................................................................................35 Afferent arteriole diameter ..................................................................35 Rho-kinase inhibitor Y-27632 .............................................................37

Study III ...................................................................................................38 Blood pressure and renal parameters ...................................................38 Fractional Na+ excretion and transported Na+ .....................................39 In vivo oxygen consumption................................................................40 Micropuncture parameters: filtration pressures ...................................40 In vitro oxygen consumption ...............................................................41

Study IV ...................................................................................................42 Plasma insulin concentrations..............................................................42 Blood glucose concentrations ..............................................................43

Discussion .....................................................................................................45 Penta-peptide effects ................................................................................45 C-peptide and glomerular hyperfiltration.................................................45

Glomerular filtration rate and tubuloglomerular feedback ..................46 The tubular hypothesis of glomerular filtration...................................47 Separate effects on capillary and proximal pressure ...........................48 Renal glomerular arteriolar vascular tone and blood flow...................51

Cellular C-peptide signaling.....................................................................51 C-peptide and Rho-kinase....................................................................51 C-peptide and oxygen consumption ....................................................54 Potential tissue or state-specificity of C-peptide .................................54 Lack of effect on normoglycemic animals ..........................................55

C-peptide-induced reduction of blood glucose.........................................55 Intradermal insulin treatment ...................................................................56

Summary and conclusions ............................................................................57

Future perspectives .......................................................................................58

Sammanfattning på svenska..........................................................................59 Njurskador vanliga vid diabetes ...............................................................59 Inte bara insulin........................................................................................60 C-peptid skyddar ......................................................................................61 En sorts plåster med miniatyrnålar ...........................................................62

Acknowledgements.......................................................................................63

References.....................................................................................................66

Abbreviations ANOVA analysis of variance bw body weight C-peptide proinsulin connecting peptide ��onc oncotic pressure FF filtration fraction GFR glomerular filtration rate IU international unit IV intravenous MAP mean arterial blood pressure MAPK mitogen-activated protein kinase Na+/K+-ATPase sodium/potassium ATPase NO nitric oxide PBS hydrostatic pressure in Bowman’s space Pcap hydrostatic pressure in the glomerular capillary Pff free-flow tubular pressure Pnet glomerular net filtration pressure Pstf stop-flow tubular pressure Ptub hydrostatic pressure in early proximal tubulus PKC protein kinase C QO2 oxygen consumption RIA radioimmunoassay RBF renal blood flow RVR renal vascular resistance SC subcutaneous SEM standard error of the mean STZ streptozotocin TGF tubuloglomerular feedback TNa transported sodium TNa/QO2 transported sodium per consumed oxygen

Homo prudens non contra ventum mingit

11

Introduction

Diabetes mellitus Diabetes mellitus is a chronic disease where blood glucose concentrations increase as a consequence of insufficient secretion of insulin from the pan-creatic beta cells. Insulinopenic diabetes mellitus involves autoimmune de-struction of the insulin-secreting cells. The diagnosis therefore mostly means being subject to a life with multiple daily insulin injections, and also being exposed to considerable risks for complications. There are several long-term complications associated with diabetes mellitus. Among the complications are retinopathy, circulatory problems, neuropathy, and nephropathy1. The incidence of insulinopenic diabetes mellitus is constantly increasing in Scan-dinavia, and in addition approximately 30% of all hyperinsulinemic diabetic patients develop insulinopenia.

Renal anatomy and physiology Our kidneys consist of approximately 1,000,000 structural and functional units known as nephrons (Fig. 1). Blood arrives at the nephron through the afferent arteriole of the glomerulus. In the glomerulus, low molecular weight constituents of the plasma are filtered across the glomerular membrane and into Bowman’s space, creating a filtrate known as primary urine. In the hu-man kidneys, approximately 180 l of primary urine and 1.5 kg of NaCl are formed daily, but the normal urinary excretion equals only 1-2 l and 10-15 g of NaCl2. This means most of the constituents of the primary urine must be reabsorbed by the tubular structures following Bowman’s space, before the urine exits the kidneys and enters the urethra leading to the urinary bladder. To achieve this, the different nephron segments contain specialized cells, which via interconnected segments regulate kidney function in order to maintain electrolyte homeostasis, blood pressure, blood volume and acid-base balance, to name a few. Approximately 80% of the total oxygen con-sumed by the kidneys is related to electrolyte transport3, primarily by proxi-mal tubular cells that are responsible for about 2/3 of all tubular electrolyte transport.

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Figure 1 A schematic overview of the nephron: “Scheme of renal tubule and its vascular supply”4.

To maintain proper kidney function, it is essential to regulate the rate by which the blood is filtered across the glomerular membrane. This rate is referred to as the glomerular filtration rate (GFR). There are several factors influencing the GFR: the filtration area, the membrane permeability, and the hydrostatic and oncotic pressures. The resistance of the afferent and efferent arteriole determines the blood flow, and the relative constriction between the two regulates the hydrostatic pressure within the glomerulus (Pcap), and thus the driving force for the GFR. A factor opposing filtration is the hydrostatic pressure in Bowman’s space (PBS). This pressure is influenced by the rate by which the filtered primary urine is reabsorbed from the early proximal tubule back to the peritubular capillaries. In the glomerular capillaries, the oncotic

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pressure (�onc) is made up by proteins in the blood. Since proteins are not filtered across to Bowman’s space, the �onc in the primary urine is generally assumed to be close to zero. Development of diabetic nephropathy Diabetes is the most common cause for chronic and end stage renal failure5. More than 30% of all diabetic patients will eventually develop diabetic neph-ropathy. The first quantifiable change in this development is thickening of the glomerular basement membrane. Later, mesangial expansion, known as diffuse glomerulosclerosis, occurs, inducing glomerular enlargement and clinical manifestations such as glomerular hyperfiltration5. Through positive feedback, the decrease in GFR will increase the capillary pressure, inducing glomerular damage.

Podocyte integrity and numbers are related to diabetes-induced alterations in glomerular membrane permeability, although it is not elucidated what is cause and what is effect. However, hypertrophy of the glomerular capillaries might cause the podocytes to stretch. If so, when the hypertrophy of the glomerular capillaries would exceed the stretching ability of the podocytes, spaces would form between the podocytes and result in unwanted leakage.

After the initial phase with renal hyperfiltration and hypertrophic altera-tions, the diabetic patients experience a quiet, non symptomatic phase, with microscopic renal alterations. Further on in the development of diabetic nephropathy follows a phase of incipient nephropathy with microalbuminu-ria. The diabetic nephropathy is considered manifest when this phase aggra-vates, albuminuria is persistent, blood pressure increases, and GFR declines5. For some patients, this stage results in renal failure and the need for dialysis or transplantation. It has been demonstrated that diabetic patients requiring hemodialysis have a decreased survival rate compared to other hemodialysed patients6.

Alterations in glomerular filtration rate There are several hypotheses for the mechanisms leading to the onset and progression of diabetes-induced, progressive renal dysfunction. Early in the development of diabetic nephropathy, glomerular pressure increases and the glomerular filtration rate is augmented. The glomerular hypertension and hyperfiltration may play a crucial role in the development of diabetic neph-ropathy5, 7, and thus, prevention or inhibition of these increases may have beneficial effects on renal function in diabetes.

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In healthy kidneys, the glomerular capillary pressure is maintained by autoregulation5. However, in the development of nephropathy, it is possible that autoregulated hyperfiltration helps to maintain overall GFR short-term, but concomitantly augments glomerular hydrostatic pressure, which acceler-ates the renal damage5. This is thought to be mediated mainly by afferent vasodilation5. It has been suggested that autoregulation is defect due to hy-perglycemia in the diabetic state, but data is unclear5.

An increased filtration surface area is also associated with the develop-ment of diabetic hyperfiltration8. It seems that patients with glomerular enlargement secondary to mesangial expansion, are more susceptible to dia-betic nephropathy if they are unable to compensatory preserve their glome-rular filtration surface area5.

Diabetes is associated with an increase in renal oxygen metabolism9. The

increased oxygen consumption (QO2) could be an essential component of renal hypertrophy, hyperperfusion, and hyperfiltration commonly observed in the diabetic kidney. Sodium/potassium adenosine triphosphatase (Na+/K+-ATPase) is ubiquitously expressed in the basolateral membrane of tubular cells, where it creates the driving force for Na+ reabsorption10. Since the Na+/K+-ATPase is responsible for a majority of the cortical oxygen con-sumption, it has been proposed that the increased oxygen utilization occurs secondary to increased Na+/K+-ATPase protein expression. Numerous stud-ies have shown an increased renal Na+/K+-ATPase activity in the kidneys of streptozotocin (STZ)-induced diabetic animals9, 11-13, and in other diabetes models14-16.

It is possible that the increased Na+/K+-ATPase activity could partially be

responsible for diabetes-induced glomerular hyperfiltration, since an in-creased Na+/K+-ATPase could affect PBS by decreasing the hydrostatic early proximal tubular pressure (Ptub), thus opposing filtration across the glomeru-lar membrane.

Prevention and treatment of diabetic nephropathy Relationships between diabetic duration and renal pathology are imprecise, due to variability in susceptibility as well as treatment5. However, the degree of metabolic control is clearly associated with the risk of developing diabetic nephropathy. The Diabetes Control and Complication Trial concluded that the degree of hyperglycemia predicts long-term diabetic renal complica-tions17, making a proper glycemic control of utmost importance to prevent renal damage. It seems clear that strict glycemic control is an effective way to reduce hyperfiltration5. Hence, combinations of slow- and fast-acting in-

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sulin, as well as improved delivery via continuous, subcutaneous insulin pumps have been developed to achieve optimal glycemic control. Recently, transplantation of pancreatic beta cells in order to restore islet mass has be-come a reality for diabetic patients, but mainly for those already on the list for a kidney transplant. In the future, human embryonic stem cells may be-come a novel source for generation of pancreatic beta cells for the treatment of diabetes mellitus18.

Since Pcap appears to be implicated in the development of diabetic neph-

ropathy, reducing glomerular hypertension protects against the development of diabetic nephropathy5. It has been shown that dilators of the efferent arte-riole, such as angiotensin-converting enzyme inhibitors, as well as afferent constrictors, such as low protein diets, improve long-term function in similar manner5.

Even patients with good glycemic control may develop complications. Despite strict metabolic control, more than 15% of the patients in The Diabe-tes Control and Complication Trial developed microalbuminuria during the 9 year duration of the study5, 17. Thus, it seems that a factor other than insu-linopenia is at least partially, involved in the development of renal complica-tions.

Prevention by proinsulin C-peptide C-peptide is the 31-residue cleavage product of insulin synthesis, and is se-creted from the islets of Langerhans, along with insulin in equimolar amounts. In the early 1980s, it was suggested that proinsulin C-peptide may exert biological functions19, and during the past few years, studies have re-ported reno-protective effects from C-peptide20-24. When, as in insulinopenic diabetes, insulin synthesis is impaired, C-peptide will be affected to the same extent. In STZ-treated rats, administration of physiological doses of C-peptide has been shown to reduce diabetes-induced glomerular hyperfiltra-tion23, 25-28, decrease albuminuria and renal hypertrophy, and normalize glomerular volume25, 26. Recently, the normalizing effect of C-peptide on diabetic hyperfiltration was confirmed in conscious, unrestrained rats (Fig. 2). In that study, conscious GFR was measured as plasma clearance of a single bolus injection of fluorescein isothiocyanate inulin29. It has been shown that pancreatic transplantation reverses glomerulopathy in insu-linopenic diabetic patients30, an observation that fits in well with the benefi-cial and reno-protective effects of C-peptide on renal function.

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Figure 2 Glomerular filtration rate in conscious, normoglycemic control and strep-tozotocin-diabetic Sprague-Dawley rats before and after exogenous C-peptide ad-ministration. C-peptide reduces diabetes-induced glomerular hyperfiltration in con-scious rats29.

It has also been reported that C-peptide decreases hyperfiltration in pa-tients with insulinopenic diabetes31, and that C-peptide supplementation to insulinopenic patients during a period of 1-3 months is accompanied by im-proved renal function27, 32. Notably, peptides with the same amino acid com-position as C-peptide, but in randomized sequences, have no such effects23,

27, 33. Hence, the lack of C-peptide has successively emerged as a possible mechanism for the development of the disproportionate burden of complica-tions affecting insulinopenic diabetes patients.

Mechanisms for C-peptide in diabetic nephropathy Although it seems clear that C-peptide reduces most diabetes-induced altera-tions, the exact mechanisms mediating these beneficial effects of C-peptide remain unclear34. Experimental data supports that C-peptide binds to and exerts its effects via a G-protein-coupled receptor, there is still no conclusive evidence34, 35. Also, the fact that hyperinsulinemic, diabetic patients develop similar complications and at a similar rate as insulinopenic diabetes patients, may speak in favor of effects dependent on the insulin receptor. Another

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factor suggesting insulin-like behavior are the metabolic effects of C-peptide, such as increased glucose utilization and lowering of blood glucose concentrations24, 36, 37.

There are reports of C-peptide-induced intracellular Ca2+ increases, acti-vation of phospholipase C, RhoA, and Akt, as well as stimulation of Na+/K+-ATPase via protein kinase C (PKC)-induced activation of mitogen-activated protein kinase (MAPK)33, 38, 39. It is possible that downstream C-peptide ef-fects induce transcriptions factors, but although several intracellular effects of C-peptide have been described in vitro, no fully explanatory model has been presented so far34.

Reducing diabetic glomerular hyperfiltration GFR is determined by the glomerular membrane permeability, filtration sur-face area, differences in hydrostatic pressures, and the difference in ��onc. Thus, to lower the diabetic hyperfiltration, C-peptide must alter at least one of the parameters determining GFR.

Although an increased filtration area is associated with diabetic hyperfil-

tration5, there are few reports of acute alterations in filtration surface area40. One possibility is that C-peptide constricts afferent arterioles and/or dilates efferent arterioles (Fig. 3). By constricting the afferent arteriole or dilating the efferent, C-peptide would reduce the capillary pressure. This would lower the net filtration pressure (Pnet) and reduce GFR. However, altered vascular tone of the afferent or efferent arterioles should induce alterations in renal blood flow, which has not been reported for C-peptide26.

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Figure 3 Constriction of the afferent or dilation of the efferent arteriole will reduce the capillary pressure. Thus, filtration pressure is lowered, inducing a reduction in glomerular filtration rate.

Another parameter that may affect filtration is early Ptub41-43. Diabetes is

associated with increased proximal tubular Na+ reabsorption44. Changes in the proximal fluid reabsorption rate may induce changes in GFR through an effect on Ptub

45. If increased Na+ excretion is due to a decreased proximal Na+ reabsorption that could increase the hydrostatic pressure in the proximal tubule, and thereby affect PBS. An increase in PBS would decrease the net driving force for filtration across the membrane, leading to a reduction in GFR. Thus, any factor reducing fluid reabsorption in the early proximal tu-bule could reduce GFR. C-peptide-treated diabetic animals have been re-ported to display increased urinary Na+ excretion27, implying an inhibitory effect of C-peptide on tubular Na+ reabsorption.

In addition, a C-peptide-mediated effect on Na+ reabsorption could have

further beneficial consequences. When glomerular hyperfiltration increases

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the tubular Na+ load, this increases the oxygen demand and thereby tissue QO2. This is amplified by the enhanced Na+ reabsorption due to increased glucose co-transport in diabetes. Numerous studies have reported increased renal Na+/K+-ATPase activity and increased QO2 during the early hyperfiltra-tion phase in STZ-induced diabetes46, 47 9, 11, 12, 15, and in other models of ex-perimental diabetes48. Since Na+/K+-ATPase consumes most of the oxygen in the renal cortex49, an influence of C-peptide on cortical Na+/K+-ATPase activity could influence not only GFR, but also QO2 and oxygen availability.

The importance of glycemic control Optimal glycemic control is essential to minimize the risk for diabetes-

induced complications, but the majority of diabetic patients fail to achieve proper long-term glucose levels even in clinical trials, and even more so in clinical practice17. The majority of diabetic patients are reported not to be confident to manage their disease themselves50, and one out of five diabetic children place their injections inappropriately51. By achieving good glycemic control, a subcutaneous insulin pump markedly reduces the risk of complica-tions in diabetic patients17.

A major benefit of the insulin pump is that it reduces the need for multi-

ple daily injections. Injections are painful and cause skin trauma, which causes complications for patients to comply with their treatment. Every fourth diabetes patient taking insulin reports anxiety regarding self-injection50, 52. This reluctance and suboptimal compliance in patients using multiple daily injection regimens, together with the perceived risk of hypo-glycemia, are factors which reduce the glycemic control53. Finally, patients express a preference for discrete, easy to use treatments54.

Compliance to a treatment regimen is likely to be higher if the procedure is simple, painless, and discrete. Insulin has so far been suggested for nasal, gastrointestinal, as well as inhalation therapy52, 55. Although gastrointestinal and nasal administrations have been unsuccessful so far, an insulin inhaler was recently developed and approved56. However, since insulin is also a potent growth factor, there is concern that intra-alveolar deposition of insulin could adversely affect pulmonary function57, 58. Other routes are therefore to prefer, which do not compromise the comfort and future health of diabetic patients.

Intradermal delivery of insulin For drugs that easily diffuse across the skin barrier, patch-based intradermal drug delivery provides convenient drug administration without the draw-

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backs of subcutaneous injections. Recently, a device with miniaturized nee-dles was developed that combines the simplicity and discretion of patch-based treatments, but with the potential of peptide and protein administra-tion59-62.

Our largest organ, the skin, has two main functions: it is an excellent

physical barrier for protection, and a sensitive receiver of external sensory stimuli. The skin can be divided into three sections; the epidermis, dermis and hypodermis, or subcutaneous tissue (Fig. 4). The epidermis is approxi-mately 100 μm thick and consists of an outer barrier part, stratum corneum, where stacked, dead cells are continuously replaced by outward moving, new cells produced in the more basal layer. The underlying dermis is more than 1000 μm thick and constitutes a thick supportive layer, containing nerve endings, blood vessels, sweat glands, and hair follicles. The subcutaneous tissue, finally, is a layer of adipose tissue serving as an energy reservoir and thermal insulation63.

Figure 4 Schematic illustration depicting a cross section of the skin, with epidermis, dermis, and subcutaneous tissue 64.

The standard insulin treatment involves subcutaneous injections, which includes that the syringe is inserted through the nociceptor-rich dermis into the subcutaneous tissue (Fig. 4). Microneedles, on the other hand, are shorter

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than 1 mm62, and are also considerably thinner (Fig. 5), making microneedle delivery of any substance a minimally-invasive injection technique. Since the microneedles only penetrate part of the dermis, they do not reach most of the nociceptors. Thus, microneedle insertions are perceived as painless62.

Figure 5 Scanning Electron Microscopy picture showing a complete microneedle device displayed next to two standard, 20G and 27G syringes65.

If sufficient bioavailability can be obtained using this newly developed technique for intradermal administration, one could achieve the advantages of subcutaneous drug delivery, but in a discrete and minimally invasive manner (Fig. 6). Since studies report needle size and fear of pain as two ma-jor reasons for injection anxiety52, such a device could improve patient ac-ceptance, and the development of a “controlled release”-design could further prevent long-term complications. Due to the small size and user comfort, the microneedle approach is a potentially very good candidate for future insulin delivery.

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Figure 6 Photomicrograph showing a pattern in the skin of a human subject after dye injection through the patch-like microneedle device62.

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Aims of the investigation

Study I investigates and compares the effects of a small C-peptide frag-ment, EVARQ, with that of the intact C-peptide on diabetes-induced glome-rular hyperfiltration in rats.

Study II investigates vascular reactivity of isolated afferent arterioles

from diabetic C57/Bl mice to endogenous administration of C-peptide, in order to understand the mechanisms of C-peptide-induced vasoconstriction.

Study III investigates if altered renal afferent-efferent arteriole tonus me-diates the reduction of diabetes-induced glomerular hyperfiltration, and how this relates to blood flow alterations. Furthermore, this study investigates the effect of C-peptide on renal sodium handling and oxygen usage in diabetic rats.

Study IV evaluated an integrated patch-like microneedle system in vivo

for delivery of insulin.

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Methods

Animals and induction of diabetes The animals were housed in a facility at the Laboratory Animal Resources of the BioMedical Centre, Uppsala University in Uppsala. All animals were fed standard chow (R3, Ewos, Södertälje, Sweden) and water ad libitum. All experiments were approved by the Uppsala Ethical Committee for Animal Experiments, and were performed in accordance with national guidelines for the care and use of laboratory animals.

For all diabetic animals, body weight (bw) and blood glucose concentra-

tions were monitored every other day throughout the entire experimental period, in order to evaluate the degree of hyperglycaemia and severity of diabetes. Blood samples were obtained from the cut tips of the tails and ana-lysed by a glucose oxidase method (15-20 μl, Precision QID, MediSense, Bedford, MA, USA). Animals were excluded if they developed a weight loss of more than 10%, or if the blood glucose concentrations exceeded 30 mM (560 mg/dl) on more than two consecutive measurements. Age-matched normoglycemic animals served as controls.

All chemicals used in these experiments were purchased from Sigma-

Aldrich, St Louis, MO, USA and were of the highest grade available unless otherwise stated.

Rats (Studies I, III, and IV) In Study I, III and IV, 160 male Sprague-Dawley rats were used. Two

weeks prior to the acute experiment, the rats were made diabetic by means of a single intravenous injection of STZ (50-55 mg/kg bw) dissolved in 0.2 ml saline. This resulted in blood glucose concentrations above 20 mM66. STZ is derived from Streptomyces achromogenes. It was developed in the 1960s as an antibiotic, but was soon found to induce beta cell destruction67-69. STZ consists of a glucose moiety attached to nitrosourea, and the toxicity has been shown to be due to the nitrosourea decomposing to methyl ions after being transported by specific glucose transporters into the beta cells70-72. Today, STZ is widely used to induce diabetes in experimental models of diabetes mellitus.

25

In Study III and IV, the rats were treated daily with a subcutaneous injec-

tion of long-acting insulin (Insulatard, Novo Nordisk, Hjørring, Denmark; 5 IU/kg bw/day).

Mice (Study II) In Study II, 73 male C57BL6 mice were used. The mice were made dia-

betic by means of a single intravenous injection of alloxan (75 mg/kg) two weeks prior to the experiment, increasing blood glucose concentrations to above 18 mM. Diabetes was induced with alloxan. Alloxan, originally used as raw material for a purple dye, is one of the oldest organic compounds named73, 74, and has been used for induction of diabetes for more than 60 years75. Although not toxic to human beta-cells76, it destructs rodent beta cells by interactions with the oxidative metabolism by the formation of superoxide radicals77, 78.

Experimental protocols, treatment regimens, drugs and preparations

Study I Five groups of animals were studied for two consecutive 20 minute-periods before and after 60 minutes of drug administration: control rats given vehicle only (n=10), control rats given rat C-peptide (50 pmol/kg bw/min; n=9), diabetic animals given vehicle (n=8), diabetic rats given rat C-peptide (50 pmol/kg bw/min; n=9), and diabetic rats given the C-peptide fragment EVARQ (500 pmol/kg bw/min; n=9).

Study II Seven groups of mouse afferent arterioles were studied before and after drug administration: a normoglycemic group administered vehicle only (n=4), a normoglycemic group receiving scrambled C-peptide (5 nM, n=5), a normo-glycemic group receiving C-peptide (5 nM, n=9), a hyperglycemic group receiving vehicle (n=4), a hyperglycemic group receiving scrambled C-peptide (5 nM, n=5), a hyperglycemic group receiving C-peptide (0.5 nM, n=7), and a hyperglycemic group receiving C-peptide (5 nM, n=7).

The involvement of Rho-kinase in C-peptide-induced vasoconstriction

was investigated by administering the Rho-kinase inhibitor Y27632 (1 μM, n=7)79.

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Study III In vivo, hyperglycemic (n=9) and normoglycemic (n=8) animals were meas-ured at baseline and after 40 minutes of C-peptide or vehicle infusion. For measurements of tubular pressures, 6 hyperglycemic animals were measured at baseline and after 40 minutes of C-peptide infusion.

In vitro, four groups were studied. The effect of C-peptide on QO2 was measured in isolated proximal tubular cells from normoglycemic (n=9) and hyperglycemic (n=12) animals during baseline and after incubation with ouabain (1 mM) or N�-nitro-L-arginine-metyl-ester (L-NAME; 37 μM).

Study IV Blood glucose concentrations were measured at baseline, and after 60, 120, 180, and 240 minutes of insulin infusion. Plasma concentrations of insulin lispro were measured after 30, 90, 150, and 210 minutes of infusion.

Seven groups of diabetic animals were studied before and after drug ad-

ministration: intravenous saline solution only (n=9), intravenous insulin in-fusion (0.14 IU/h, 70 IU/ml, n=7), subcutaneous insulin infusion, (0.20 IU/h, 100 IU/ml, n=9), microneedle-aided intradermal low-rate insulin infusion (0.20 IU/h, 100 IU/ml, n=9), microneedle-aided intradermal high-rate insulin infusion (0.40 IU/h, 100 IU/ml, n=9), microneedle-aided passive intradermal insulin diffusion (100 IU/ml, n=9), and insulin applied onto microneedle-penetrated skin (100 IU/ml, n=9).

Preparations and measurements Anesthesia (Study I, III, and IV) and euthanasia 13-15 days after the induction of diabetes, the animals were anaesthetized with an intraperitoneal injection of sodium-5-sec-butyl-5-ethyl-2-thiobarbiturate (Inactin).

All mice were sacrificed by cervical dislocation. All rats were sacrificed

under anesthesia by an intravenous injection of saturated potassium chloride.

Surgical procedure (Study I, III and IV) In studies I, III and IV, the anaesthetized rats were placed on a servo-controlled heating pad in order to maintain temperature at 37.5°C, and tra-cheotomized to facilitate spontaneous breathing. The right femoral artery was catheterized for monitoring of mean arterial blood pressure (MAP) and for blood sampling. The right femoral vein was catheterized for substance

27

administration and infusion of isotonic saline at a rate of 5 ml/kg bw/h for non-diabetic rats and 10 ml/kg bw/h for diabetic rats. The urinary bladder was catheterized for the collection of urine.

In Study III, the left kidney was exposed by a left subcostal flank incision,

immobilized in a plastic cup, embedded in pieces of saline-soaked cotton wool, and the surface covered with paraffin oil (Apoteksbolaget, Gothen-burg, Sweden). Kidney weights were determined at the end of the experi-ment.

Blood pressure (Studies I, III and IV) Blood pressure was continuously measured by connecting the arterial cathe-ter to a pressure transducer (Statham P23dB; Statham Laboratories, Los An-geles, CA, USA), and recorded on a polygraph (Model 7D Polygraph, Grass Instrument Co., Quincy, MA, USA) or a MacLab instrument (AD Instru-ments, Hastings, UK).

Tissue isolation, dissection and perfusion (Study II) After cervical dislocation, the kidneys were removed and sliced along the corticomedullary axis. The afferent arterioles were dissected using sharpened forceps under a stereoscopic microscope at +4°C in Dulbecco’s Modified Eagle Medium enriched with 1% albumin. With intact glomeruli, the affer-ent arterioles were transferred into a thermoregulated chamber. The use of a flexible perfusion system allows for adjustments of holding and perfusion pipettes (Fig. 7).

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50 μm50 μm50 μm

Figure 7 The setup of the renal afferent arteriole experiments. The photomicro-graph shows a glomerulus and its afferent arteriole held by two holding pipettes. The perfusion pipette is inserted into the right holding pipette.

To achieve physiological pressure and flow, the perfusion pressure was set to 100 mmHg at the pressure head of the perfusion pipette. The diameter of the perfusion pipette was 26 μm at the tip, and the holding pipette, which was put into the lumen of the afferent arteriole, had a diameter of 5 μm. The experiments were recorded by a video system with a Nikon water immersion lens and a digital camera.

Measurements of oxygen consumption (Study III)

In vitro Proximal tubular cells were isolated as previously described9, 46. In brief, renal cortical tissue was minced through a metallic mesh strainer and imme-diately placed in an ice-cooled buffer solution and incubated in collagenase (0.05% wt/vol) at 37°C for 60 minutes while equilibrated with 95% O2/5% CO2. Thereafter, the cell suspension was cooled to +4°C and filtrated through graded filters with pore sizes of 180, 75, 53 and 38 μm, in order top separate isolated tubular cells from glomerulus. The isolated cells were then pelleted by slow centrifugation (100 g, 4 minutes) and re-suspended in a collagenase-free buffer, a procedure that was repeated three times. The sus-

29

pension was kept on ice until QO2 was measured according to a previously described procedure46. A custom-made thermostatically-controlled (+37°C) gas-tight plexiglas chamber was filled with buffer solution (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, osmolality 298±2 mOsm), and con-tinuously stirred with an air-driven magnetic stirrer. The glucose concentra-tion in the medium was 5.8 mM (100 mg/dl) for cells from normoglycemic controls and 23.2 mM (400 mg/dl) for cells from diabetic animals. A modi-fied Unisense 500 oxygen-sensing electrode (Unisense, Aarhus, Denmark), calibrated with air-equilibrated or Na2S2O5 saturated buffer solution was used. 100 μl of cell suspension was then injected into the chamber, and the rate of oxygen disappearance was measured.

At the end of each experiment, a sample was taken to determine protein

concentration. In all groups, the rate of oxygen disappearance was adjusted for protein concentration. The effect of C-peptide on QO2 was estimated with and without preincubation with ouabain or L-NAME.

In vivo The QO2 of the whole kidney in vivo was measured as the artero-venous dif-ference in blood oxygen content, multiplied by the renal blood flow. Blood gas parameters were analyzed in samples drawn from the left renal vein and femoral artery (ISTAT, Abbott, Solna, Sweden).

Blood flow estimations (Study III) Total renal blood flow (RBF) was measured using an ultrasound probe (Transonic Systems, Ithaca, NY, USA) placed around the left renal artery, and cortical RBF was measured with laser-Doppler flowmetry (PF 4001–2; Perimed, Stockholm, Sweden). All parameters were continuously recorded with a MacLab instrument (AD Instruments) connected to a Macintosh Power-PC 6100.

Measurements of tubular pressures (Study III) Pnet was estimated by stop-flow technique at the baseline and 40 minutes after tha infusion of C-peptide was commenced (bolus 5 nmol/kg bw + con-tinuous infusion 50 pmol/kg bw/min). Early proximal tubular segments (5/rat) were randomly chosen on the kidney surface for each measurement. A pipette, filled with 1 M NaCl and Lissamine green, connected to a servo-null pressure system (World Precision Instruments, New Haven, CT, USA) was used to determine proximal tubular free-flow (Pff) and stop flow pres-sure (Pstf), the latter after tubular flow was interrupted with a wax blockade distal to the pressure pipette. Pnet was calculated according to Pstf – Pff.

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Intradermal infusion of insulin (Study IV) A patch-like microneedle device was attached on depilated skin (potassium thioglycolate, Veet Creme, Reckitt Benckiser, Slough, UK) using moderate hand force. The device was then fixated onto the skin with surgical tape.

The microneedles were fabricated in monocrystalline silicon by Deep Re-

active Ion Etching. To allow active delivery, the needles are hollow with the needle bore-opening located on the side of the needle, and are organized on a chip in arrays (Fig. 8). A sharp and well-defined needle tip allows micronee-dle insertion into the skin by hand. The system also includes a small, active dispenser mechanism based on a novel thermal actuator consisting of highly expandable microspheres. When actuated, these microspheres expand into a liquid reservoir, dispensing of stored liquid through the microneedles.

Figure 8 Drawing showing an exploded view of the setup of the used drug delivery micro system. When voltage is passed through the heater, the expandable composite heats up and expands into the drug reservoir, consequently ejecting the liquid through the hollow needles on the chip62.

Analyses and calculations Estimation of glomerular filtration rate (Study I and III) 3H-inulin (3-5 �Ci/ml; American Radiolabeled Chemicals, St. Louis, MO, USA) was infused at a rate of 5 ml/kg bw/h for non-diabetic rats and 10

31

ml/kg bw/h for diabetic rats. GFR was estimated by urinary clearance of 3H-inulin before and after the infusion of vehicle, C-peptide, or EVARQ. The 3H activities in urine and plasma were measured using a standard liquid scin-tillation technique. The GFR was calculated according to GFR=U�V/P, where U and P denote the activity of 3H-inulin in urine and plasma respec-tively, and V denotes the urine flow rate (ml/min).

Electrolyte analysis (Study I and III) Urine volumes were measured gravimetrically. Urinary sodium and potas-sium concentrations were measured by flame photometry (Model IL 543, Instrumental Laboratory, Milan, Italy). Osmolality was measured by freezing point depression (Model 3MO, Advanced Instruments, MA, USA). Urine excretion rates were calculated by multiplying urinary concentrations with urinary flow rates.

Measurements of arteriole contraction (Study II) The video sequences were digitalized, and the vessel luminal diameters es-timated using customized software. At the end of a recovery period, baseline control values were obtained for each experiment. Hereafter, luminal diame-ter was measured every 5 minutes for a total of 30 minutes.

Calculations in Study III The filtration fraction (FF) was estimated as FF = GFR/RBF�(1-Hct). Renal vascular resistance (RVR) was calculated as MAP divided by RBF. In vivo renal QO2 (μmol/min) was estimated from the arterio-venous difference in O2 content (O2ct = [Hb]�sO2�1.34+PO2�0.22) multiplied by total RBF80. Tubular Na+ transport (TNa; μmol/min) was calculated using TNa = [PNa]�GFR. TNa per QO2 was calculated according to TNa/QO2. Estimates of the fractional Na+ excretion were obtained from the calculation [UNa]�[Pinulin]/[PNa]�[Uinulin].

Insulin analysis (Study IV) Plasma insulin lispro concentration was determined using a 125I-monoiodinated, competitive-binding radioimmunoassay analysis (Lispro insulin RIA kit, Linco Research, MO, USA).

32

Statistical method and presentation of data In all studies, when analyzing two data sets with normal distribution, un-paired or paired Student’s t-tests were applied for comparisons between or within the same group respectively.

In Study I, multiple data sets between groups were analyzed using analy-ses of variance (ANOVA) followed by Tukey’s post hoc test when appropri-ate.

In Study II, multiple data sets between groups were analysed with non pa-rametric Kruskal Wallis followed by Dunn’s post hoc test, or ANOVA fol-lowed by Fisher’s post hoc test when appropriate. Multiple data sets within groups were analysed with repeated measurements ANOVA followed by Dunnett’s post hoc test for paired comparisons.

In Study III, multiple comparisons between different groups were per-

formed using ANOVA followed by Fisher’s protected least significant dif-ference test. Multiple comparisons within the same group were performed using repeated measures ANOVA followed by Dunnett’s post-hoc test for paired comparisons.

In Study IV, multiple data sets between groups were analyzed with

ANOVA followed by Tukey’s post hoc test, when appropriate. Multiple data sets within groups were analyzed with ANOVA followed by Dunnett’s post hoc test, when appropriate.

All statistical analyses were performed using GraphPad Prism software

(GraphPad Software Inc., San Diego, CA, USA). Descriptive statistics are presented as mean values ± standard error of the mean (SEM). For all com-parisons, P <0.05 was considered to be statistically significant.

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Results

Study I Glomerular filtration rate All diabetic groups displayed a pronounced glomerular hyperfiltration com-pared to the non-diabetic rats (Fig. 9). The hyperfiltration persisted during the infusion of vehicle, whereas C-peptide and EVARQ reduced GFR to baseline levels. Infusion of C-peptide in control rats had no effect.

Figure 9 C-peptide and EVARQ decrease glomerular hyperfiltration in diabetic rats. Modified from28.

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Blood glucose concentrations All diabetic animals displayed elevation of blood glucose concentrations (Fig. 10). When compared to baseline values, C-peptide, as well as EVARQ, reduced the blood glucose in diabetic animals. Control animals were unaf-fected by C-peptide, and vehicle treatment did not affect blood glucose in any group.

Figure 10 C-peptide and EVARQ decrease blood glucose in diabetic rats.* denotes P<0.05 vs. vehicle-treated control group. Modified from28.

Urinary sodium excretion Baseline urinary excretion of Na+ was similar in all groups (Fig. 11). During the experiments, urinary Na+ excretion increased in all groups, except for the vehicle-treated control group.

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Figure 11 C-peptide and EVARQ increase urinary sodium excretion rate. * denotes P<0.05 vs. vehicle-treated control group. Modified from28.

Study II

Afferent arteriole diameter C-peptide administered to afferent arterioles from hyperglycemic mice re-duced afferent arteriolar diameter within 10 minutes. After 30 minutes, C-peptide had decreased afferent arteriolar diameter by -27±8% (Fig. 12 and 13). Normoglycemic animals given C-peptide showed a minor, but signifi-cant decrease in arteriolar diameter after 30 minutes only (4±1%; Fig. 14). Scrambled C-peptide or vehicle had no effect.

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A.

B.

C.

Figure 12 C-peptide-induced constriction of an isolated renal afferent arteriole from a diabetic mouse. A. the arteriole at baseline, B. the arteriole after 15 minutes of C-peptide perfusion, and C. the arteriole after 30 minutes of C-peptide perfusion. Photos from Study II.

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Figure 13 The effect of C-peptide, scrambled peptide, and vehicle on afferent glomerular arterioles from hyperglycemic C57-Bl mice. * denotes p<0.05 vs. all other groups. Modified from Study II

Figure 14 The effect of C-peptide, scrambled peptide, and vehicle on afferent glomerular arterioles from normoglycemic C57-Bl mice. Modified from Study II.

Rho-kinase inhibitor Y-27632 The effect of C-peptide was prevented by the Rho-kinase inhibitor Y-27632. The inhibitor did not affect vessel diameter per se (data not shown).

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Study III

Blood pressure and renal parameters Diabetic rats had increased GFR and FF compared to controls (Fig. 15). C-peptide selectively reduced these parameters in the diabetic animals. MAP and RVR (Fig. 16) were similar during baseline, and C-peptide decreased both parameters in both groups.

Figure 15 Glomerular filtration rate (left panel) and filtration fraction (right panel) in normoglycemic controls and diabetic rats before and after the infusion of C-peptide. Figures from Study III.

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Figure 16 Renal vascular resistance in normoglycemic controls and diabetic rats before and after the infusion of C-peptide. Figure from Study III.

Fractional Na+ excretion and transported Na+ Diabetic rats had similar baseline fractional Na+ excretion to control animals, but C-peptide increased the fractional Na+ excretion selectively in diabetic animals (Fig. 17). Baseline TNa was higher in diabetic animals compared to controls, and was only reduced by C-peptide in the diabetic group (Fig. 17).

There was no difference in either total or cortical RBF in the diabetic

animals compared to the normoglycemic controls. C-peptide did not alter RBF in any of the investigated groups. The infusion of vehicle did not alter any of the investigated parameters (GFR, FF, MAP, RVR, fractional Na+ excretion, or RBF).

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Figure 17 Fractional urinary sodium excretion (left panel) and transported sodium (right panel) in normoglycemic controls and diabetic rats before and after the infu-sion of C-peptide. Figure from Study III.

In vivo oxygen consumption Diabetic animals displayed increased total in vivo QO2, although TNa/QO2 did not differ between diabetic and normoglycemic control rats. C-peptide or vehicle did not affect either total QO2 or TNa/QO2 in any of the two groups.

Micropuncture parameters: filtration pressures C-peptide reduced the Pff by 5.2% (11.6±0.2 vs. 11.0±0.2 mmHg), Pstf by 8.4% (41.9±0.3 vs. 38.4±0.3 mmHg), and the calculated Pnet by 9.6% (30.3±0.4 vs. 27.4±0.3 mmHg) in diabetic rats (Fig. 18).

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A.

B.

C.

Figure 18 Tubular free-flow pressure (A.), stop-flow pressure (B.), and calculated glomerular net filtration pressure (C.) in diabetic rats before and after C-peptide. Figures from Study III.

In vitro oxygen consumption Isolated proximal tubular cells from diabetic animals displayed increased baseline QO2 compared to controls (39±3, vs. 28±2 nmol/mg protein/min) (Fig. 19). Treatment with C-peptide reduced QO2 in cells from STZ-treated rats (28±3 nmol/mg protein/min), but had no effect on cells from normogly-cemic controls (26±5 nmol/mg protein/min). The transport-independent QO2

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was higher in diabetic cells compared to controls (18±2 vs. 13±3 nmol/mg protein/min). C-peptide had no effect on ouabain-pretreated cells (17±6 vs. 18±2 nmol/mg protein/min). Pretreatment with L-NAME had no effect on any of the investigated groups, and did not alter the effect of C-peptide on the diabetic cells. .

Figure 19 Oxygen consumption by isolated proximal tubular cells from normogly-cemic controls and diabetic rats during baseline and after incubation with C-peptide, ouabain, L-NAME or a combination of C-peptide and either ouabain or L-NAME. * denotes P<0.05 when compared to baseline in the control group. Figure from Study III.

Study IV Plasma insulin concentrations The subcutaneous and microneedle infusions displayed similar increases in plasma insulin concentrations (Fig. 20). Animals receiving the intravenous infusion did not show any changes in insulin concentrations between the first and last measurement.

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Figure 20 Effects of different administration routes of insulin lispro on plasma insulin concentration IV = intravenous, SC = subcutaneous, microneedle = patch-like microneedle device. * denotes a difference compared to the first measurement in that same group. Modified from65.

Blood glucose concentrations All animals displayed a pronounced elevation of blood glucose after the induction of diabetes (Fig. 21). Sixty minutes after the start of the insulin infusion, blood glucose was lowered in the intravenously-infused and the microneedle-infused groups. Intravenous, subcutaneous, and intradermal insulin infusion all lowered blood glucose concentration after 180 minutes.

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Figure 21 Effect of different administration routes of insulin lispro on blood glu-cose concentration. IV = intravenous, SC = subcutaneous, microneedle = patch-like microneedle device. * denotes P<0.05 vs. baseline in corresponding group. Modi-fied from65.

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Discussion

It has now been established that diabetes-induced glomerular hyperfiltration is reduced by C-peptide in patients23, 32 as well as in animal models of insu-linopenic diabetes20, 22, with concomitant reduction of diabetes-induced renal damage22, 23, 25, 31. The main new findings in this thesis are that acute admini-stration of C-peptide reduces GFR in diabetic rats via more than one mecha-nism, and that this effect can be achieved by infusion of only the C-terminal penta-peptide sequence. The results also suggest that the vascular effects of C-peptide involve Rho-kinase. The final part of this thesis demonstrates a new administration route for insulin which may have several benefits com-pared to today’s standard therapy.

Penta-peptide effects In Study I, the infusion of the intact C-peptide, as well as the carboxy-terminal penta-fragment EVARQ, abolished diabetes-induced glomerular hyperfiltration. The effect of EVARQ on diabetic hyperfiltration was a re-duction of the GFR within an hour from the start of administration, while vehicle-infused diabetic rats maintained a substantial hyperfiltration throughout the experiments. Hence, it is possible that the five amino acid sequence of the carboxy-terminal of C-peptide is the sequence, or one of the sequences, mediating the normalizing effect on GFR in these diabetic rats.

It is common that proteins have defined functional sites. A fragment of

gonadotropin-releasing peptide, displays increased activity compared to the intact peptide81. Gastrin, CCK and osteogenic growth peptide, have active sites in their C-terminal penta-peptides82-85.

C-peptide and glomerular hyperfiltration Evidence is increasing of the beneficial effects of C-peptide on the diabetic kidney, and it seems clear that C-peptide can reduce diabetic hyperfiltration. But what causes this effect of C-peptide on hyperfiltration? A possible mechanism is a direct effect of C-peptide on the renal afferent arteriole. It was concluded in Study II that C-peptide constricts the afferent arteriole

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from diabetic mice, but not from normoglycemic animals. A constriction of the afferent arteriole will lower glomerular filtration pressure and thus de-crease GFR.

In study III, two seemingly different mechanisms are reported for the C-

peptide-induced decrease of diabetic glomerular hyperfiltration. First, C-peptide dilated the efferent arteriole in vivo, as evident from the decreased RVR, FF, and Pstf in the absence of altered RBF in the diabetic rats. Sec-ondly, C-peptide inhibited the tubular Na+ reabsorption as evident from the increased fractional Na+ excretion in diabetic rats and reduction in transport-dependent QO2 by the isolated proximal tubular cells from diabetic rats.

Glomerular filtration rate and tubuloglomerular feedback It has previously been postulated that diabetic hyperfiltration occurs due to alterations in tubuloglomerular feedback (TGF)86. TGF is an intrarenal mechanism that stabilizes GFR, and thus the tubular Na+-load to match the tubular Na+ handling capacity (Fig. 22). The anatomical prerequisite for TGF is the return of the tubule to its own glomerulus. These, together with the macula densa (MD) make up the juxtaglomerular apparatus. The MD con-sists of specialized epithelial cells localized where the returning tubule passes the glomerulus, and constitutes a sensor mechanism for Na+, but pref-erentially Cl-. Increased tubular flow rate will increase the tubular NaCl load, which is sensed by the MD, and results in a constriction of the afferent arte-riole. The results from Study III show that C-peptide inhibits Na+/K+-ATPase, and this inhibition will increase the NaCl load to the MD, activating the TGF to constrict the afferent arteriole.

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Figure 22 The hypothesized effect of C-peptide on the tubuloglomerular feedback mechanism resulting in reduced glomerular filtration rate: Na+/K+-ATPase inhibi-tion (1) causes increased NaCl-load to macula densa (2), resulting in TGF activa-tion and constriction of the afferent arteriole (3).

Even though the data may seem to fit into the hypothesis that C-peptide decreases hyperfiltration via a TGF-altering mechanism, this conclusion is not entirely self-evident. Although it is likely that Na+/K+-ATPase is inhib-ited, the afferent constriction from Study II occurs in isolated arterioles, where the arteriole is not set up with an intact tubulus. In addition, it was recently shown by Sällström et al. that TGF does not mediate diabetes-induced hyperfiltration41, since diabetes-induced glomerular hyperfiltration occurs in adenosine A1-receptor-deficient mice known to lack a functional TGF mechanism41, 42, 87. If TGF is not the mediator of diabetic hyperfiltra-tion, it is unlikely that C-peptide would exert the effect on filtration via a TGF-dependent mechanism. That would render it unlikely that the two ef-fects seen on the nephron in this thesis are related or interlinked. Therefore, a possible alternative conclusion might include that C-peptide exerts its effect on glomerular filtration via two separate mechanisms.

The tubular hypothesis of glomerular filtration As an alternative to TGF-mediated regulation of filtration, “The Tubular Hypothesis of Glomerular Filtration” has been stipulated as a possible model for the regulation of GFR43. This hypothesis postulates that proximal tubular

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reabsorption will determine GFR through alterations in Ptub that directly in-fluence PBS. It has been shown that sustained hyperglycemia in patients44 as well as experimental animal models9, 88, results in increased proximal tubular Na+ reabsorption secondarily to increased Na+-linked glucose reabsorption42. Furthermore, it has also been shown that early distal tubular Na+ concentra-tions decrease during hyperglycemia42, together with increased cortical Na+/K+-ATPase expression89. Conversely, decreased Na+ reabsorption in the early proximal tubule will increase Ptub and thereby augment PBS, which in turn lowers Pnet and therefore reduces GFR. This hypothesis is consistent with reports of a linear correlation between GFR and Na+/K+-ATPase activ-ity in the kidney cortex in several models of experimental diabetes48, 89. Therefore, a reduced or normalized Na+ reabsorption in the early proximal tubule is likely to reduce GFR in the diabetic kidney.

Separate effects on capillary and proximal pressure If the effects of C-peptide on the glomerular afferent arteriole and the inhibi-tion of Na+/K+-ATPase are not related, that opens the possibility of three separate filtration lowering mechanisms: constriction of the afferent arteri-ole, dilation of the efferent arteriole, and proximal tubular reabsorption (Fig. 23). Although a reduction in the filtered electrolyte load could also reduce reabsorption, it will not in itself affect the fractional reabsorption (as seen in Study III), and it cannot explain the findings on the isolated proximal tubular cells.

Figure 23 Three plausible, separate mechanisms by which C-peptide may decreases diabetes-induced glomerular hyperfiltration supported by results presented in this thesis.

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As stated in the introduction, a main factor determining acute alterations in the GFR is Pnet (Fig. 24 and 25), which is determined by the Pcap and PBS. Changes in Pcap are a result of alterations in the interplay of afferent and ef-ferent arteriolar resistance, which alter the pressure transmitted into the glomerular capillaries. Ptub is mainly determined by proximal tubular reab-sorption, and under certain conditions by the hydraulic resistance in the more distal nephron segments, e.g. tubular obstruction45. Since Ptub will affect PBS, and thus is one of the determinants of Pnet

87, changes in the proximal reab-sorption rate have the potential to influence the GFR45.

tubcapnet PPP �� Figure 24 Relationship between net filtration pressure (Pnet), glomerular capillary pressure (Pcap) and early proximal tubular pressure (Ptub).

GFR is also influenced by differences in ��onc (Fig. 25). ��onc is deter-mined by differences in the protein concentration between the blood in the glomerular capillaries and the primary urine in Bowman’s space.

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Figure 25 Hydrostatic and oncotic pressures affecting the net filtration pressure, and thus GFR.

The main intrarenal factor influencing acute changes in ��onc is the FF90,

91. A reduced FF will decrease the difference between the blood in the glomerular capillaries and the primary urine, thereby decreasing the protein concentration in the blood that leaves the glomerulus through the afferent arteriole. Thus, FF can reduce the driving force for reabsorption along the proximal part of the nephron into the peritubular capillaries92. The decrease in FF seen in Study III supports the involvement of reabsorption in the mechanism of C-peptide-reduced reduction of GFR.

No reduction was seen in total Na+ excretion after C-peptide, a finding

that is consistent with previous studies36. However, the reduction in tubular Na+-load, due to reduced GFR after C-peptide, will conceal the inhibitory effect on total urinary Na+ excretion. The calculated fractional Na+ excretion increased in the diabetic animals after C-peptide administration, which shows that C-peptide directly inhibits Na+ reabsorption exclusively in the

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diabetic rats. The observed differences in the reduction of Pstf and Pff in this study may also indicate a component of proximal tubular reabsorption in the decrease in GFR.

Renal glomerular arteriolar vascular tone and blood flow Renal glomerular arterioles exert critical of Pcap, thereby influencing GFR. However, a pronounced constriction of the afferent arterioles alone would reduce GFR, FF and RBF, and concomitantly increase RVR. Indeed, C-peptide reduced the elevated GFR and FF in the present study, but without altering RBF whatsoever. Although an unaltered RBF is consistent with previous reports24, 26, these results suggest that C-peptide reduces hyperfiltra-tion partly via constriction of the afferent glomerular arteriole, but without affecting renal blood flow, findings that impliy there must be a simultaneous dilation of the efferent arteriole that counterbalances the effect on RBF. The finding reduced RVR and Pstf after C-peptide administration even indicates a net dilation of the efferent arteriole in vivo.

Cellular C-peptide signaling

The vasoconstrictive effect of C-peptide takes considerable time to develop (10 minutes), compared to angiotensin II or norepinephrine. An explanation could be a sequential cascade of intracellular events leading to vasoconstric-tion, e.g. through the activation of a receptor with subsequent effects on pro-tein synthesis. Although it seems clear that C-peptide possesses a reducing effect on diabetic hyperfiltration, there is still no effector, no receptor, and no downstream signalling cascade reported for this phenomenon. So what causes this effect of C-peptide on hyperfiltration?

C-peptide and Rho-kinase The G-protein Rho and its downstream effector Rho-kinase play important roles in mediating vasoconstriction in the kidney93. They regulate glomerular blood flow and GFR, as well as the function and structure of renal cells such as tubular epithelial cells and mesangial cells94. Inhibition of Rho-kinase in constricted renal afferent arterioles, as well as in other vessels, causes a re-laxation93, 95, 96. Rho/Rho-kinase seems to act in the tonic phase of constric-tion. Although this phase is stimulated by Ca2+ influx and Rho/Rho-kinase is activated by Ca2+, vasoconstriction is maintained at modestly elevated Ca2+ levels, a phenomenon referred to as Ca2+-sensitization97-99. Except for Ca2+ influx or Ca2+ sensitization Rho-kinase activity has also been shown to be stimulated independently of Ca2+96, 100, 101.

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We show in this study that the Rho-kinase inhibitor Y-27632 prevents the vasoconstrictive effects of C-peptide in vitro. These results are in line with previous data by Zhong et al., showing translocation and binding of RhoA by C-peptide in renal tubular cells38. A C-peptide-induced activation of Rho-kinase could be attributable to an increase in Ca2+ sensitivity96, 102.

However, the preventive effects of Y-27632 on C-peptide-induced vaso-

constriction of isolated afferent arterioles do not necessarily imply that C-peptide activates Rho-kinase. C-peptide is known to increase intracellular Ca2+ in human tubular cells33, smooth muscle cells103, and aortic endothelial cells104, and it has been reported that C-peptide activates PKC via increased intracellular Ca2+ in opossum proximal tubular cells105. C-peptide also acti-vates MAPK through a PKC-dependent mechanism38. Importantly, PKC and MAPK both mediate contraction, e.g via phosphorylation of CPI-17 and caldesmon, respectively106-108. Additionally, the MAPK cascade has been linked to phosphorylation of the regulatory subunit of the myosin light chain in the same location as myosin light chain kinase109. Inhibition of Rho-kinase will allow for greater myosin phosphatase activity, which in turn decreases the phosphorylation status of the regulatory subunit of the myosin light chain. Therefore, it is possible that C-peptide induces phosphorylation of the myosin light chain in a non-Rho-A-dependent manner, such as PKC or MAPK (Fig. 26).

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Figure 26 A. Vasoconstriction is dependent on the phosphorylation of the myosin light chain, which is controlled by myosin phosphatase, mitogen-activated protein kinase (MAPK) and myosin light chain kinase (MLCK). B. The myosin phosphatase activity is regulated by Rho kinase and protein kinase C (PKC).

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C-peptide and oxygen consumption Electrolyte transport via Na+/K+-ATPase activity accounts for approximately 80% of total renal QO2

110. Therefore, a diabetes-induced increase in Na+/K+-ATPase activity is likely to influence total renal QO2, possibly limiting renal O2 availability46, 47. The finding of increased cellular QO2 in diabetic rats in Study III is in good agreement with previous reports9, 111. However, Study III shows that C-peptide directly inhibits QO2 in isolated proximal tubular cells, but the in vivo total kidney QO2 did not differ before and after C-peptide ad-ministration, even though TNa decreased by approximately -17%. A possible explanation can be a shift of Na+ reabsorption to other sites along the nephron after the inhibition of proximal tubular Na+/K+-ATPase activity. The QO2 per TNa in the proximal tubulus is significantly less compared to more distal parts of the nephron, since a notable part of the Na+ reabsorption in the proximal tubulus is achieved via paracellular transport112. This expla-nation would account for the lack of reduced QO2 in vivo in C-peptide-treated diabetic rats.

Ouabain inhibits Na+/K+-ATPase, although complete inhibition cannot be

achieved in rats113, 114. Ouabain-treatment reduced cellular QO2 in both inves-tigated groups, but C-peptide had no further effect on ouabain-treated cells. This strongly indicates that C-peptide reduces Na+/K+-ATPase activity. No-tably, this would not be unique for C-peptide, since insulin also has been shown to influence tubular electrolyte transport115.

Sustained hyperglycemia is closely associated with a reduced nitric oxide

bioavailability, which might influence QO2. A possible mechanism for the observed reduction in QO2might therefore be C-peptide-mediated effects on NO production. However, in Study III, inhibition of NO production by L-NAME had no effect on the C-peptide-induced decrease in QO2 by the iso-lated tubular cells.

Potential tissue or state-specificity of C-peptide Although the beneficial effects of C-peptide in diabetes mellitus have been observed in various tissues116, 117, no complete mechanism, receptor, or downstream signalling cascade has yet been proposed to fully explain these effects, and existing data seem contradictory. In this thesis, C-peptide in-duced a constriction of the renal afferent arteriole. Blood flow seems to be differentially affected in the kidney compared to peripheral tissues in the diabetic state118-120, and when previous studies have showed normalizing effects of C-peptide on diabetes-impaired blood perfusion and blood cell velocity116, 121, C-peptide has had a normalizing effect on diabetes-induced decreases in blood perfusion 122-124, indicating a dilating effect. In renal ves-

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sels, sustained diabetes will cause increased blood flow118. Thus, it is likely that C-peptide exerts tissue-specific effects.

Data sometimes seem contradictory also on the molecular level. Neurons,

heart and skeletal muscles from diabetic animals, as opposed to diabetic proximal tubules, display reduced Na+/K+-ATPase activity117, 125. C-peptide has been reported to improve autonomic nerve function via stimulation of Na+/K+-ATPase activity126, 127, and seems to increase erythrocyte Na+/K+-ATPase in diabetes128, 129. A diabetes-induced decrease in Na+/K+-ATPase activity also occurs in the renal medulla after long-term hyperglycemia130, and C-peptide seems to stimulate medullary Na+/K+-ATPase activity after an extended period of hyperglycemia131. In glomerular tissue from diabetic rats, Na+/K+-ATPase activity may decrease as well as increase as a result of long-term hyperglycemia 132, 133. Taken together, most studies report pronounced effects of C-peptide on the Na+/K+-ATPase activity, but C-peptide seems to have tissue-specific effects during insulinopenic diabetes. Furthermore, most of the previous studies on C-peptide-induced effects on Na+/K+-ATPase activity in the kidney, have been conducted during normoglycemia and by applying pharmacological doses86, 131. Thus, it cannot be excluded that the effects are crucially dependent on hyperglycemia or the length of C-peptide deficiency.

Lack of effect on normoglycemic animals In neither Study I, nor III was there any effect of C-peptide on normoglyce-mic animals with normal GFR. In Study II, the afferent arterial diameter was unaffected by C-peptide until the very last measurement. The fact that nor-moglycemic mice and rats did not respond to C-peptide is consistent with previous studies23, 134, 135. Also, as reported previously, perfusion with scram-bled C-peptide did not produce any effect27, 38, 117, 136, 137.

The lack of effect in normoglycemic animals can be explained by C-peptide, or a residual effect of C-peptide, being present in these animals be-fore the start of exogenous administration. This explanation is supported by the observation that the full effect of C-peptide is obtained already at nano-molar concentrations136.

C-peptide-induced reduction of blood glucose In Study I, C-peptide as well as EVARQ reduced blood glucose concentra-tions in diabetic rats. The effect of C-peptide on blood glucose has previ-ously been described134. Sato and co-workers have reported that EVARQ increases glucose turnover to the same extent as intact C-peptide37, which

56

suggests that the active site within the C-peptide that mediates these meta-bolic effects is located within the EVARQ sequence.

Intradermal insulin treatment Good glycemic control is essential in order to minimize the risk of diabetes-induced complications17. Study IV shows no disadvantage using the in-tradermal patches as compared to the standard, subcutaneous delivery route. Subcutaneous injections are known to cause a substantial variability in dose delivered53. Intradermal delivery showed a controlled distribution to the sys-temic circulation, and even displayed a trend towards lower variability, pos-sibly due the size of the infusion area, which minimizes the influence of the infusion site. The novel, patch-like microneedle-assisted intradermal infu-sion, showed a pronounced effect on blood glucose before the effect on blood glucose was obvious using subcutaneous infusion. This could also be due to the larger infusion area of the microneedle array, compared to the single-needle subcutaneous infusion.

Previously, few data exist from in vivo studies on diabetic animals, and

never on animals given the time to develop the skin characteristics of diabetes mellitus138. Previous studies have been performed on small groups of anaesthetized rats without facilitated breathing, compensation for fluid losses or monitoring of hematocrit and blood pressure to guarantee physio-logically relevant conditions139. No previous reports describe an integrated system where the microneedles are attached to a drug dispenser, and there is little previous research on active microneedle delivery, where insulin can be administered in a controllable fashion. The results for Study IV show that the newly developed device has all these characteristics.

It is not at all unlikely that compliance to insulin treatment would be

higher if the treatment procedure was simple and painless, as with this patch-like microneedle device61. In parallel with avoiding multiple injection regi-mens, needle anxiety, and social difficulties associated with self-injecting, this novel administration route requires minimal training and attention. Thus, the patch-like microneedles are a feasible option to improve glycemic con-trol, and might reduce the development of long-term diabetes complications.

Study IV presents a possibility of controllable and patient-friendly insulin

delivery. The results indicate that microneedle arrays used as intradermal patches should be used clinically, and might introduce a more efficient treatment for diabetic patients. This microneedle technology could also prove to be efficient in systemic as well as local delivery of other macromo-lecular drugs, such as DNA vaccines, and endocrine substances140-144.

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Summary and conclusions

Taken together, the results from these studies support the conclusion that C-peptide affects diabetes-induced glomerular hyperfiltration via several seem-ingly different mechanisms. These include constriction of the afferent arteri-ole and dilation of the efferent arteriole, but possibly also reduction of the proximal tubular electrolyte reabsorption, all factors influencing GFR.

The results from Study I show that the in vivo effects of C-peptide can be

mimicked by its carboxy-terminal penta-fragment EVARQ. The infusion of EVARQ decreased filtration to the same extent as the intact C-peptide. This suggests at least one active site within the EVARQ region of C-peptide.

The main new findings from Study II, are that C-peptide has a pro-

nounced vasoconstrictive effect on afferent arterioles from diabetic mice. This effect seems to be dependent on Rho-kinase, since the Rho-kinase in-hibitor Y27632 prevented any C-peptide effect.

In Study III, acute administration of C-peptide reduced GFR in diabetic

rats via possibly two separate mechanisms. First, C-peptide caused a net dilation of the efferent arteriole. Secondly, C-peptide inhibits tubular Na+ reabsorption in diabetic rats, likely via a direct effect on the Na+/K+-ATPase.

Study IV showed that intradermal microneedle insulin administration re-

duced plasma glucose and increased plasma insulin comparably to standard, subcutaneous administration, indicating that the microneedle approach should be tested clinically, and might provide a more efficient treatment of diabetic patients.

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Future perspectives

The results from these studies display several opportunities for new drug targets and research directions.

Since it theoretically should be simpler to develop agonists to a penta- than a 31-amino acid peptide, the in vivo effect of EVARQ may simplify the development of agonists for the clinical settings. Research should aim at investigating the potential of EVARQ treatment via less invasive delivery systems, such as preparations for inhalation, intradermal delivery or oral administration. Finally, the development of antagonists against C-peptide targets could provide valuable tools in the search for the pathophysiological mechanisms mediating the development of diabetic nephropathy.

Since the reducing effect of C-peptide on diabetes-induced glomerular

hyperfiltration is achieved without affecting blood flow, further studies should be undertaken, investigating the effect of C-peptide on the intricate interplay between the afferent and the efferent arteriole and on renal Na+ reabsorption, e.g. on expression and trafficking of Na+-transporters.

Concomitantly, the effectiveness of C-peptide on the prevention and re-

versal of diabetic complications, should be more thoroughly investigated in human subjects. To achieve this, long-term, large-scale studies should be conducted in order to evaluate safety and health benefits of long-term ad-ministration of C-peptide or a C-peptide derivate.

Finally, this thesis shows that the newly developed microneedles are a

possible treatment strategy for the commonly used, fast-acting insulin lispro. Further studies should aim at investigating the microneedle device in terms of increased flow rate, biocompatibility and develop devices with incorpo-rated blood glucose sensing and self-controlled insulin rate.

Meum cerebrum nocet

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Sammanfattning på svenska

Trots noggrann insulinbehandling utvecklar fortfarande omkring 30 procent av alla diabetiker njurskador. Dessa njurskador leder i sin tur till bland annat hjärt- kärlproblem och är också den vanligaste orsaken till för tidig död bland dessa patienter. På senare tid har forskare upp-täckt att en tidigare förbisedd molekyl, C-peptid, har fördelaktiga effek-ter vid behandling av diabetiker. Kanske börjar vi nu även förstå hur dessa positiva effekter uppkommer.

Njurskador vanliga vid diabetes Ofta drabbas diabetiker av komplikationer som ytterligare försämrar deras hälsa. Båda typerna av diabetes leder under årens lopp många gånger till livshotande följder, t.ex. njurskador. Så mycket som 30% av de diabetiker som haft sjukdomen i 10-20 år uppvisar påtagliga njurskador.

Njuren spelar en mycket viktig roll inte bara för utsöndringen av diverse

skadliga ämnen från kroppens ämnesomsättning, utan också för regleringen av kroppens blodtryck. Denna reglering sker på så sätt att kroppens vätske-volym bestämmer blodtrycket. Ju större vätskevolym, desto högre blodtryck. I njurarna filtreras allt blod kontinuerligt över från små kapillärer, glomeruli, till ett rörsystem, tubuli. Regleringen av kroppens blodtryck sker sedan ge-nom att njurarna reglerar hur mycket vätska som tas tillbaka till kroppen igen och hur mycket som ska stanna kvar i tubuli för att så småningom ledas ut ur njuren till urinblåsan.

Skador på njuren innebär att skadliga ämnen och nedbrytningsprodukter

från ämnesomsättningen inte kan utsöndras och att blodtrycket inte längre regleras optimalt. Sådana skador kan därför leda till allvarliga hjärt- och kärlsjukdomar. Dessutom ökar skadorna på njurarna risken för nedsatt kän-sel, synskador och blindhet. Då njursvikten förvärras och den sjuka njuren inte längre förmår utsöndra de skadliga ämnena, kan så småningom urinför-giftning uppstå. Tillståndet kan bli så allvarligt att patienten inte överlever utan njurtransplantation eller regelbunden rening av blodet genom dialys.

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Idag vet man att minutiöst övervakade blodsockernivåer minskar riskerna för skador och att själva skadeprocessen kan bromsas med noggrann insulin-behandling och en aggressiv blodtryckssänkande behandlingsstrategi. Någon egentlig bot finns dock fortfarande inte och heller ingen annan behandling som i längden kan bromsa sjukdomsförloppet. Detta medför inte bara myck-et lidande för de drabbade, utan kostar även sjukvården och samhället i öv-rigt stora summor pengar, eftersom patienterna ofta tvingas till sjukpension och omfattande vård.

Inte bara insulin Det saknas fortfarande mycket kunskap om hur diabetes drabbar våra njurar. Målet är nu att försöka komma fram till ett sätt att förhindra diabetespatien-ters njurskador, så att patienternas livslängd ökar och deras livskvalitet för-bättras. Under mitt avhandlingsarbete har detta huvudsakligen skett genom forskning kring en substans, C-peptid, som produceras hos friska individer, men som saknas hos vissa diabetiker. Hypotesen är att det är kombinationen av insulinproblematiken och bristen på C-peptid som tillsammans ger de njurskador som så ofta drabbar diabetiker.

Ett av de första tecknen på att njurarna påverkats hos en diabetespatient är

att njurarna tillväxer och att de filtrerar alltför mycket, något som belastar njuren och de kapillärer, glomeruli, där filtrationen sker. Senare övergår denna ökade filtration istället till en underfiltration, och proteiner börjar läcka ut till urinen. Alla dessa parametrar anses vara viktiga markörer för begynnande njurskador. Det är inte klarlagt vad som orsakar dessa fenomen, men tidigare studier har visat att C-peptid kan minska den ökade filtrationen till normala nivåer hos både försöksdjur och diabetiska patienter. Mekanis-men bakom detta är dock okänd.

Ett steg i att kartlägga C-peptids verkningsmekanism och bana vägen för

nya läkemedel, är att undersöka vilken del av molekylen som ger effekt. I avhandlingsarbetets första studie undersökte vi om en kortare bit av C-peptid, EVARQ, kan ge samma effekt som hela molekylen. Studien visade att EVARQ sänker den ökade filtrationen hos diabetiska råttor lika effektivt som hela C-peptidmolekylen. På sikt skulle detta kunna underlätta utveck-lingen av nya läkemedelsmolekyler som, till skillnad från C-peptid, inte bryts ned i magtarmkanalen och därmed kan tas i exempelvis tablettform.

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C-peptid skyddar Ett annat viktigt steg för att förstå hur C-peptid skyddar den diabetiska nju-ren är att undersöka hur den ökade filtrationen sänks. En möjlighet är att det lilla blodkärl som leder till varje filtrationsställe dras ihop, så att mindre vätska i taget når fram och kan filtreras. I delstudie två visade vi att sådana små kärl från diabetiska djur kraftfullt dras ihop då C-peptid tillförs. Ett för-virrande faktum i detta fynd är dock att vare sig vi eller andra sett någon minskning i blodflöde, något som borde vara en naturlig följd av en kärl-sammandragning. Resultaten från delstudie tre antyder dock att det blodkärl som leder från filtrationsstället vidgas av C-peptid, vilket kan förklara varför njurens blodflöde förblir konstant. En sådan vidgning kommer att leda till att blodet snabbare lämnar filtrationsstället och kommer alltså även det att bidra till en minskning av den ökade filtrationen. Det är alltså möjligt att C-peptid på mer än ett sätt skyddar njuren från diabetesorsakade skador.

En ytterligare fråga är om C-peptid verkar på ytterligare flera sätt än des-sa. Därför undersökte vi vad som händer om friska celler eller celler från diabetiska råttor får tillgång till C-peptid. Den tredje studien visar att vissa njurceller från råttor med diabetes använder mer syre än njurceller från friska råttor, men att C-peptid kan minska syrgasanvändingen mot normala nivåer. Detta innebär två potentiella fördelar: dels minskar en lägre syrgasanvänd-ning risken för syrebrist i njuren vid diabetes, dels kan syrgasanvändningen även den kopplas till att den ökade filtrationen sänks.

Så hur kan syreanvändningen tänkas hänga samman med en minskad filt-ration? Fyra femtedelar av syrgasförbrukningen i de njurceller vi undersökte går åt till att hålla igång saltsparande pumpar, Na+/K+-ATPas. En minskad användning av syre skulle alltså kunna innebära att dessa pumpar hämmas av C-peptid, så att de saktas ned. Denna hypotes stärktes av att C-peptids effekt uteblev då Na+/K+-ATPas-pumparna redan i förväg hämmats av en annan substans, ouabain. Eftersom Na+/K+-ATPas-pumparna tar tillbaka natrium-joner från urinen till blodet, borde en hämmande funktion leda till att en större andel natrium följer med urinen ut ur kroppen. Mätningar i delstudie tre visade att behandling med C-peptid faktiskt minskade den andel natrium som återupptas till blodet och ökar den del som stannar kvar i urinen. Det är detta återupptag som kan påverka filtrationen genom att det natrium som finns kvar i nefronet i den blivande urinen håller kvar vätska och därigenom indirekt minskar drivkraften att filtrera ytterligare blod till nefronet. Ett minskat återupptag till blodet, som vid administrering av C-peptid, kan alltså minska filtrationen.

Sammantaget antyder försöken att C-peptid minskar belastningen på nju-rens nefron genom att dra ihop de tillförande kärlen, vidga de frånledande

62

samt eventuellt skapa ett mottryck via en hämmande effekt på Na+/K+-ATPas.

En sorts plåster med miniatyrnålar För att förebygga uppkomsten av njursjukdom hos diabetiker krävs att blodsockernivåerna noggrant hålls stabila, något som kräver ständigt förbätt-rade behandlingsformer och att patienten är följsam och använder sina läke-medel på ett optimalt sätt. I den fjärde och avslutande studien testade vi en sådan ny behandlingsform, en sorts plåsterliknande beredning med mycket korta och tunna, smärtfria mikronålar på undersidan, som bara är en tiondels millimeter tjocka.

I våra studier sänkte mikronålarna blodsockret mer förutsägbart än tradi-tionell behandling. Resultaten visade att mikronålarna mycket väl borde kunna användas för tillförsel av insulin till diabetiker. Eftersom sådana smärtfria ”nålplåster” inte gör ont och alltså torde vara mindre obehagliga att använda än de injektioner diabetiker är beroende av idag, borde följsamheten också öka, så att fler patienter stabiliserar sina blodsockernivåer och på så sätt minskar risken för följdsjukdomar. Dessa nålar öppnar dessutom för ett helt nytt sätt att tillföra peptidbaserade läkemedel, såsom insulin och C-peptid.

Ett fortsatt vetenskapligt arbete kan ge en fördjupad insikt i de sjukdoms-processer som ligger bakom de njurskador och därmed också hjärtkärlsjuk-domar som ofta uppstår hos diabetespatienter. I bästa fall kan de bidra till att bana vägen till nya behandlingsstrategier. Kanske skulle följdsjukdomar kunna stoppas om insulinbehandlingen förändrades samtidigt som C-peptid gavs redan vid insjuknandet, allt som en naturlig del av behandlingen.

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Acknowledgements

This work was performed at the department of Medical Cell Biology, Upp-sala University, Sweden. It was financially supported with grants from The Swedish Medical Research Council, The Swedish Heart and Lung Founda-tion, The Swedish Society for Medical Research, The Fredrik and Ingrid Thuring Foundation, The Marcus and Amalia Wallenberg Foundation, The Magnus Bergvall Foundation, NIH K-99 grant (DK-077858), The Wallen-berg Foundation, The Swedish Foundation for Strategic Research (SFF), The Ingabritt and Arne Lundberg Foundation, The Wenner-Gren-Foundation, Medartuum, Stiftelsen Sveriges Farmacevtiska Sällskap, Svensk Njurmedi-cinsk Förening, Stiftelsen Petersenska Hemmet, Fredrika Bremer Stiftelsen, Håkanssons stipendiefond, Rönnows stipendiefond, Kvinnliga Akademikers Förening (KAF), Rektors Wallenbergmedel, The Swedish Pharmaceutical Society, and The Scandinavian Physiological Society.

Sincere thanks and gratitude to everyone who, in different ways, has con-tributed to this thesis by helping, encouraging, guiding, nodding or shaking your heads during these years. I utterly hope you already know how much I have appreciated your support, but if I haven’t showed it enough, don’t ever think I didn’t notice!

In particular, I would like to thank: My supervisor, Mats Sjöquist, for introducing me to research, and for in-

spiring me to think independently. Thanks also to my co-supervisor, Peter Bergsten, for enthusiastic, bold ideas.

Erik Persson, for thoughtful and invaluable help. Leif Jansson for sharing

much needed wisdom and inspiration, Britta Isaksson, for always taking the time to help, and for helping to perfection. It simply wouldn’t have been possible without you. Angelica Fasching, for untiring help, for encourage-ment, and for laboratory skills beyond belief. All the other corridor seniors; Peter Hansell, Johanna Henriksnäs, Lena Holm, Annika Jägare, Örjan Käll-skog, Mia Phillipson, Göran Ståhl, Mats Wolgast et al. For your vast knowl-edge in practical as well as theoretical aspects of research, for challenging and stimulating discussions, for surströmming, kräftskivor, and november-

64

fester. I have enjoyed working with you. A special thanks to Astrid Nordin for your excellent technical assistance.

A great thank-you also to my co-authors and contributors: To Enyin Lai

for excellent skills and company and for being a good friend, to Niclas Rox-hed for changing faculty over night and becoming a life scientist instead of being a civil engineer. Erika Moe for making it worth it in the very begin-ning, Russell Brown for unimaginable speed, Göran Stemme for making the insulin-patch project a reality, Patrick Griss for turning Swiss fondue and wine into science, and of course to Marco Seiz for cooking it in the first place.

My warmest thanks to Carolyn Ecelbarger for welcoming me to your lab

and for involving me in your research. Thanks also to Swasti Tiwari, Shahla Riazi and Xinqun Hu for fun and hard-working summers at the lab, for in-spiring collaboration, and for taking the time to share your experience.

Thanks also to the C-peptide group at Karolinska Institute, Stockholm.

For rat C-peptide and scrambled C-peptide kindly provided from the Dr Ek-berg lab. To Emma Lindahl, the catcher in the rye. And of course to profes-sor John Wahren for helpful discussions and encouragement.

Birgitta Klang, Gunno Nilsson, and all students for making teaching in-

teresting, uncomplicated, worthwhile, and fun. Agneta Bäfwe, Kärstin Flink, Marianne Ljungkvist, Karin Öberg, and Gun-Britt Lind, for all your help with the practicalities regarding employment, conferences, leaves of ab-sence, courses, et cetera. I am seriously impressed by your organizing skills.

Thanks also to the BMC Animal Facility for taking good care of my rats

and mice, to the BMC Reception for numerous new key cards, and to BMC Foto for helping me make posters and prints, even though I was always late and often confused.

Daniel Damper Färnstrand, for untiring, kind and skillful revision of my

at times slightly overcomplicated Swenglish language. For taking away most of the Who Dunnit? from the manuscripts. Thank you.

Joel, Mattias and Tommy, Johan Fiasko, Olerud, Andrei, Professor Enyin,

Zofu, Jenny, Malou-Malou, Olof, Lazlo Deputy Brown, Micke Fluff, Louise, Nina, Sara, Åsa, Ulrika and Gustav: Thank you all for help, coffee breaks, conference professionalism, relaxed Friday beers, spontaneous partying and lab intrigues. And of course to Magnus – best friend and PhD-student in arms.

65

A special thought to Göran Odmark for forcing me to think: you are truly one of a kind, and without you, I would probably have ended up at a com-pletely different faculty.

Crull, Thérese, Emil, Malin, e-Rolf and Tanya: thanks for nothing but

headaches, you bastards. And possibly some friendship. To M., Odli, HMS T. and the Winers; closer than thee. And to Emelie F-

C, for sometimes spontaneously pulling me away for a fast gallop. For helping me through statistics, setbacks and endless lab sessions: my

frank gratitude to Dionne Warwick, Aretha Franklin, Maria Callas, Georg Friedrich Händel, Dolly Parton, Marvin Gaye, Kasey Chambers, Monica Zetterlund, Eric Cartman, Georges Bizet, Björn&Benny, Diana Ross, Anto-nio Carlos Jobim, Roger Whittaker, Lill Lindfors and, of course, Stevie Wonder.

For the essentials of life: wet kisses to Daniel, Erica, Isabelle, Lilla Gris,

Mårten, Saris, Snibben, Tossa (as well as Naken-Janne and Metro-Christer), Åsan, Aida, and all other friends. Thank you for your support, friendship, and for valuing one another for everthing but academic merits. I cannot be-lieve I am finally here! Thank you too, Mårten, even though you discuss science and urine samples after midnight.

Mams och paps, Saris och Larz: thank you for your unconditional love,

handiwork, midnight korvmackor, patience and support. Moreover, Saris, sorry for all the lattes and Sven Dufva lunches I ran away from to get back to being boring. Kommissarie Rex next week? Och så klart en stor kram till världens bästa mormor och morfar!

Soon, the storm has finally faded. Thank you, Fredrik, for being construc-

tive when I was not. For challenges, for encouragement, and for keeping my feet on the ground. For covered-up Trollhål. For sharing your life with me. For making me happy. For loving me back.

In utter panic, Uppsala, October 2007

Ad astra per alas porci

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