Renin–angiotensin system blockade in diabetic nephropathy

Diabetes & Metabolic Syndrome: Clinical Research & Reviews (2008) 2, 135—158

http://diabetesindia.com/

REVIEW

Renin—angiotensin system blockade in diabeticnephropathy

Jamal Ahmad *

Centre for Diabetes & Endocrinology, Faculty of Medicine, Jawaharlal Nehru Medical College Hospital,Aligarh Muslim University, Aligarh 202002, India

KEYWORDSAngiotensin-convertingenzyme inhibitor;Angiotensin II receptorblockade;Diabetic nephropathy

Summary Diabetes nephropathy (DN) is a clinical syndrome characterized by arelentless decline in GFR, persistent albuminuria, arterial hypertension and highlyelevated risk for cardiovascular morbidity and mortality. Increased urinary albuminexcretion rate (UAER) in the range of microalbuminuria (20—200 mg/min) predictsovert nephropathy in patients with type 1 and type 2 diabetes. The renin—angiotensinaldosterone (RAAS) system has been implicated in the patho-physiology of diabeticrenal disease, based mainly upon the ability of angiotensin-converting enzymeinhibitors (ACEI) and angiotensin II type 1 receptor blockers to reduce proteinuriaand the progression of diabetic glomerulosclerosis. The RAAS is a co-ordinatedcascade of proteins and peptide hormones, the principal effector of which isangiotensin II. The RASS is a key player in the progression of diabetic renal disease.The systemic RAAS is generally suppressed in DN, whereas the intrarenal RAAS may beactivated even early in the course of diabetes. All of the components of the RAAS arepresent within the kidney. Together with the observations that angiotensin II recep-tors are localized to renal arterioles, glomerular mesangial cells and the basolateraland apical membranes of proximal tubule cells, these findings are consistent with aprimary role for ANG II as a paracrine substance in the control of renal function. RenalANG II is therefore, a strong determinant of glomerular haemodynamics and intra-glomerular pressure and stimulates mesential cell proliferation by increased expres-sion of TGF-b and other cytokines and growth factors independent of blood pressure.Furthermore, prosclerotic effects of high glucose concentrations are most likelymediated by interaction with angiotensin II and autocrine production of TGF-b.

Several studies have clearly shown that both in type 1 and type 2 diabetic patientswho already have microalbuminuria, ACE inhibition is effective in reducing renalprogression. It has been proposed that the benefit is independent of blood pressure.Therefore, inhibition of RAAS plays an essential role in the treatment of hypertensionand diabetes related complications. Studies focusing on renal end-points suggest thatACE are more effective than other traditional agents in reducing the onset of clinicalproteinuria in both type 1 and type 2 diabetic patients with incipient nephropathy,mainly in normotensive ones (secondary prevention). However, several small trials intype 2 diabetic patients with overt nephropathy (tertiary prevention) failed todemonstrate a specific renoprotective role for ACE inhibitors, at variance withtype 1 diabetes. Three recent large trials addressed the question of whether ARB

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136 J. Ahmad

prevented the development of clinical proteinuria or delayed the progression ofnephropathy in type 2 diabetes. The IRMA study showed that irbesartan is moreeffective than conventional therapy in preventing the development of clinicalproteinuria and favouring the regression to normoalbuminuria for comparable BPcontrol in patients with incipient nephropathy. The IDNT and RENAAL trials showedthat ARB are more effective than traditional antihypertensive therapies in reducingprogression towards end-stage renal failure in type 2 diabetic patients with overtnephropathy independently of changes in BP. Moreover, a reduction in hospitalizationfor heart failure was demonstrated by ARB-treated patients compared with placebo.The time course of antihypertensive and antialbuminuric effects after initiation ofARB treatment shows that the maximal antialbuminuric effect is seen within 7 daysand therefore, primarily consequence of systemic and renal haemodynamic changes.However, non-haemodynamic effects such as reduced renal expression of TGF-b andother cytokines are likely to contribute to the long-term reno-protective effect.Furthermore, elevated levels of sVCAM-1 are independently associatedwith increasedcardiovascular mortality in type 2 diabetes and blockade of RAAS may have anantiatherogenic effect in diabetic patients with elevated albumin excretion asevaluated by a reduction in concentrations of circulating adhesion molecules.

A number of studies in diabetic renal disease have found an increased risk for renalfunction loss in patients homozygous for the D allele of the ACE/ID polymorphismcompared to II patients, even during ACE inhibition. ARB treatment in hypertensivetype 1 diabetic patients with DN homozygous for I or D allele of the ACE/ID poly-morphism conferred similar beneficial effect on changes in urinary albumin excretionand rate of decline in GFR in patients with different genotypes. A large-scale trialcomparing the effects of ARB treatment and ACE inhibition should be performed tofurther investigate the pharmacogenetic possibilities of ACE/ID polymorphism intreatment of diabetic renal disease.

Preliminary studies have suggested that dual blockade of the RAAS by combinedtreatment with ACE inhibition and ARBs is well tolerated and reduces albuminuria andblood pressure in patients with type 2 diabetes and DN responding insufficiently toantihypertensive combination therapy, including the recommended dose of ACE inhi-bitor. However, long-term studies of dual blockade in incipient and overt DN should beinitiated. Increasing evidence suggest an implication of aldosterone in the pathogenesisof progressive renal disease, which may suggest a beneficial effect of aldosteroneblockade. Therefore, selective and non-selective aldosterone blockers are being inves-tigated as future treatment options in DN. The increased expression of renal TGF-b andother cytokines in diabetic glomerulopathy may be lowered by RAAS blockade, butprobablynot completelyabolishedevenbyhigh-dose treatment.Therefore,agents thatmodulate other pathogenic pathways are being evaluated as potential treatmentmodalities in diabetic renal disease. These studies include agents that interfere information of advanced glycation end products, compounds targeting the activity ofprotein kinase C and antibodies/receptor blockers to cytokines such as TGF-b.

After establishing the concept of renin uptake as the underlying cause of tissueangiotensin generation, focus is now on the mechanism that mediates this uptakeprocess. Several renin receptorshavealreadybeendescribed.These receptors alsobindprorenin, and such binding results in prorenin activation, either proteolytically or non-proteotytically. This is important in view of earlier observations that prorenin levels indiabetic subjects are an indication of microvascular complications. Now that renininhibitors will soon be clinically available, it will be of great interest to investigate howthese drugs affect these mechanisms in comparison with other RAAS blockers. Even-tually a new class of drugsmight emerge, the renin receptor blockers, which selectivelyblock angiotensin generation at tissue sites and/or renin receptor-mediated effects.# 2008 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137The renin—angiotensin-aldosterone system in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138The intrarenal RAAS system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Renal effects of angiotensin II in diabetic nephropathy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Renin—angiotensin system blockade in diabetic nephropathy 137

Haemodynamic effects of angiotensin II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139Non-haemodynamic effects of angiotensin II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139Angiotensin II receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140New aspects of the RAAS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141Glomerular permselectivity in diabetic nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142Antihypertensive treatment in incipient diabetic nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143Type 1 diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143Type 2 diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145Optimal renoprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146Non-haemodynamic effects of angiotensin II receptor blockade in diabetic nephropathy . . . . . . . . . . . . . . . 146

Effect on TGF-b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146Effect on circulating adhesion molecules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147Patient’s fact ors in therapy resistance in diabetic and non-diabetic nephropathy. . . . . . . . . . . . . . . . . . 147ACE insertion/deletion polymorphism and diabetic nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147Dual blockade of renin—angiotensin system in diabetic nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . 149Pathophysiology and rationale for dual blockade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149Effect of dual blockade of RAAS in diabetic nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149Renin inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Introduction

The proportion of patients with end-stage renaldisease (ESRD) caused by diabetes has progressivelyincreased during the last few decades and diabeticnephropathy (DN) is the leading cause of chronickidney disease in patients starting renal replace-ment therapy [1]. It is associated with increasedcardiovascular mortality [2]. Diabetic nephropathyhas been classically defined by the presence ofproteinuria >0.5 g/24 h or persistent albuminuria(>300 mg/24 h or 200 mg/min) if the following addi-tional criteria are fulfilled: presence of diabeticretinopathy and the absence of clinical or laboratoryevidence of other kidney or renal tract disease [3,4].This clinical definition of DN is valid in both type 1and type 2 diabetes [3,5]. During the last decadeseveral longitudinal studies have shown that raisedurinary albumin excretion (based on single measure-ment) below the level of clinical albuminuria, socalled microalbuminuria, strongly predicts thedevelopment of DN in both type 1 [6—10] and type2 diabetes [11—14]. Microalbuminuria is defined asurinary albumin excretion greater than 30 mg/24 h(20 mg/min), and less than or equal to 300 mg/24 h(200 mg/min) irrespective of how the urine iscollected [15]. The natural course of DN is charac-terized by a mean rate of decline in GFR of 10—15 mL/min/year ranging from 0 to 25 mL/min/year[16—18]. Experience of the past two decades con-vincingly demonstrates that intensive control ofhyperglycemia and adequate lowering of hyperten-sive blood pressure are the key components ofDN management. The aggressive antihypertensive

treatment through the last decade at Steno-Diabetes Centre has reduced the mean rate ofdecline in GFR to 4.0 mL/min/year in type 1 diabeticpatients [19].

Recently, several trials have addressed the role ofrenin—angiotensin system (RAAS) in this disease andthe therapeutic utility of blocking this system.Accordingly, blockade of RAAS is first line therapyin the treatment of DN [20]. The primary effect ofACE inhibitors is mediated through reduced genera-tion of angiotensin II, but additional effects, per-haps mediated through bradykinin accumulation,may also contribute to the beneficial effect. Onthe other hand, specific blockade of the AT1 recep-tor inhibits actions of angiotensin II generated byany enzyme pathway and may overcome the sug-gested interaction between ACE inhibition and theACE/ID polymorphism, ACE inhibitors are currentlyfirst line therapy in type 1 diabetic patients with DNaccording to the Captopril Collaborative Study [21]but the effect of ARBs has not been investigatedthoroughly.

The pathogenesis of DN is complex and involvesthe direct action of high extracellular glucose onglomerular, tubular, vascular and interstitial cells.This stimulates the production of cytokines andgrowth factors, including transforming growth fac-tors-b (TGF-b), which plays a major role in glomer-ular and tubulointerstitial fibrosis [22]. High glucosemediates these effects via activation of proteinkinase C, stimulation of polyol pathway and forma-tion of advanced glycosylation end products. Thisarticle will review the aspects of pathophysiologicalmechanisms responsible for albuminuria in early DN,

138 J. Ahmad

Fig. 1 The renin—angiotensin aldosterone system.

the effect of RAAS blockade in DN and the role ofindividual patient factors in response to renopro-tective treatment including genes. Furthermore,expression of RAAS in DN, antihypertensive treat-ment and dual blockade of the RAAS in diabeticrenal disease will also be discussed.

The renin—angiotensin-aldosteronesystem in diabetes

Renin—angiotensin-aldosterone system (RAAS) playsan essential role in the pathogenesis of hypertensionand diabetes related complications. The status ofthe RAAS in diabetes has been extensively studied.Suppression of the systemic RAAS is most oftenobserved in DN [23]. A physiologic clinical rationale,therefore, exists for RAAS blockade. Among severalmechanisms involved in the development and pro-gression of diabetic kidney disease, renal haemody-namic factors have been shown to play an importantrole in its pathogenesis; among these, high intra-glomerular pressure is affected by the RAAS system.The functional role of RAAS in the regulation ofblood flow, epithelial Na+, bicarbonate and watertransport, and cell growth and differentiation havealso been clarified. The classic RAAS is a co-ordi-nated cascade that begins with the biosynthesis andsecretion of renin in the juxtraglomerular cells ofthe afferent renal arteriole. Formerly, renin wasconsidered to have no direct actions of its own otherthan the enzymatic cleavage of ANG I from angio-tensinogen (AGT). ANG I is degraded to biologicallyactive ANG II by ACE, which also stimulated the

degradation of bradykinins. Renin action on AGTto form ANG I is the rate-limiting step in the RAASCascade. Renal angiotensin II derived from the cir-culation or formed intrarenally from angiotensin I isthe main biological effector molecule of the RAASand acts as a circulating vasoconstrictor hormone aswell as paracrine and autocrine peptide tomodulaterenal function (Fig. 1). Another ANG II receptor, AT2has recently been cloned, but its functional signifi-cance was unknown until recently.

The intrarenal RAAS system

In the recent years, the intrarenal RAAS has been thefocus of extensive studies. It has a critical role in theparacrine regulation of renal function [24], asdemonstrated by presence of mRNA and proteinsof RAAS components in glomeruli and tubules incultured cells from healthy rats and humans [25—29]. Furthermore, recent immunohistochemical stu-dies have demonstrated an abundance of AT1 recep-tors in afferent and efferent arteriolar vascularsmooth muscle cells, mesangial cells of the glomer-uli and on the luminal surface of tubular cells [30].These findings are consistent with a primary role ofANG II as a paracrine substance in the control ofrenal function. In the lumen of the proximal tubules,angiotensin II levels are approximately 1000-foldgreater than levels in plasma, probably due tosecretion from tubular cells to activate epithelialAT1 receptors and thereby stimulate sodium andwater reabsorption [31]. The intrarenal RAAS maybe activated early in the course of diabetes and its


activation would be expected to generate increasedlevels of angiotensin II. A recent study in a model ofearly experimental diabetes suggested an interac-tion between activation of the intrarenal RAAS andhyperglycemia as demonstrated by significantlyincreased expression of renin mRNA in proximaltubules and whole kidney angiotensin II [32]. Bal-lermann et al. [33] first reported a decrease inglomerular angiotensin II receptors in diabetic rats3—4 weeks after induction of diabetes. Data from aclinical study in type 2 DN, demonstrated thatdespite the presence of a low plasma renin at base-line, renal vasodilation in response to ARB treat-ment was enhanced compared to control persons,which may reflect an increased intrarenal angioten-sin II level in the diabetic patients [34]. An indica-tion of intrarenal RAAS activation in type 1 diabeteswas demonstrated by Miller [35], who found thatmoderate hyperglycemia was associated with anincreased renal vascular resistance and filtrationfraction compared to euglycemia. Accordingly,renal haemodynamic responses to high blood glu-cose were blunted by administration of ARB,whereas stimulatory effects of angiotensin II infu-sion were abolished during hyperglycemia comparedto euglycemia.

Renal effects of angiotensin II indiabetic nephropathy

Renal angiotensin II is derived from the circulationor formed intrarenally from angiotensin I [24].Increasing evidence suggests that angiotensin II actsas a circulating vasoconstrictory hormone as well asa paracrine and autocrine peptide tomodulate renalfunction [36].

Haemodynamic effects ofangiotensin II

Hemodynamic mechanisms such as hyperfiltrationand increased intraglomerular pressure are asso-ciated with the development and progression ofrenal disease [37]. Early experimental studies ofglomerular ultrafiltration and infusion of angioten-sin II [38,39] demonstrated by direct micropuncturemeasurements, that angiotensin II increases effer-ent and afferent arteriolar or at least preglomerularresistance, the latter primarily due to an autoregu-latory response to an increased systemic bloodpressure. As a consequence of a greater efferentthan afferernt arteriolar effect of angiotensin II, theglomerular capillary pressure increases. The semi-

nal studies of the renoprotective effect of ACE1were performed in rats subjected to 5/6 nephrect-omy, a well-established model of progressive renaldisease in which extensive renal mass ablationresults in a compensatory increase in single nephronglomerular filtration rate (SNGFR) and elevatedglomerular capillary hydraulic pressure, with sub-sequent development of proteinuria and focal glo-merulosclerosis. Electron microscopic studiesrevealed that these changes in glomerular hemody-namics were associated with evidence of injury tovisceral epithelial and glomerular endothelial cellsand mesangial expansion, as early as 7 days afterextensive renal mass ablation. Normalization ofglomerular hemodynamics by feeding rats a low-protein diet resulted in protection against theseearly structural lesions [40]. These observationsled Brenner et al. to propose that the glomerularhemodynamic adaptations consequent uponnephron loss ultimately prove maladaptive andresult in damage to remaining nephrons, therebyestablishing a vicious cycle of progressive nephronloss [41].

In diabetic rats, glomerular capillary hyperten-sion, proteinuria and renal structural abnormalitieswere found 14 months after induction of disease[42], however, ACE inhibitor treatment reducedglomerular capillary pressure to a level comparableto a non-diabetic control group which preventeddevelopment of proteinuria and structural abnorm-alities. These observations indicate the central roleof angiotensin II in the pathogenesis of diabeticglomerulosclorosis. Similar results have also beenfound in non-diabetic rat models [43]. In type 2diabetes, calculated glomerular pressure and effer-ent arteriolar resistance were found to be increasedin patients with elevated urinary albumin excretionrate compared to normoalbuminuric patients [44]and ACE inhibitor treatment lowered glomerularcapillary pressure and albuminuria. Therefore,human and experimental data suggest that haemo-dynamic effects of angiotensin II are crucial in thepathogenesis of diabetic glomerulosclorosis.

Non-haemodynamic effects ofangiotensin II

Non-haemodynamic effects of angiotensin II are alsoimportant (Fig. 1) Angiotensin II induce expressionof TGF-b, collagen and fibronectin and stimulatesmesangial cell proliferation resulting in increasedsynthesis of extracellular matrix [45,46]. The effectappears to be mediated through activation of PKCand P38 MAPK pathways which stimulate TGF-b geneexpression [47]. Increased gene expression of TGF-b

140 J. Ahmad

induced by hyperglycemia in experimental andhuman studies [48,49] as well as in cell culturestudies [49,50] appears to be mediated by the samePKC and P38MAPK pathways [47]. The hypothesis issupported by the observation that increased TGF-bsecretion induced by hyperglycemia may be inhib-ited by addition of an ARB [51].

Other cytokines like CTGF and VEGF which act toinduce angiogenesis and markedly increased micro-vascular permeability have got a key role in thepathogenesis of diabetic microvascular complica-tions [52,53]. Angiotensin II has also been shownto increase PAI-I in cultured mesangial cells, whichmay lead to increased matrix accumulation [54].

Angiotensin II has been identified as a majormodulator of endothelial function. Endothelial cellsproduce vasoconstrictor substances such asendothelin-1 and vasodilator substances such asnitric oxide (NO). The NO system may be up-regu-lated in DN, but according to James et al. [55]angiotensin II also activates superoxide anion pro-duction and due to interaction of superoxide anionwith No, the net effective NO availability in DNappears to be reduced. The interaction betweenRAAS and endothelin-1 is emphasized by demonstra-tion of reduction of endothelin-1 by ACE inhibition inhuman and experimental studies [56].

Angiotensin II receptors

The two major types of angiotensin receptors, AT1and AT2 [57], belonging to the seven-transmem-brane G-protein-coupled receptor family and havinga similar affinity for ANG II have been identified.Further, receptor subtypes have also been identi-fied, but the physiological significance of these areunknown [58]. AT1 receptors are widely distributedthroughout the kidney, and are localized on vascularsmooth muscle cells (VSMC) throughout the renalvasculature, including the afferent and efferentarterioles and glomerular mesangial cells [59]. Theyare also found on the proximal tubule cell brushborder and basolateral membranes, thick ascendinglimb epithelia, distal tubules, cortical collectingducts, glomerular podocytes and mascula densacells [59], and on the luminal surfaces of bothproximal and distal segments of the nephron. mRNAcoding the AT1 receptor has also been localized tothe proximal convoluted and straight tubules, thickascending limb, cortical and modullary collectingducts, glomeruli, arterial vasculature, vasarectaand juxtaglomerular cells [60].

The AT1 receptors are responsible for the majoractions of angiotensin II in adults. In addition to itsactions as a circulating vasoconstrictor hormone and

stimulator of aldosterone secretion via the AT1receptor, ANG II has multiple direct intrarenalactions, including renal arterial vasoconstriction,stimulation of tubule epithelial Na+ reabsorption,augmentation of tubulo-glomerular feedback sensi-tivity and inhibition of pressure-natriuresis,mediated by the AT1 receptor. Activation of AT1re-ceptors in mesangial cells promote growth factorssuch as TGF-b [45,46] and decrease matrix degrada-tion due to PAI-1 activation [54]. In proximaltubules, AT1 receptor activation stimulates trans-port of sodium and water and induces expression ofTGF-b [61] thereby stimulating the fibrogenic pro-cess. AT1 receptor activation reduces renal bloodflow and causes efferent arteriolar vasoconstriction[62].

The AT2 receptor is highly expressed in fetalkidney, but decreases markedly during the neonatalperiod [63]. In the adult kidney, it is synthesized inthe afferent arteriole, glomerular endothelial andmesangial cells, proximal tubule cells and intersti-tial cells [59,63]. The functions of AT2 receptor areincompletely understood. Experimental data sug-gest that activation of AT2 receptor opposes theactions mediated via the AT1 receptors, by endothe-lial cell-mediated renal vasodilation and exert anti-proliferative effects [64,65]. Renal vasodilationinduced by AT2 receptor activation may also bemediated through release of bradykinin and nitricoxide [66]. The evidence regarding the effect of AT2receptor activation in diabetes is presently limitedand conflicting. AT2 receptor stimulation mightinduce both a reduction in renal vascular resistanceand natriuresis [67]. At least some of the beneficialrenal effects of AT1 receptor blockade (e.g. withlosartan or valsartan) are the results of AT2 receptorstimulation [66,68], probably because of increasedANG II production resulting from the interruption ofAT1 receptor-mediated tonic inhibition of reninrelease at the juxtaglomerular cell. The AT2 recep-tor also increases pressure natriuresis, probably bystimulating cGMP production [69,70]. The AT2 alsohas a role in endothelium-dependent renal vasodi-lation via the cytochrome P-450 pathway, possiblyby increasing synthesis of epoxyeicostetranoic acid[71]. Unlike the AT1 receptor, AT2 receptor does notinternalize in response to agonist stimulation [72].

Angiotensin II is primarily generated by conver-sion of angiotensin I through the ACE pathway, butalternative enzymes such as chymase, also contri-bute to formation of angiotensin II [73]. Thus, inhi-bition of ACE will probably not induce a completeblockade of the RAAS. After initiation of ACE inhi-bitor treatment [74,75], angiotensin II levels inplasma are lowered, however, during prolongedtreatment, the degree and duration of angiotensin


II suppression may be reduced in agreement withpartial escape of ACE inhibition [75]. IncompleteRAAS blockade during chronic ACE inhibitor treat-ment due to ACE-escape and angiotensin II forma-tion by alternative pathways may be overcome byinhibiting the action of angiotensin II at the site ofthe AT1 receptor by an ARB.

New aspects of the RAAS

New information regarding the composition of theRAAS and the specific function of its various com-ponents has recently accumulated (Fig. 2).

Renin is an aspartyl protease synthesized as pro-renin, a proenzyme that has an additional 43-aminoacid N-terminal fragment. Prorenin is convertedinto active renin exclusively in the juxtraglomerularcells of the kidney. Binding of renin to human renalmesangial cells in culture induces cellular hypertro-phy and an increase of plasminogen activator inhi-bitor-1 [76,77]. Recently, the cDNA for the humanrenin receptor, which encodes a 350-aminoacid pro-tein with a single transmembrane domain, wascloned [77], and is functional in that renin binding

Fig. 2 The new renin—angiotensin system. Solid arrows depicBroken arrows indicate enzymatic activities or heterodimerizaAGT, angiotensinogen; ANG, angiotensin; AT1 receptor, type 1receptor.

induces a 4-fold increase in angiotensinogen (AGT)conversion to ANG I. The receptor was localized inthe heart, brain placenta, liver and kidney. In thekidney it was found in the glomerular mesangiumand the subendothelium of the renal arteries [77].This discovery provides a new perspective of therole of the cell membrane in angiotensin genera-tion, the intrarenal RAAS and the possibility thatrenin acts independently of ANG II formation.

ACE promotes degradation of bradykinin, whichacts as an endogenous vasodilator via stimulation ofNO and cGMP and also via release of vasodilatorprostaglandins (PGE2 and prostacyclin). When ACEinhibitors are used, the synthesis of ANG II is inhib-ited and the formation of brdykinin, NO and pros-taglandins is facilitated. ACE inhibitor inducescrosstalk between the brodykinin actions [78]. Allof these effects serve to promote vasodilation andlower blood pressure. Experimental studies gener-ally support the hypothesis that the beneficialeffects of ACE inhibitors are mediated both bydecreased ANG II formation and by increased bra-dykinin concentrations. Deletion of ACE causes astriking reduction of blood pressure, serum electro-lyte abnormalities and renal pathology, indicating

t biochemical pathways and ligand—receptor interactions.tion. Abbreviations: ACE, angiotensin converting enzyme;angiotensin II receptor; AT2 receptor, type 2 angiotensin II

142 J. Ahmad

the many crucial roles of RAAS. A new ACE enzyme,ACE-2 has recently been discovered [79,80] that, inconstrast to ACE, hydrolyzes ANG I to ANG (1—9) andANG II to ANG (1—7), does not covert ANG I to ANG IIand is not inhibited by ACE inhibitor. Thus, ACE-2reduces ANG II formation by competitively stimulat-ing alternative pathways for ANG I degradation.ACE-2 is localized on cell membranes of cardiacmyocytes, renal endothelial and tubule cells andin the testis [80]. Interestingly, ACE-2 gene ablationseverely impairs cardiac contractivity and causesmild ventricular dilation and increased ANG II levels,but no alteration in blood pressure [81]. Ablation ofboth ACE and ACE-2 prevented the cardiac abnorm-alities and the increase in ANG II formation [81].Thus ACE and ACE-2 probably counter balance eachother in terms of enzymatic activity and function.

The newly described peptide ANG 1—7 is formedfrom either ANG I or ANG II by the enzymatic actionof tissue-specific endopeptidases, including neutraland propyl endopeptidases [82]. It is also formed bythe action of ACE-2 on ANG II, which is possibly themajor pathway of ANG (1—7) formation. ANG (1—7)is degraded by ACE to inactive peptides and itsplasma levels markedly increase in response toACE inhibitor [83]. It inhibits ACE by 30—70%. Angio-tensin-converting enzyme 2 (ACE 2) has emerged asa noval regulator of cardiac function and arterialpressure by converting angiotensin II into the vaso-dilator and antitrophic heptapeptide, angiotensin(1—7) [84]. ANG (1—7) appears to oppose the actionsof ANG II via stimulation of NO and vasodilatorprostaglandins, and can act synergistically withbradykinin [85]. It can also protentiate bradykininaction on the B2 receptor by binding to the activesite (C-domain) of ACE [86]. In addition to its vaso-dilator and natriuretic actions, ANG (1-7) has anantiproliferative action of VSMC [87].

ANG II is a hexapeptide ANG II metabolite forwhich a specific binding site exists in the outer stripeof the renal medulla [88,89]. Renal arterial infusionof ANG II elicits a dose-dependent increase in renalcortical blood flow associated with the generation ofNO [80]. ANG II also increases renal cortical bloodflow and induces natriuresis via a tubule mechanismin vivo.

Glomerular permselectivity in diabeticnephropathy

The glomerular capillary walls behave as high-capa-city ultrafiltration membranes. Glomerular perme-ability has been studied by determining fractionalclearance to exogenous non-reabsorbable macro-molecules. The fractional clearance of macromole-

cules is defined as the clearance of molecule dividedby the glomerular filtration rate of water deter-mined by insulin clearance [90] Dextran, a polymerof glucopyranose, has been most widely employedas a test macromolecule to probe glomerular perms-electivity [91,92]. Size-selectivity in type 1 diabeticpatients with DN and different levels of proteinuriahas been thoroughly investigated by clearance ofneutral dextran test molecules of graded size (30—60 A) [93,94,90,95—99]. Myers et al. [90] originallydemonstrated that advanced DN with nephroticrange proteinuria is associated with elevated sievingcoefficients above normal levels for large, nearlyimpermeant molecules >50 A. Similar results ofsize-selectivity in DN have been found in type 2diabetes [100]. Effects of RAAS blockade on chargeselectivity in microalbuminuric type 1 diabeticpatients were investigated by Hansen et al. [101]but a favourable influence of ACE inhibition couldnot be demonstrated. Two studies in non-diabetickidney disease similarly suggested that reduction inproteinuria induced by ARB treatment was asso-ciated with reductions of glomerular shunt volume[102,103]. Furthermore, comparison of ACE inhibi-tion and ARB treatment in one of the studies [103]demonstrated similar effects on membrane para-meters. Hence, the beneficial effect of RAAS block-ade of the RAAS on glomerular size-selectivefunction is likely to be a consequence of inhibitionof the action of angiotensin II. In summary, func-tional studies of size-selectivity in type 1 diabeticpatients have demonstrated that the existence of adefect in the large shunt-like pores in early andadvanced DN which may be partly restored by ARBor ACE inhibitor treatment. However, loss of barriersize selectivity may be of minor importance in thegenesis of modest albuminuria, thus, contributionsfrom charge or shape selectivity, and the influenceof RAAS blockade on these should be further inves-tigated.

It is generally acknowledged that the glomerularbasement membrane restricts large plasma proteinsbut there is an increasing evidence indicating thatthe ultimate barriers for proteins larger than albu-min are the cellular layers and slit diaphragms [104—106]. Foot processes are joined laterally by slitdiaphragm. It represents a tiny membrane bridgingthe 30—40 nm filtration slit. The transmembraneprotein, nephrin as a major component of the slitdiaphragm complex, provided a decisive progress inpodocyte biology and pathophysiology [107—109].Human and experimental studies of diabetic glomer-ulopathy have demonstrated podocyte injury andloss, along with broadening of podocyte foot pro-cesses [110,111]. Bonnet et al. [112] demonstratedthat expression of the nephrin gene and protein was


significantly reduced in spontaneously hypertensivestreptozotocin-diabetic rats compared to controlanimals. However, whether the decrease in nephrinexpression is the cause of proteinuria or a conse-quence of advanced renal injury is unknown, butchanges in nephrin expression were blunted by ARBtreatment, which also prevented proteinuria. Simi-lar results have been found in animal studies of non-diabetic kidney disease, in which changes in nephrinexpression were abolished by ARB and ACE inhibitortreatment [113]. Furthermore, ACE inhibition hasbeen shown to be associated with preservation ofslit diaphragm function and protein zonula occlu-dens-1, a component of slit diaphragin [114].Changes observed during RAAS blockade in nephrinexpression and slit diaphragm function could berelated to the reduction in intraglomerular bloodpressure rather than a direct action on podocytes.Thus, renoprotective effects by RAAS blockade maybe partly related to podocyte and slit diaphragmfunctionmediated directly via AT1 receptors locatedon podocytes on glomeruli.

Antihypertensive treatment inincipient diabetic nephropathy

Treatment of hypertension dramatically reduces therisk of cardiovascular and microvascular events inpatients with diabetes. Hypertension is common indiabetic patients, even when renal involvement isnot present. About 40% of type 1 and 70% of type 2diabetic patients with normoalbuminuria haveblood pressure levels >140/90 mmHg [115]. In theUKPDS a reduction from 154 to 144 mmHg on systolicblood pressure reduced the risk for the developmentof microalbuminuria by 29% [116]. Blood pressuretargets for patients with diabetes are lower (130/80 mmHg) than those for patients without diabetes[117]. In the Hypertension Optimal Treatment Study(HOT), a reduction of diastolic blood pressure from85 to 81 mmHg resulted in a 50% reduction in the riskof cardiovascular events in diabetic but not non-diabetic patients [118].

Reduction in proteinuria represents an addi-tional filtration parameter in antihypertensivetreatment [119]. The initial reduction in protei-nuria after initiation of antihypertensive treat-ment is predictive of the long-term efficacy ofsubsequent renoprotection in diabetic and non-diabetic renal disease [120,121]. Furthermore,residual proteinuria during treatment predicts rateof decline in GFR [19]. Therefore, in addition toblood pressure reduction, antihypertensive ther-apy should be titrated upon maximal antiprotei-nuric effect.

The concept of incipient nephropathy or micro-albuminuria is based upon the observation thatelevated urinary albumin excretion, defined as per-sistent urinary albumin excretion rate between 30and 300 mg/24 h or 20—200 mg/min, predicts pro-gression to overt DN in type 1 and type 2 diabetes[8,122—124]. Furthermore, microalbuminuria isassociated with increasing blood pressure andhyperfiltration [8]. Early, intervention studies innormotensive type 1 diabetic patients demon-strated beneficial effects of antihypertensive ther-apy with beta blockers [125] on regression ofmicroalbuminuria. Numerous subsequent studiesin type 1 as well as type 2 diabetes have confirmedthe renoprotective effects of antihypertensivetreatment in microalbuminuria, even though thedrug of choice has been debated [126].

Type 1 diabetes mellitus

Antihypertensive treatment in DM has become mostsuccessful since the first studies by Mogensen et al.[127] and Parving et al. [128] demonstrated thataggressive antihypertensive treatment with betablockers and diuretis lowered rate of decline inGFR from more than 10 mL/min/year to less than5 ml/min/year. ACE inhibitors have been recom-mended as renoprotective therapy for all type 1diabetic patients regardless of blood pressure byMathiesen et al. [129], Marre et al. [130], Vibratiet al. [131]. In 1992, Bjorck et al. [132] reported anopen prospective study with 40 type 1 diabeticpatients treated with either enalapril or metaprololfor a mean of 2.2 years, GFR declined by 5.6 mL/min/year in the metaprolol group, but only 2.0 mL/min/year in the enalapril group ( p = 0.02). A similarconclusion was made by the Captopril CollaborativeStudy Group [21] who randomized 409 type 1 dia-betic patients with DN to treatment with eithercaptopril or placebo in combination with conven-tional antihypertensive treatment for 3 years. Asignificant risk reduction by captopril treatmentof 68% (39—83; 95% CI) ( p < 0.01) in time to dou-bling of serum creatinine was found in 102 patientswith a baseline serum creatinine above 133 mmol/L,whereas the risk reduction for the 307 patients withbaseline serum creatinine below 133 mmol/L of 33%(�44—69) did not reach statistical significance(p = 0.31). The risk reduction for advanced nephro-pathy remained significant after adjustment for adifference in mean arterial blood pressure of4 mmHg in favour of the captopril group. In a recentreview [133] of randomized placebo controlledtrials investigating the effect of ACE inhibitors indiabetic and non-diabetic kidney disease, 115 of 700

144 J. Ahmad

patients in the ACE group developed ESRD or dou-bling of serum creatinine, as compared to 193 of 689individuals in placebo group. The aggregate relativerisk for these end points was 0.60 (0.49—0.70; 95%CI) for individuals treated with an ACE inhibitorcompared with placebo. In our own study of a 5-year randomized, double blind, placebo-controlled,the effect of ACE inhibitor enalapril was determinedon the progression of renal function and histology insubjects with type 1 diabetes and microalbuminuj-ria [10]. The enalapril treatment resulted in abso-lute risk reduction of 22.4% for the development ofclinical proteinuria over a 5-year period (p < 0.01).After 5 years of treatment glomerular basementmembrane thickness showed a consistent, thoughstatistically insignificant, increase in placebo group,whereas it remained stable in the enalapril group.These data indicated a specific renoprotectiveeffect of ACE inhibition on kidney function in addi-tion to what might be expected from blood pressurereduction alone. The accumulating data shows thatACE inhibitors prevent progression of microalbumi-nuria to overt nephropathy and must be regarded asfirst-line therapy for all type 1 diabetic patients withmicroalbuminuria regardless of blood pressure.

The reduced formation of angiotensin II is theprimary action of ACE inhibition. Multiple studies

Table 1 Comparison of three clinical trials with angiotensin

RENAAL ID

Design Usual care (placebo) vs.losartan 50, 100 OD

Uvsir15

Primary endpoints Doubling of serumcreatinine,ESRD or death

Docr

Target BP a140/90 a1Number of patients 1513 (750 losartan

and 750 usual care)175555

Condition Hypertensive andnon-hypertensive SCr1.3—3.0 mg/L UAE > 0.5 g/24 h

H1.U

Duration Average 3.4 years AvProteinuria Decreased DeRenal function Stagilized StIncidence of clinical

nephropathyNo data N

Incidence of ESRD Decreased NDeath No change NIncidence of first

hospitalizationfor heart failure

Decreased N

Reference [146] [1

All trials were multicenter, multinational, double-blind, randomizedUAE, urinary albumin excretion.

have suggested the existence of ACE independentpathways for angiotensin II generation, as reviewedby Hollenberg et al. [73]. Thus, angiotensin II for-mation may not be completely suppressed by ACEinhibition. On the other hand, specific blockade ofthe action of angiotensin II at the receptor levelomits the possibility of contribution from bradykininaccumulation to the antihypertensive effect.Anderson et al. [74] compared the short-term reno-protective effects of the ACE inhibitor enalapril tothe effect of specific intervention in the RAAS by theARB losartan in 16 hypertensive type 1 diabeticpatients with DN. It was observed that blockadeof the RAAS at the angiotensin II receptor level bylosartan, induces similar renoprotective effects asinhibition of ACE by enalapril as evaluated by reduc-tion in albuminuria and blood pressure in hyperten-sive type 1 diabetic patients with DN. The studyfurther suggested that the primary action of ACEinhibition is mediated by interference in the RAAS,i.e., reduced formation of angiotensin II.

Anderson et al. [134] investigated the long-termrenoprotective effect of losartan 100 mg o.d. on aprincipal renal end-point, i.e., GFR in a prospectivestudy of 54 hypertensive type 1 diabetic patientswith DN homozygons for the Ior D allele of the ACE/ID polymorphism with an average follow-up time for

AT1 receptor antagonists in patients with type 2 diabetes

NT IRMA 2

sual care (placebo). amlodipine vs.besartan 75,0, 300 OD)

Usual care (Placebo) vs.irbesartan 150, 300 OD

ubling of serumeatine, ESRD or death

Time to occurrence ofclinical proteinuria and " ofUAER from baseline by 30%

35/85 a135/8515 (550 irbesartan,0 amlodipine and0 usual care)

610 (200 irbesartan 150,200 irbedsartan 300 and200 usual care)

ypertensive SCr0—3.0 mg/dLAE > 0.9 g/24 h

Hypertensive SCr< 1.5 mg/dL UAE < 0.2 g/24 h

erage 2.6 years 2 yearscreased Decreasedabilized No differenceo data Decreased

o change No datao change No datao change No data

47] [148]

and placebo-controlled. OD, every day; SCr, serum creatinine;


36 months. Considering the total cohort disregard-ing genotypes, the annual rate of decline in GFR was3.2 (2.2—4.7) (95% CI) mL/min/year with a 24 hmean arterial blood pressure of 95 � 1 mmHg(mean � S.E.M.) during 3 years treatment withlosartan 100 mg o.d, in combination with conven-tional antihypertensive treatment other than ACEtreatment if required. A rate of decline in GFR of3.2 mL/min/year was considerably lower comparedto the outcome of the largest ACE inhibitor study,the Captopril Collaborative Study [21] in which theaverage rate of decline in creatinine clearance was8.0 mL/min/year in the captopril group and10.8 mL/min/year in the patients treated with anti-hypertensive drugs other than ACE-inhibitor or cal-cium channel blockers. Long-term renoprotectiveeffect of the ACE-inhibitor lisinopril with the long-acting dihydropyridine calcium channel blockernisodipine was evaluated by Tarnew et al. [135] ina randomized double-blinded (single-blinded after12 month) design lasting for 4 years. During thestudy period, mean rate of decline in GFR wassimilar in treatment groups, 6.0 mL/min/1.73 m2,mean arterial blood pressure levels 100 � 2 mmHg(mean � S.E.M.) and 103 � 2 mmHg in the ACE inhi-bitor and calcium channel blocker group, respec-tively. Albuminuria was lowered by approximately50% by ACE inhibitor treatment, but remainedunchanged in the calcium channel blocker groups.However, levels of albuminuria were not signifi-cantly different in the two groups during the study.

In summary, ACE inhibition may confer renopro-tective effects beyond the effect of lowering arter-ial blood pressure, however, blockade of RAAS byARB treatment induce similar short-term renopro-tective effects as inhibition of ACE. Long-term ARBtreatment implies beneficial effects on preservationof GFR, but large-scale studies of ARBs in type 1diabetes on a principal renal and cardiovascular endpoint should be performed. Therefore, ACE inhibi-tors must be considered the first line drug in thetreatment of DN in type 1 diabetes. However,increasing evidence suggest that the effect of ACEinhibitor is mediated by interference in the RAASwhich indicate that ARBs represent an effectivealternative to ACE inhibition. Calcium channelblockers may represent an effective class of drugsand long-term studies, e.g., in supplementary treat-ment of RAAS blockade in DN, should be performed.

Type 2 diabetes

Several studies have demonstrated beneficial reno-protective effects of antihypertensive treatmentin type 2 diabetic patients with microalbuminuria

[14,136—144]. Originally, Ravid et al. showed thatnormotensive patients treated with enalapril fora 7-year period, experienced an absolute riskreduction in progression from microalbuminuria tomacroalbuminuria by 42 percentage points [136].Subsequently, we have also reported that 5 years oftherapy with enalapril, compared with placeboin normotensive subjects with type 2 diabetesresulted in significant less progression (risk reduc-tion = 66.7%, p < 0.001) of microalbuminuria toclinical albuminuria, reduced albumin excretionrate, and preserved GFR [14]. Evidence has beenconflicting in hypertensive patients with microalbu-minuria regarding the existence of a specific reno-protective effect beyond the hypotensive effects ofACE inhibitors [137—145]. Superior renoprotectiveeffects of ACE inhibitors compared to conventionaltreatment have been reported by Lebovitz et al.[140], Trevisan et al. [144] and Chan et al. [145],whereas similar effects were found in the FACET[137], the ABCD (140) and the UKPDS [138] studies.The reasons for these conflicting results may in partbe explained by short duration of antihypertensivetreatment, blood pressure differences betweentreatment groups and small sample size. An excep-tion from this is the long lasting UKPDS study sug-gesting equal effect of ACE inhibition and betablockade.

Angiotensin AT1 receptor antagonists representthe newest class of antihypertensive drugs thatblock the RAAS. They selectively block the angio-tensin AT1 and thereby inhibit the vasoconstrictiveand tissue effects of ANG II, including growth, cellmovement, inflammation and thrombosis. Twolarge, long-term outcomes studies designed to testthe effects of AT1 receptor antagonists on renal andcardiovascular events in patients with type 2 dia-betes have now been completed and published[146,147]. These studies examined the effects ofLosartan [146] and irbesartan [147] on the compo-site end points of doubling serum creatinine, theonset of ESRD or death (from any cause) in patientswith type 2 diabetes and overt nephropathy. A thirdstudy reported the effects of irbesartan on theprogression of microalbuminuria to macroalbumi-nuria in type 2 diabetic patients [148]. A comparisonof the three studies is shown in Table 1. All threestudies reported that AT1 receptor antagonistsreduce baseline proteinuria 33—38% more than con-ventional non-RAAS blocking therapy. Consequently,these studies confirm that blockade of the RAASconfers renal benefits above and beyond those asso-ciated with blood pressure reduction. Similar resultsmay be obtained by ACE inhibitors, but wouldrequire investigation in a large-scale head-to-headcomparative study with an ARB. Therefore, first line

146 J. Ahmad

therapy of newly diagnosed hypertensive type 2diabetic patients with DN should be ARBs withinthe recommended range. However, blood pressurecontrol in hypertensive type 2 diabetic patients israrely obtained by monotherapy. In the IDNT study,patients received an average �3 non-study antihy-pertensive drugs to control the blood pressure.

Optimal renoprotection

Lowering of arterial blood pressure has been themain strategy for renoprotection and reduction ofalbuminuria a secondary benefit from decreasingblood pressure, rather than a primary goal of treat-ment during the past two decades. Numerous stu-dies in diabetic and non-diabetic kidney diseasehave documented that albuminuria is an importantprogression promoter as reviewed by Rossing [149]and Remuzzi [150]. Furthermore, albuminuria is aputative predictor of progression during treatment,i.e., the more albuminuria is lowered, the betterrenal function outcome will be [120,121]. Doseresponse studies of losartan in primary hypertensionhave demonstrated that doses above 50 mg o.d.provide no further antihypertensive effect [151].However, in non-diabetic renal disease, dose—response relationship of RAAS blockade for reduc-tion of blood pressure and proteinuria may be dif-ferent [152]. Anderson et al. [74] in his initial studyof the renoprotective effect of losartan in DNdemonstrated a similar blood pressure reductionby losartan 50 and 100 mg, whereas high dose treat-ment was insignificantly more effective in loweringalbuminuria. Andersen et al. [153] performed adose-escalation study with the aim to determinethe optimal dose of losartan for renoprotection andblood pressure reduction in 50 type 1 diabeticpatients with DN. Patients received increasing dosesof losartan initially 50 mg o.d. followed by 100 and150 mg in three treatment periods each lasting 2months. All doses of losartan significantly loweredarterial blood pressure and albuminuria. Albumi-nuria assessed by at least 24 h urine collection,was significantly reduced from baseline by 30%(15—41) (95% CI) by losartan 50 mg compared to48% (35—57) and 44% (32—56) by 100 and 150 mg,respectively. Hence, high dose treatment was sig-nificantly more effective in reducing albuminuriacompared to 50 mg (p < 0.05 losartan 50 mg vs.100 mg), without differences between the benefi-cial effects of 100 and 150 mg. Moreover, analysis ofdata separated by the median albumin value atbaseline, revealed a similar antialbuminuria effi-cacy of losartan in low and nephritic range albumi-nuria. Losartan 100 mg was more effective in

reducing 24 h systolic blood pressure ( p = 0.05 losar-tan 50 mg vs. 100 mg) and diastolic blood pressure(p = 0.02) as compared to 50 mg, whereas no dif-ference was found between the blood pressure low-ering effects of the two high doses. Separateanalysis of day- and night-time blood pressuresrevealed a similar blood pressure lowering efficacyof the three doses of losartan during the day andnight. Hence, the beneficial effect of blood pressurereduction by losartan persists during the night eventhough the medication is administered once daily inthe morning. These results are consistent with sys-temic levels of RAAS hormones during treatment,which indicated a maximum blockade of RAAS bylosartan 100 mg. GFR declined by 4 mL/min/1.73 m2 in the two high-dose treatment periods,probably reflecting a haemodynamic reversible con-sequence of blood pressure reduction. Thus, opti-mal dose of losartan for renoprotection is 100 mgo.d. in type 1 diabetic patients with DN. Further, themaximal antialbuminuric and antihypertensiveeffects of losartan 100 mg o.d. in hypertensive type1 diabetic patients with DN appear within 7 daysafter onset of treatment and remain stable there-after [154]. Similar results have been reported inmicroalbuminuric type 1 diabetic patients treatedwith losartan 50 mg [155].

Urinary TGF-b may represent a future supple-mentary target in treatment of DN. Recent studieshave suggested that over production of TGF-b sti-mulated by angiotensin II and high glucose may bereduced by RAAS blockade [156—158]. A dose-esca-lation study targeting TGF-b expression in a ratmodel of non-diabetic kidney disease demonstratedthat the maximal reduction in pathological glomer-ular TGF-b expression was seen at doses of enalapriland losartan higher than those known to controlblood pressure [159]. In summary, the importanceof dose titration of ARBs and ACE inhibitors aimed atachieving the optimal renoprotective effect,assessed by reduction of albuminuria have beendocumented. Doses might be higher than thosedefined or required for optimal treatment of hyper-tension.

Non-haemodynamic effects ofangiotensin II receptor blockade indiabetic nephropathy

Effect on TGF-b

Increased renal production of TGF-b is a feature ofdiabetes expressed by elevated TGF-b excretion intype 1 and type 2 diabetic patients with nephro-pathy [160—162]. Therefore, reduced stimulation of


TGF-b expression may be part of the renoprotectiveeffect of RAAS blockade in DN. ARB treatment ofstreptozotocin diabetic rats revealedmarked reduc-tion of renal TGF-b and collagen III mRNA comparedto untreated animals 12 weeks after induction ofdisease [163]. Furthermore, RAAS blockade by ACEinhibition in streptozotocin diabetic rats normalizedexpression of TGF-b type II receptor mRNA andproteins compared to untreated animals 30 daysafter induction of diabetes mellitus [164]. Effectof ARB treatment on plasma levels of TGF-b inhumans was investigated in patients with chronicallograft nephropathy by Campistol et al. who founda significant reduction of plasma TGF-b by losartantreatment for 8 weeks [165]. In an open study byEsmatijs et al. [157], fourteen type 2 diabeticpatients with mild hypertension and microalbumi-nuria were investigated. Plasma TGF-b was signifi-cantly lowered by Losartan treatment in sevenpatients with TGF-b levels at baseline above themean. Levels remained unchanged in patients withTGF-b levels below the mean value. In conflict withthese data, plasma TGF-b was unaffected by losar-tan in a recent study, whereas urinary TGF-b wassignificantly reduced [158]. Furthermore, urinaryTGF-b excretion at base line correlated closely withindices of glycaemic control, possibly reflecting arelation between glucose concentration and TGF-b.In the Collaborative Study Group Captopril Trial[21,166] in 58 type 1 diabetic patients, captoprilsignificantly lowered serum TGF-b compared to anincrease in the placebo group 6 months after initia-tion of treatment. It is possible that blockade of theRAAS is likely to reduce glomerular scarring, indi-cated by reductions in urinary TGF-b levels.

Effect on circulating adhesion molecules

The endothelial attachment of leukocytes ismediated by an increased expression of adhesionmolecules such as intercellular adhesion molecule-1(ICAM-1), vascular adhesion molecule-1 (VCAM-1),and E-Selection. Soluble forms (e.g.(s ICAM-1) havebeen detected in plasma and are thought to reflectshedding of membrane,in bound form [167]. Angio-tensin II induces expression of VCAM-1 on endothe-lial cell surfaces in animal studies, which may beinhibited by ARB treatment, thus mediated by theAT1 receptor [168]. Andersen et al. [169] in double-blind five-armed crossover study investigated theeffect of ARB treatment by losartan 50 and 100 mgand ACE inhibition by enalapril 10 and 20 mg com-pared to placebo on circulating adhesion molecules.Plasma levels of sVCAM-1, SE-selection and vWFwere elevated during the placebo period as com-pared to the control persons ( p < 0.05). However,

sVCAM-1 and sE-Selection were significantlydecreased by RAAS blockade. Thus, inactivation ofangiotensin II by ARB or ACE inhibition may reducethe proatherogenic leukocyte—endothelial adhe-sion in type 1 diabetic patients with DN.

Patient’s fact ors in therapy resistance indiabetic and non-diabetic nephropathy

Patients responding favourably to one class of reno-protective drugs, perform with a similar positiveoutcome to other agents, whereas these with a poorresponse to one class of drugs are not likely benefitfrom changing to another agent [170]. Analyses ofthe individual antiproteinuric responses to two dif-ferent doses of ACE inhibition revealed a significantpositive correlation between responses to the twodoses in both populations ( p < 0.05). These ana-lyses support the hypothesis of individual patientrelated factors as determinants for response torenoprotective treatment. The patients-relatedfactors need to be analyzed. These may includeto activation and respensiveness of the RAAS, highdietary sodium intake suppressing RAAS, therebyreducing the efficacy of RAAS blockade, which canbe restored by addition of a diuretic [171]. Suscept-ibility to treatment may depend on genetic poly-morphism. The ACE/ID polymorphism has beensuggested as a major determinant of therapyresponsiveness to ACE inhibition [172,173], i.e.,type 1 diabetic patients with DN homozygoms forthe D-allele, have a reduced antialbuminuricresponse to ACE inhibitior treatment compared topatients homologous for the I allele.

ACE insertion/deletion polymorphism anddiabetic nephropathy

The association between the ACE/ID polymorphismand the development of DN has been thoroughlyinvestigated and received in four meta-analyses.Staessen et al. [174] reported a general increasedrisk for DN in individuals carrying the D-allele. Fuji-sawa et al. [175] suggested based on a meta-analysisof 18 studies that the D-allele was significantlyassociated with development of DN in Caucasianand Asian Type 1 and type 2 diabetic patients. Incontrast, a meta-analysis by Kunz et al. [176] failedto confirm this association. In a meta-analysis byTarnow et al. [177], a trend towards a protectiveeffect of the II genotype on development of ele-vated urinary albumin excretion was found in Cau-casian type 1 diabetic patients. In type 2 diabetes,this association was confined to Japanese popula-tion. Thus, the D-allele may be a risk factor fordevelopment of DN, even though the data are con-

148 J. Ahmad

Table

2Sh

ort-term

effectsoflosartan

onkidneyfunction,bloodpressure

andRAASin

hyp

ertensive

typed1diabeticpatients

withdiabeticnephropathyan

dhomozygo

sity

fortheI(n

=26

)ord(n

=28

)allele

oftheACE/IDpolymorphism

ACEge

notype

Baseline

Losartan

50Lo

sartan

100

IIDD

IIDD

IIDD

Albuminuria(m

g/24

h)

1123

(821

—15

37)

1210

(886

—16

55)

755a(511

—11

15)

802a(558

—11

52)

508a(314

—82

0)64

5a(434

—95

9)Frac

tional

albumin

clearan

ce(10�6)

309(205

—46

5)33

2(223

—49

5)21

4a(133

—34

5)21

9a(137

—35

1)16

2a(94—

281)

212a(135

—33

3)GFR

b(m

Lmin�11.73

m�2)

86(4)

88(4)

84(4)a

84(4)a

81(4)a

84(4)a

24hsystolicBPb(m

mHg)

156(3)

153(3)

145(3)a

145

(3)a

144(4)a

143(4)a

24hdiastolicBPb(m

mHg)

82(2)

80(2)

77(2)a

76(2)a

76(2)a

76(2)a

S-renin

c(m

U/m

L)40

(1)

38(1)

49(1)a

57(1)a

69(1)a

72(1)a

S-ACEc(IU/L

)17(1)

25(1)d

16(1)

24(1)d

17(1)

24(1)d

P-angiotensinIIc(pmol/L)

8(1)

12(1)

15(1)

17(1)

21(1)a

22(1)a

Geometric

mean

(95%

CI).

ap<

0.05

vs.baseline.

bMean

(SE).

cGeometric

mean

(SE).

dp<

0.05

IIvs.DD.

flicting. Results from recent morphological studiesin type 1 [178] and type 2 diabetes [179] have shownthat the DD genotype is associated with progressionof DN. Several studies have also reported an asso-ciation between the D-allele, progression of dia-betic kidney disease [172,180—182] or a trend fora worse renal prognosis [183] and also associationsbetween the D allele and decline in renal function innon-diabetic renal disease [184—186].

Although ACE inhibitors may be renoprotective intype 1 diabetic patients with DN beyond the effectof lowering blood pressure [21], there is consider-ably variation in response to treatment betweenindividuals. Individuals with the II genotype exhibitlower plasma levels of ACE compared to the DDgenotype [187,188], which obviously could influ-ence the individual efficacy of ACE inhibitor treat-ment. The EUCLID trial tested the hypothesis thatACE/ID polymorphism modulates the therapeuticefficacy of ACE inhibition in normoalbuminuric type1 diabetic patients in a randomized prospectivedesign [189] and that progression from normoalbu-minuria to microalbuminuria did not differ betweenII and DD genotypes. However, a beneficial effect oflisinopril on urinary albumin excretion rate wasobserved only in the II group. Jacobsen et al.[190] investigated in an observational study, thereduction in albuminuria after initiation of ACEinhibitior treatment in type 1 diabetic patients withDN and observed that in patients homozygous for the1 allele, albuminuria was reduced by 61% as com-pared to only 31% in the DD patients (p < 0.01).Hence, the beneficial effect of ACE inhibition maybe lower in type 1 diabetic patients with DN homo-zygons for the D allele compared to the 1 allele.

Specific blockade of the AT1 receptor may over-come interaction between ACE genotype and ACEinhibition and potentially induce similar renopro-tective effects independent of genotypes. Andersenet al. [191] performed a study to evaluate the short-term renoprotective effect of ARB treatment bylosartan assessed by reduction in albuminuria inhypertensive type 1 diabetic patients with DN(Table 2) Fifty four patients homozygous for 1(n = 26), or D (n = 28) allele of the ACE/ID poly-morphism were investigated. Patients and the pri-mary investigator were blinded to genotypes. Thestudy consisted of two treatment periods each last-ing 2 months and patients received two doses oflosartan starting with 50 mg followed by 100 mg o.d.in the second treatment period. No significant influ-ence of the ACE/ID polymorphism on the antipro-teinuric effect of losartan was found. Albuminuria,assessed by at least 24 h urine collections, waslowered by 55% (35—68; 95% CI) (p < 0.01 mg vs.baseline) in the II groups and 46% (28—61)


( p < 0.01 mg vs. baseline) in the DD group by treat-ment with losartan 100 mg ( p < 0.01) (NS betweengroups). Thus, blockade of the action of angiotensinII at the AT1 receptor may be independent of the ACEgenotype. Several studies have found an increasedrisk for renal function loss in DD patients comparedto II patients, even during ACE inhibition. HoweverARB treatment may confer similar long-term reno-protective effects in both genotypes. It is suggestedthat long-term comparative trials with ACE inhibi-tion should be performed before recommendationsof selective therapy in different ACE/ID genotypescan be justified.

Dual blockade of renin—angiotensinsystem in diabetic nephropathy

Several clinical studies have shown similar effectswith ACE inhibitors or ARB in reducing albuminuria,retarding progressive loss in renal function andimprovements in survival [192—194] above andbeyond any such effects attributable to reductionin blood pressure alone [13,21]. Blockade of RAAS byeither ACE inhibitors or ARBs shows but does notcompletely arrest the progression of renal diseasetowards ESRD. Since these agents act at differentsites on the RAAS, they may have additive effectwhich results in greater reno-protection when usedin combination [195] and it is being reported to beeffective in diabetic patients withmicroalbuminuria[196] and macroalbuminuria [197,198].

Pathophysiology and rationale for dualblockade

Despite maximal recommended doses of ACE inhi-bitors, several patients with DN do not reach bloodpressure target, have persistent albuminuria andrapid progression of renal disease. Insufficientresponse during sustained therapy may be partlyrelated to under-dosing or the ACE-escape phenom-enon [75], generation of angiotensin II though alter-nate enzyme pathways such as chymase [73] orindividual factors. The dual blockade will alsodepend on the different sites of action of ACEinhibitors and ARBs leading to synergestic blockadeof RAAS not obtainable by either drug alone. ACEinhibition decreases the formation of Angiotensin IIby blockade of ANG II at both A1 and A2 receptors andthe degradation of bradykinin, which is a powerfulvasodilator [199]. However, Ang II may be generatedthrough ACE independent pathways such as chy-mase, tonin, cathepsin G leading to incompleteblockade and thus formation of Ang II [73]. Up-regulation of chymase has recently been reportedin hypertensive patients with type 2 diabetes and

nephropathy [200]. Such incomplete blockade mayexplain the observation that plasma Ang II levelsreturn to normal after chronic ACE inhibitor treat-ment, the so-called ‘ACE-escape’ phenomenon[201].

ARB treatment leads to reduced Ang II binding atANG 1 receptors and increased ligand availability atAng II receptors. Treatment with ARBs may result inmore complete blockade of the unfavourableactions of Ang II type 1 receptors. However, newdata from animal studies indicate that some of thedeleterious effects of angiotensin II on glomerularcell migration, tubular cell proliferation and thedevelopment of urine protein excretion may bemediated through the Ang II type 2 receptor[202—204] which is not blocked by currently usedARBs.

A recent study of combined treatment with ACEinhibition and ARB in healthy rats demonstrated thatrenal angiotensin II levels were lowered by mono-therapy with either agent [205] in accordance withprevious experimental studies of ARBs [24]. Dualblockade of RAAS caused a further reduction ofkidney angiotensin II levels, whereas separatedose-escalation studies of both agents were unableto demonstrate similar degree of suppression ofkidney angiotensin II levels. According to theseexperimental data also, combined treatment withACE inhibition and ARB may induce a supplementaryeffect on the intrarenal RAAS, unavailable by mono-therapy with either agent.

Effect of dual blockade of RAAS in diabeticnephropathy

Several studies have reported at the effects of dualblockade therapy on blood pressure and urinaryprotein excretion in experimental models of kidneydiseases. Although some of these studies have shownconflicting results, beneficial effects of dual block-ade in humans have been demonstrated in short-term clinical studies of diabetic patients with micro-albuminuria [196] and macroalbuminuria [197,198]and also patients with non-diabetic nephropathy[206,207]. Combination therapy with candesartan16 mg o.d. and lisinopril 20 mg o.d. in CALM Study[196] was significantly more effective in reducingboth systolic and diastolic blood pressure comparedto monotherapy with either agent. Reduction ofmicroalbuminuria tended to be greater with combi-nation therapy, though only reaching statisticallysignificance compared to candesartan monotherapy.Jacobsen et al. [208] performed the first short-termtrial with dual blockade of the RAAS in patients withtype 1 diabetes and DN. In a randomized, double-blind cross-over trial, they included 21 patients with

150 J. Ahmad

type 1 diabetes, hypertension and albuminuria (pro-tein excretion > 1 g/24 h) who received 2 monthstreatment with either 300 mg irbesartan or placeboin addition to prior antihypertensive treatments(including ACE inhibitors, diuretics and a thirddrug). The main result of dual blockade therapywas a drop in BP of 8/5 mmHg (24-h ambulatory)and an additional significant (37%) drop in albumi-nuria in comparison with that of ACE inhibitors usedalone [208]. Further, Rossing et al. [197] demon-strated a 25% reduction in albuminuria and a reduc-tion of 10 mmHg in 24 h ambulatory systolic BP withthe use of 8 mg candesartan in addition to ACEinhibitors in 18 patients with type 2 diabetes andDN. Earlier observations of Jacobsen et al. [208]were further extended in patients with type 1 dia-betes in a randomized, double-blind, cross over trialof 18 patients with DN (albuminuria >300 mg/24 h)who received 8 weeks of placebo, 20 mg benazeprilor 80 mg valsartan in a random order [209]. In thisstudy,monotherapywith an ACE inhibitor or ARBwasequally effective with regards to reduction in albu-minuria and BP. In addition, short-term dual block-ade of RAAS caused a significant (43%) reduction inalbuminuria and a reduction of 7/6 mmHg BP incomparison with bothmonotherapies [209]. Agarwalet al. [210] reported that addition of 50 mg oflosartan daily to 40 mg of lisinopril had no effecton BP and proteinuria in a small heterogenous groupof predominantly obese, hypertensive, proteinuric,diabetic African—Americans with advanced renalfailure. Surprisingly, this study showed a low plasmarenin activity and worsening of GFR during dualblockade treatment. In this study only 50 mg oflosartan was used as it is known that 100 mg is moreeffective on both albuminuria and BP in patientswith DN [210]. Furthermore, the findings of Agarwalet al. cannot be extrapolated to other ethinic groupsdue to difference in RAAS activity and known vari-able response of RAAS blockade amongst differentethnic groups [211,212]. However, subsequent ana-lysis of urinary excretion of the profibrotic growthfactor TGF-b in the same group of patients showedan elevated urinary TGF-b excretion during monoblockade with maximal doses of ACE inhibitor, but a38% reduction with the addition of losartan that wasindependent of changes in BP or albuminuria [213].

The COOPERATE Study [214] investigated theeffect of monotherapy with ACE inhibitor or ARBcompared to dual blockade with combined treat-ment in 263 patients with non-diabetic kidney dis-ease followed for 2.9 years. In the combinationgroup, 11% of patients reached the combined pri-mary endpoint, time to doubling of serum creatinineor end-stage renal disease compared to 23% in bothmonotherapy arms ( p � 0.018). Parvanova et al.

[215] in their study at the Steno-Diabetes Centredid not find additional antihypertensive and anti-albuminuric effects of dual blockade relative totreatment with an ACE inhibitor in 39 hypertensivepatients with type 2 diabetes and DN. However, thisstudy compared combination therapy of an ACEinhibitor (10 mg enalapril) and an ARB (50 mg losar-tan) with monotherapy using twice the dose of thesame inhibitors [215]. Because of the halving ofdoses, the study investigated the synergistic, notthe superior effect of dual blockade of RAAS. This isa very relevant observation but should not lead tounnecessary rejection of the new treatment con-cept of dual blockade. Cooper et al. [216] havesuggested that treatment of hypertensive patientswith DN should be started with an ACE inhibitor orARB, often in fixed combination with a low-dosethiazide diuretic. Calcium channel blockers andbeta-blockers may be added if required as secondline agents. In patients not responding to this com-bination, doses of ACE inhibitor or ARB should betitrated upwards in order to obtain maximal ther-apeutic effect. However, if BP control is still insuffi-cient, duel blockade of RAAS should be consideredand even an aldosterone-receptor blocker may needto be added to the therapeutic regimen. Wade et al.[217] in their review of literature concerning dualblockade of RAAS in DN showed that in comparisonto monotherapy, dual blockade using ACE inhibitorsand ARBs (in approximately 300 patients studied)resulted in an additional 11—43% reduction in albu-minuria and that the combination was well toler-ated except that it required additional monitoringfor hyperkalemia.

Therefore, preliminary studies have suggestedthat dual blockade of the RAAS by combined treat-ment with ACE inhibition and ARBs may offer anadditional effect compared to monotherapy. Thus,long-term studies of dual blockade in diabetic kid-ney disease should be initiated, however, dose—response relationships for optimal renoprotectiveeffects of monotherapy with both agents shouldbe clarified before further action is taken.

Sengul et al. [218] evaluated the long-termeffectsof dual blockade using the ACE inhibitor lisinopril andthe long-acting angiotensin II receptor blocker tel-misartan on blood pressure and albumin excretionrate in patients with type 2 diabetes, microalbumi-nuria and hypertension. It was observed that dualRAAS blockade using the ARB telmisartan and an ACEinhibitor lisinopril was associated with a greaterreduction in blood pressure and improvement inurinary albumin excretion rate than that observedwith either lisinopril or telmisartan monotherapy intype 2 diabetic hypertensive patients with microal-buminuria. Combined treatment for 28 weeks


revealed additive effects on the normalization ofmicroalbuminuria.Thecombination therapywaswelltolerated and safe,withnoevidence of hyperkalemiaor hypotension. Fernandez-Juarez et al. [219] in amulticenter, prospective, open, active controlled,and parallel-group trial have designed to comparethe effects of an ACE inhibitor versus an ARB or itscombination on renal disease progression, protei-nuria, and cardiovascular events in type 2 DN. Theresults of this trialwill beavailable in2009.Until now,there has not been any reference to a beneficialeffect on progression of the dual blockade in type2 DN, which is the most frequent cause of ESRD.

Renin inhibitors

RAAS has been a highly successful pharmacologictarget, as the system is strongly implicated in thedevelopment of DN. However ANG II reactivation andaldosterone escape or breakthrough during eitherACE inhibitor or AT1 antagonist treatment, due tocompensatory increase in plasma renin levels thatlead to higher angiotensin production and conversionas well as aldosterone secretion and sodium reab-sorption presents limitations for existing renin—angiotensin-aldosterone system inhibitors [220].Renin and angiotensin 1 accumulate during ACE inhi-bition, and might overcome the ability of an ACEinhibitor to effectively suppress ACE activity. Thereare also data suggesting that 30—40% of ANG II for-mation in the healthy human during RAAS activationis formed via renin-dependent, but ACE-independentpathways. Moreover, ACE gene polymorphisms con-tribute to the modulation and adequacy of the neu-rohormonal response to long-term ACE inhibition, atleast in patients with CHF (up to 45% of CHF patientshave elevated ANG II levels despite the long-termuseof an ACE inhibitor) or diabetes. The reactivated ANGII promotes aldosterone secretion and sodium reab-sorption. Aldosterone breakthrough also occurs dur-ing long-term AT1 antagonist therapy, mainly by anAT2 dependent mechanism.

After establishing the concept of renin uptake asthe underlying cause of tissue angiotensin genera-tion, focus is now on the mechanism that mediatesthis uptake process. Several renin receptors havealready been described. These receptors also bindprorenin, and such binding results in prorenin acti-vation, either proteolytically or nonproteotytically.This is important in view of earlier observations thathigh prorenin levels in diabetic subjects are anindication of microvascular complications [221].Now that renin inhibitors will soon be clinicallyavailable [222], it will be of the greatest interestto investigate have these drugs effect thesemechanisms in comparison with other RAAS block-

ers. A recent analysis of six clinical trials of aliski-ren, a promising renin inhibitors, involving morethan 5000 patients with mild to moderate hyperten-sion indicates that aliskiren is no more effectivethan ACE inhibitors or diuretics for lowering bloodpressure. Although aliskiren suppresses plasmarenin activity, it causes much greater reactive risesin plasma renin concentration that does any otherantihypertensive class tested. Three studies (AVOID,ALOFT and ALLAY) are ongoing with aliskiren toassess end-organ protective properties [223]. Even-tually, a new class of drugs might emerge, the reninreceptor blockers, which selectively block angioten-sin generation at tissue sites and/or renin receptor-mediated effects [224].

Conclusions

Enormous progress haas been made in the under-standing of the risk factors and mechanisms of dia-betic nephropathy, the stages of renal involvementin diabetes, and the treatment strategies to preventor interrupt the progression of diabetic nephropathy.RAAS is a coordinated cascades of proteins and pep-tide hormones, the principal effector of which isangiotensin II. Evidence now indicates that the kid-ney regulates its function via a self-contained RAASin a paracrine fashion. In diabetic nephropathy, theintrarenal generation of ANG II is increased, in spiteof suppression of the systemic RAAS. This increasecan contribute to the progression of DN via severalhemodynamic, tubular and growth promotingactions. ANG II induces insulin resistance. ANG IItype-1 (AT1) and type 2 AT2) receptors are downregulated in chronic diabetes, but decreased AT2receptor expression might contribute to early DNby reducing AT2 receptor mediated beneficial actionsthat are counter-regulatory to those of the AT1receptor. AT2 receptor stimulation might accountfor part of the renal protection seen with AT1 recep-tor blockade. Furthermore, dual RAAS blockade pro-vides additional renoprotection by further reducingboth blood pressure and albuminuria and is welltolerated. These findings point to a new therapeuticapproach to the prevention of overt nephropathy inpatients with type 1 and type 2 diabetes, hyperten-sion and microalbuminuria.

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Renin–angiotensin system blockade in diabetic nephropathy

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