Prof. Gunter Wolf MD.
Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany
Wolf@uke.uni-hamburg.de

Introduction

The number of patients with end-stage renal failure is steadily, if not exponentially, increasing¹. The majority of patients have a chronic decline of kidney function and dialysis is required months or even years after initial diagnosis². Such a continuous decline of renal function is called progression of renal disease and is characterized by the common pathomorphological endpoint of tubulointerstitial fibrosis and tubular atrophy, irrespectively of the primary etiology of the renal disease. Clinical and experimental evidence accumulating over the last decade convincingly suggests that the renin-angiotensin aldosterone system (RAAS) is a key player in this complex events of progression of chronic renal failure.

The classical function of ANG II is the maintenance of extracellular volume and blood pressure during volume depletion³. Activation of the RAAS causes an ANG II-dependent increase in renal vascular resistance leading consequently to a decrease in renal plasma flow (RPF). However, micropuncture studies have shown that ANG II preferentially raises efferent glomerular resistance⁴. The consequence is an increase in the glomerular capillary filtration pressure that results in a protected glomerular filtration rate (GFR) despite an ANG II-induced decrease in RPF⁴. Micropuncture investigations in the rat demonstrated that infusion of exogenous ANG II reduces the capillary ultrafiltration coefficient (K_f;⁵).

It has been traditionally argued that ANG II-mediated contraction of mesangial cells is responsible for the reduction in K_f by decreasing available glomerular capillary surface area⁶. This somewhat mechanistic explanation has been more recently questioned because electron microscopy failed to detect changes of ANG II on the area of the capillary surface⁷, and the geometrical arrangement of the mesangial contractile fibers compresses the capillary interface in case of mesangial cell contraction, but leaves the peripheral filtration area unchanged⁷.

Alternative explanations how ANG II reduces K_f may be a decline in the hydraulic permeability of the capillary for water or constriction of only defined subset of glomerular capillaries resulting in a reduction in effective filtration surface⁷. Hostetter and colleagues found an increase in glomerular capillary pressure in many animals models of progressive nephron loss⁸. This increase was seen as an adaptive response how the kidney tries to maintain GFR after irreversible destruction of renal tissue by increasing the single-nephron GFR of surviving nephrons⁹.

Although these adaptive mechanisms may maintain indeed renal function in the early phase of chronic renal injury, glomerular hypertension and hyperfunction are ultimately detrimental to renal function and structure⁹. An increase in capillary pressure may directly stimulate in glomerular cells through mechanical forces injury, proliferation, and production of extracellular matrix¹⁰. ANG II is an important mediator of glomerular hemodynamic changes and the increase in tubular transport in chronic renal injury. The RAAS is activated in chronic renal disease. The landmark study by Anderson and co-workers demonstrated that an ACE inhibitor limited glomerular injury in rats with experimentally induced reduction in renal mass¹¹. This protective effects of ACE inhibitor treatment were independent of systemic blood pressure and were then solely attributed to a reduction of the increased glomerular capillary pressure¹¹. Subsequently, it became clear that ANG II exhibit many other non-hemodynamic effects (see following paragraph) that may partly explain the protective effect of ACE inhibitor treatment on renal injury¹².

More than 15 years ago, Bertani and Remuzzi proposed that an increased amount of proteins filtered through the glomerular capillary could have intrinsic renal toxicity and contributes to the progression of renal disease¹³. The pathophysiological mechanisms have been fairly well worked out and include induction of a variety of proinflammatory and vasoactive substances in proximal tubular cells exposed to an increased concentration of proteins¹⁴. Tubular cells exposed to protein synthesize more ANG II that may in turn influence tubular transport or exhibit profibrogenic effects. However, in regards of hemodynamic mechanisms of ANG II, the peptide plays an important role in increasing proteinuria because high intraglomerular capillary pressure impairs the barrier´s size-selective function¹⁵. In addition, ANG II directly modulates glomerular permeability and increases proteinuria by a variety of mechanisms including loss and suppressed synthesis of negative charged proteoglycans, increase in glomerular basement membrane-associated extracellular matrix components, and decrease of nephrin, an important component of the podocyte slit diaphragm¹⁶.

One of the first hints that ANG II may be involved in renal growth and tissue remodeling stems from studies investigating the effects of ACE inhibitors¹⁷. Direct infusion of ANG II into naive rats leads to a tubulointerstitial injury with proliferation of distal tubules, collecting ducts, and interstitial cells, but not of proximal tubules¹⁸. We showed in the early 90s that ANG II causes a small but significant proliferation of murine mesangial cells in the absence of other factors¹⁹.ANG II also stimulates proliferation of rat glomerular endothelial cells²⁰. This mitogenesis is transduced by AT₁ receptors. However, ANG II stimulates cellular hypertrophy of a murine proximal tubular cell line (MCT cells;²¹) and in porcine LLC-PK₁ cells²². ANG II-induced hypertrophy is mediated through AT₁ receptors and requires G₁-phase arrest. ANG II treatment in vitro and in vivo enhances p27^Kip1 protein but not mRNA expression^{23, 24}. p27^Kip1 is an inhibitor of cyclin-dependent kinases and overexpression of this protein clamps cells in the G₁-phase of the cell cycle.

Oxygen radicals and activation of mitogen-activated kinases (Erk 1, 2) play an important role in the ANG II-mediated induction of p27^{Kip125, 26}. ANG II upregulates the membrane-bound NAD(P)H oxidase resulting in an increased formation of oxygen radicals. ANG II induces also in vitro apoptosis²⁷. It has been suggested that activation of AT₂ receptors is responsible for this effect, but this issue is controversial because AT₁ receptor antagonists also attenuated apoptosis in certain models^{28, 29}. The decision whether cells undergo hypertrophy or rather apoptosis may depend on the presence of additional growth factors and cytokines³⁰.

Indirect evidence that ANG II may be involved in tubulointerstitial fibrosis comes from comprehensive studies in rats with unilateral ureteral ligation³¹. Tubular TGF-β expression precedes the development of tubulointerstitial scarring and is partly reduced when animals were treated with enalapril^{32, 33}. Administration of an ACE-inhibitor or an AT₁ receptor blocker blunts the increase in TGF-β mRNA and decreases the structural manifestations of injury, indicating an in vivo relationship between ANG II, TGF-β and renal fibrosis³⁴. TGF-β expression is also elevated in the contralateral kidney of rats with 2 kidney-1 clip hypertension^{35, 36}. Probably the most direct evidence that the RAAS is involved in renal scarring stems from targeted overexpression of renin and angiotensinogen in rat glomeruli³⁷. Seven days after transfection, extracellular matrix was expanded in rats with glomerular renin and angiotensinogen overexpression without systemic hypertension³⁷.

Some of the therapeutic effects of ACE-inhibitor treatment in the prevention of renal fibrosis may be due to the inhibition of the endothelin system ³⁸. ANG II induces mRNA encoding the extracellular matrix proteins type I procollagen and fibronectin in cultured mesangial cells³⁹. ANG II also stimulates the transcription and synthesis of collagen type α1(IV), but not type I, in cultured proximal tubular cells. We have recently demonstrated that the vasopeptide additionally induces mRNA and protein expression of α3(IV) collagen which is more restricted to the kidney than the common α1 and α2 chains ⁴⁰. ANG II stimulates in cultured proximal tubular and mesangial cells the induction of TGF-β, an important profibrogenic mediator^{41, 42}. The stimulatory effects of ANG II on collagen expression in cultured proximal tubular cells depends on TGF-β expression. Interesting recent studies suggest that ANG II stimulates proliferation of cultured renal fibroblasts⁴³. Stimulation of fibroblasts with ANG II also increases mRNA expression of TGF-β, fibronectin and type I collagen (43). Moreover, recent findings indicate that ANG II also modulates the plasmin protease system and inhibits the degradation and turnover of extracellular matrix⁴⁴.

ANG II stimulates mRNA and protein expression of the chemokine RANTES, a member of the C-C chemokine subfamily with chemoattractant properties for monocytes/macrophages (M/M), in cultured glomerular endothelial cells⁴⁵. The ANG II-stimulated RANTES expression is transduced by AT₂ receptors⁴⁵. ANG II-infused rats exhibitean increase in glomerular M/M compared with controls. Treatment with an AT₂ receptor antagonist attenuates the glomerular M/M influx without normalizing the slightly elevated systolic blood pressure caused by ANG II infusion, suggesting that the effects on blood pressure and RANTES induction can be separated⁴⁵. These data were recently confirmed by Ruiz-Ortega and associates who demonstrated that ANG II infusion for 72 hours increased glomerular and interstitial inflammatory cells⁴⁶. In agreement with our findings, an AT₂ receptor antagonist but not losartan significantly reduced this inflammation⁴⁶.

The group of Jesus Egido were among the first demonstrating that ANG II activates nuclear factor κB (NF-κB;⁴⁷). NF-κB is a key proinflammatory factor involved in the transcriptional regulation of many genes including MCP-1 and RANTES. Using specific ANG II-receptor antagonists, they showed that ANG II activates NF-κB in vascular smooth muscle cells (VSMC) through AT₁ and AT₂ receptors ⁴⁷. NF-κB is strongly activated in double-transgenic rats harboring both human renin and angiotensinogen genes ⁴⁸. We used an alternative approach to study a potential role of AT₂ receptors in NF-κB activation and selectively overexpressed AT₁ and AT₂ receptors in COS7 cells that do not normally bear ANG II-receptors ⁴⁹. COS7 cells transfected with an AT₂ receptor expression construct demonstrated NF-κB activation after treatment with ANG II⁴⁹.

In the traditional concept of the RAAS and renal injury, aldosterone mediates volume-dependent hypertension and contributes through this mechanism indirectly to chronic renal injury⁵⁰. However, aldosterone exerts, similar as ANG II, directs effects on renal cells that may contribute to progression of renal disease⁵⁰. Greene and co-workers defined the role of aldosterone in the remnant kidney model⁵¹. Combined treatment with ACE-inhibitor and AT₁ antagonist totally attenuated renal pathology⁵¹. However, infusion of exogenous aldosterone reestablished kidney injury despite ACE-inhibitor and AT₁ antagonist treatment⁵¹. This strongly indicates that secondary hyperaldosteronism contributes to proteinuria and glomerulosclerosis in this model. Aldosterone also inhibits matrix turover. Interestingly, glomerular osteopontin expression is also upregulated in the deoxycorticosterone acetate (DOCA)-salt model⁵². Its is currently unclear, however, whether this induction of a chemoattractant is directly mediated by aldosterone or may be rather an effect of systemic hypertension. Observation from animals with cardiac fibrosis clearly revealed that aldosterone increased collagen expression in the heart^{53, 54}. Aldosterone also stimulates collagen type I and III expression in cultured cardiac fibroblasts^{55, 56}. A recent study suggest an important role of aldosterone in the pathogenesis of thrombotic microangiopathy in stroke-prone spontaneously hypertensive rats⁵⁷.

There is now ample evidence that the RAAS plays a central role in the progression of chronic renal disease. Although hemodynamic effects of ANG II certainly contribute to renal damage, research from the last decade clearly suggest that this vasopeptide is a multifarious cytokine engaged in may non-hemodynamic processes such as growth stimulation, apoptosis, induction of profibrogenic actions, and inflammation. In addition, animal experiments suggest that aldosterone could mediate direct renal injury independent of ANG II. Therefore, it is strongly recommended that ACE-inhibitors therapy should be used in every normotensive and hypertensive patient with clinically evident nephropathy and proteinuria who is at risk for progression.

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