Recent insights into the renin-angiotensin aldosterone system and progression of renal disease
Prof. Gunter Wolf MD.
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.
Angiotensin II and renal hemodynamic
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 (Kf;5).
It has been traditionally argued that ANG II-mediated contraction of mesangial cells is responsible for the reduction in Kf 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 Kf 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.
Trophic and inflammatory effects of ANG II in the kidney
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 p27Kip1 protein but not mRNA expression23, 24. p27Kip1 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 p27Kip125, 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 models28, 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 enalapril32, 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 hypertension35, 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 mediator41, 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.
Aldosterone as a direct mediator of renal injury
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 heart53, 54. Aldosterone also stimulates collagen type I and III expression in cultured cardiac fibroblasts55, 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.