Renal Interstitial Fibrosis and Angiotensin Inhibition

Tubulointerstitial (TI) fibrosis is a final common pathway to progressive renal injury of all forms of renal disease. However, once renal damage reaches a certain threshold, progression of renal disease is consistent, irreversible, and largely independent of the initial injury. Angiotensin (AT) II is the main effector of the renin angiotensin system (RAS) and effects that may contribute to the onset and progression of renal damage. AT II may also directly contribute to accelerate renal damage by sustaining cell growth, inflammation, and fibrosis. Interventions that inhibit the activity of the RAS are renoprotective and may retard or even halt the progression of chronic nephropathies. Unilateral ureteral obstruction suggested as a well-established experimental model of progressive interstitial expansion and fibrosis. Although technically challenging, some investigators have successfully relieved the obstruction and reported significant reduction in interstitial fibrosis severity. Drugs that modulate the RAS, such as ACE inhibitors and angiotensin type 1 (AT1) receptor antagonists, have demonstrated protective renal effects and can ameliorate fibrosis. However, neither ACE inhibitor nor AT1 receptor blockade completely suppresses progression of renal disease. Dual blockade of the RAS with ACE inhibitors and AT1 receptor blockers may provide renal benefit beyond therapy with either drug alone, due to their potential additive beneficial effect.

to pathologic events originating in these neighboring areas. Structural derangements of the TI compartment occur in virtually all progressive renal diseases 4) .
An expansion of the cortical interstitium is highly correlated with tubular lesions, especially tubular atrophy. The widening of the interstitial space in chronic renal diseases is mainly due to increased extracellular matrix (ECM), and increased cellularity (fibroblasts, macrophages, and lymphocytes) may also contribute to the tubulointerstitial fibrosis [5][6][7] . However, once renal damage reaches a certain threshold, progression of renal disease is consistent, irreversible, and largely independent of the initial injury. The renin angiotensin system (RAS) is a wellknown regulator of blood pressure (BP) and determinant of target-organ damage. It controls fluid and electrolyte balance through coordinated effects on the heart, blood vessels and kidneys. Angiotensin (AT) II is the main effector of the RAS and exerts its vaso-constrictor effect predominantly on the postglomerular arterioles, thereby increasing the glomerular hydraulic pressure and the ultrafiltration of plasma proteins, effects that may contribute to the onset and progression of chronic renal damage. AT II may also directly contribute to accelerate renal damage by sustaining cell growth, inflammation, and fibrosis. Interventions that inhibit the activity of the RAS are renoprotective and may retard or even halt the progression of chronic nephropathies 8) .
Renal injury and repair comprises a delicate balance between cell loss and proliferation and ECM accumulation and remodeling 9) . In rodents complete ureteral obstruction induces aggressive interstitial fibrosis and tubular atrophy. Although technically challenging, some investigators have successfully relieved the obstruction and reported significant reduction in interstitial fibrosis severity 10) . So it is possible to investigate and clarify the mechanism of renal repair or remodeling after renal fibrosis.

Tubulointerstitial fibrosis
Tubules and interstitium make up approximately 80% of the renal volume, and occupy the compartment referred to as the tubulointerstitium. The interstitium space takes up 10% of rat and up to 17% of dog and rabbit kidneys, including 7 to 9% of renal cortex, 3 to 5% of outer medulla and 30 to 40% of inner medulla and papillary tip [10][11][12][13] . It is made up of both cellular and matrix components. The matrix is made up of a fibrillar net of interstitial and basement membrane collagens and associated proteoglycans, glycoproteins and interstitial fluid. The interstitial compartment not only provides structural support for the individual nephrons, but also serves as a conduit for solute transport 12) . It is also the site of production of several hormones and cytokines such as erythropoietin and prostaglandins.
Renal fibrosis is pathologically characterized by interstitial fibrosis, tubular atrophy, capillary loss, and podocyte depletion. In most chronic kidney diseases there is an intimate relationship between interstitial fibrosis and tubular atrophy, both of which are closely correlated with the decline in renal function1 4) . Careful pathological analysis revealed that the impairment of renal function correlates better with the extent of TI damage than with the degree of glomerular damage 2) . It might be possible to assume that interstitial fibrosis causes tubular atrophy, although this is difficult to prove. The underlying cellular events leading to these histologic presentations are even more complicated; they include mesangial and fibroblast activation, tubular epithelial to mesenchymal transition (EMT), monocyte/macrophage and T-cell infiltration, and cell apoptosis 14,15) .
In renal fibrosis, a balance between the matrix synthesis and matrix degradation is changed due to increased matrix synthesis or decreased degradation 3) .
Renal fibrogenesis may be divided into three phases: induction, matrix synthesis, and resolution 6) . The in- and tubular atrophy appears to be irreversible 3,16) .
After the initial injury, the affected kidney tissues undergo a series of events in an attempt to repair and recover from the damage. These processes include kidney resident cell activation, which leads to the production and secretion of proinflammatory cytokines.
The gradients of chemotactic cytokines provide a directional signal for guiding the infiltration of inflammatory monocytes/ macrophages and T cells to the injured sites. Depending on the etiology of renal injury, glomerular or interstitial infiltrated inflammatory cells become activated, and produce injurious molecules such as reactive oxygen species, as well as fibrogenic and inflammatory cytokines 14) .
TI injury is mediated by massive proteinuria containing a large amount of complement components and chronic hypoxia with loss of peritubular capillaries in the tubulointerstitium 17) . Proteinuria may contribute to tubulointerstitial damage by activation of nuclear factor-kappa B (NF-κB) and upregulation of various proinflammatory and profibrotic genes. Inhibition of NF-κB improved TI injury and is also associated with the transdifferentiation of tubular cells into myofibroblasts 17) . Hypoxia can activate fibroblasts, change ECM metabolism of resident renal cells, and lead to eventual fibrogenesis 17) .

Unilateral ureteral obstruction study for renal interstitial fibrosis
In the study of human diabetic glomerular disease by Fioretto et al 18) , once diabetes was cured by successful pancreas transplantation, it took 10 years for the expanded mesangial matrix to regress.
However, diabetic renal changes in man are very slow to develop and to reverse and possible manipulations of this human model are limited by ethical constraints. Therefore, unilateral ureteral obstruction (UUO) model in the rat can be used for the exploration of these phenomena rapidly. UUO represents a well-established experimental model of progressive interstitial expansion and fibrosis 19,20) . UUO causes rapid interstitial expansion and fibrosis and tubular injury 19,20) . In the rabbit, interstitial fibrosis is seen in the affected kidney at 7 days (D) of UUO. Moreover, increased synthesis of ECM components [collagens I, III, and IV, fibronectin and heparan sulfate proteo-glycan] is detectable at 3-7D of UUO 19) . The expression of transforming growth factor (TGF)-β in rat kidneys after 1 and 7D of UUO is also increased in the interstitium and in perivascular areas 20) . These alterations are remarkably alleviated by prior or concomitant use of an angiotensin converting enzyme (ACE) inhibitor and a specific angiotensin II receptor blocker [19][20][21] . These data suggest that interstitial fibro- Several cytokines might initiate fibrogenesis, but TGF-β1 is considered a major stimulating factor.
In the animal experiment 10)  . This partial reversal was accom- . ACE inhibitor may accelerate healing process through yet to be described mechanisms but probably not through anti-inflammatory effects.
Hemodynamic independent action of and therefore may participate in renal injury 25) . AT II may promote the phenotypic change of fibroblasts to myofibroblasts. These activated fibroblasts may proliferate and invade the periglomerular and peritubular spaces, contributing to matrix deposition in the TI area. Indeed, rats chronically infused with AT II develop injury with tubular atrophy and dilation, cast formation, interstitial monocyte infiltration, and interstitial fibrosis with type IV collagen deposition 26) .
Cultured renal interstitial fibroblasts express AT1 receptors, and after AT II stimulation, there is an increase in cell proliferation and expression and synthesis of ECM proteins, such as fibronectin, via a TGFβ-mediated mechanism 27) .
Renal tubular cells also play a central role in the pathogenesis of interstitial fibrosis. Tubular cells might be stimulated by the spill over of peptides such as AT II, growth factors, and cytokines from the injured glomeruli. In cultured tubular cells, AT II causes hypertrophy and also increases the synthesis of ECM via TGF-β 28) .
Emerging evidence suggests that connective tissue growth factor (CTGF), a novel profibrogenic cytokine, may play a role in the development and progression of renal fibrosis as a mediator of TGF-β profibrotic activities 29) . Recent experimental data showed that AT II increased CTGF mRNA expression and synthesis and that ACE inhibition diminished renal CTGF expression correlated with diminution of TGF β and fibrosis. These results suggest that CTGF could be a mediator of the matrix accumulation caused by AT II 25,29) .
In using of ACE inhibitor, inhibition of ACE activity decreases the formation of AT II and potentiates the vasodilatory effects of bradykinin. As a result, ACE inhibitors are used widely to treat hypertension. These agents reduce proteinuria and delay progression of renal disease in patients with diabetic nephropathy or nondiabetic kidney disease. AT1 receptor antagonists are also effective antihypertensive agents 30,31) .

Antagonism of the AT1 receptor and binding of AT II
to the AT2 receptor probably underlies their effect. In contrast to ACE inhibitor, AT1 receptor antagonist (AT1RA) does not inhibit the breakdown of bradykinin 31) . Clinical trials suggest that AT1RAs reduce microalbuminuria and proteinuria and retard the progression of diabetic and nondiabetic kidney disease, which is similar to ACE inhibitors 31,32) .

Renoprotective benefits of renin angiotensin system inhibition
AT II, the main peptide of the RAS, is involved in the pathogenesis of renal diseases 27) . This peptide acts through its binding to two specific receptors, angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors 33) . In adult tissues, the AT1 receptor is distributed in the vasculature, kidney, adrenal gland, heart, liver, and brain 34) . In healthy adults, the AT2 receptor is present only in the adrenal medulla, uterus, ovary, vascular endothelium, and distinct brain areas 34) .
The AT2 receptor is also widely distributed in the fetus.  35) . AT II mediates both hemodynamic changes and renal growth suggesting that these processes are intimately connected. Both are thought to contribute to progression 36) . .
Treatment with AT1 receptor blocker may result in more complete blockade of the unfavorable actions of AT II mediated through the AT II type 1 receptor. AT1 receptor blocker does not reduce, but actually increases, the concentration of AT II. This excess of AT II will then bind to and activate AT2 receptors. In the past it had been assumed that activation of AT2 receptors is exclusively beneficial 33) . However, evidences have been accumulated which clearly suggest that AT2 receptors may mediate adverse effects. The activated AT2 receptor stimulates proinflammatory pathways by induction of NF-κB 37) .  38) , and improve selectivity of proteinuria in patients with glomerular diseases 39,40) . Large scale clinical trials confirmed the benefits of ACE inhibitors against a variety of renal diseases with their antiproteinuric effects 41,42) . Furthermore, recent studies demonstrated that the greater beneficial effect of ACE inhibitors in renal disease patients with higher baseline proteinuria could be explained by the greater antiproteinuric effects in these patients 43) . Thus, one of the protective mechanisms of ACE inhibitors and AT1RAs is the reduction of the amount of proteinuria.
Dual blockade of the renin angiotensin system -Promising treatment for renal protection?  48) . Although most studies suggest that dual blockade has renal benefit, maximal ACE inhibition has not been used in all studies 47,48) . These studies also have several limitations to draw firm conclusion about combination therapy in renal disease, such as small sample size, use of surrogate markers of renal protection, and short-term follow-up. Some authors suggested following recommendation; 1) titration of the single agent to maximal dose to control BP and proteinuria, 2) if proteinuria remains greater than 1 g/d, add a second agent to block the RAS further 48) .
Animal experiment with UUO showed slightly disappointing outcome after relief of UUO 49) . With ACE inhibitor (enalapril) or AT1RA (losartan) the decrease in the interstitial expansion was nearly 30%, significantly faster than with release alone (14% reduction). Unfortunately, complete recovery of interstitial damage developed after UUO could not be reached even after the treatment of ACE inhibitor plus AT1RAs. Combination therapy reduced only 20% in the interstitial expansion and was not superior to ACE inhibitor or AT1RA alone.

Conclusion
Interstitial fibrosis can be reversed by pancreas However, these treatments did not cause regression of renal damage, suggesting that novel approaches are needed.
A recent experiment showed that CTGF might be included as a potential interesting candidate for antifibrotic treatments 33) . TGF-β is an important regulator of fibrosis, however therapeutic strategies blocking TGF-β actions have not afforded the expected beneficial effects probably because of its anti-inflammatory properties. Some experimental data 37) also suggest that AT2 receptors are involved in the inflammatory cell recruitment in the kidney and the blockade of AT II generation by an ACE inhibitor or by combined blockade of both AT1 and AT2 receptors, as well as by the inhibition of the NF-κB pathway, is necessary to stop the inflammatory process fully in animal experiment.
Combination therapy with ACE inhibitor and AT1RA or combined blockage of both AT1 and AT2 receptor appears to provide further renal protection, but further clinical trials and experimental data are required to determine if combination therapy is superior.