Renal Pathophysiology Laboratory, Renal Division, Department of Clinical Medicine, Faculty of Medicine, University of São Paulo, Brazil
Introduction
The pathogenesis of progressive renal diseases leading to end-stage renal failure is still largely unknown and is therefore the subject of intense investigation. Some features have been well recognized such as the development of glomerulosclerosis, tubulointerstital injury, and the accumulation of extracellular matrix leading to fibrosis and scarring of the renal structures.
It is now clear that major events, which determine the outcome of progressive renal disease, regardless of the initial insult, are related to the tubulointerstitial compartment. Even in primary glomerulonephritis the degree of tubulointerstitial damage correlates more closely with the long-term outcome than to the extent of glomerular involvement [1,2].
Although mechanical factors such as glomerular hypertension and hypertrophy are likely to contribute to the initiation of some forms of progressive renal disease, tubulointerstitial lesions cannot be simply interpreted as an ischaemic sequela of glomerular sclerosis. There is growing evidence that the participation of cellular and inflammatory mechanisms play a critical role in the progression of renal disease. Tubulointerstitial inflammation comprises an influx and proliferation of inflammatory cells capable of producing a variety of local inflammatory mediators and activation of tubular as well as other intrinsic renal cells. The development of progressive renal damage occurs even after resolution of the initial insult suggesting that this cascade of events occurring in the tubulointerstitium, once triggered, has an autonomic and progressive pattern. Thus, the recognition of these events is of crucial importance not only in understanding the steps involved in the development of chronic inflammatory lesions but also in defining targets of therapies.
Cellular and molecular events involved in the progression of renal disease
In recent years, more attention has been focused on the inflammatory infiltrate present in various types of progressive renal diseases in humans [3] and in experimental models even of non-immunologic nature [46]. The number of inflammatory cells in the renal interstitium closely correlates with the severity of glomerular and tubulointerstitial lesions and with loss of renal function [2,6]. The exact sequence of cellular events and tubulointerstitial changes in progressive renal disease could not yet be entirely elucidated.
In early phases of renal disease, there is an influx of inflammatory cells irrespective of the nature of the initial renal insult. Immunophenotyping has confirmed the increased numbers of macrophages and of T and B lymphocytes [3,6]. The recruitment of these cells into the renal interstitium seems to be in response to injury and/or activation of some component of the tubulointerstitial compartment. Inflammatory cells migrate to the interstitium driven by the up-regulated expression of chemoattractant molecules (chemokines and adhesion molecules) produced by activated tubular cells (Figure 1).
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The infiltration of macrophages, considered the predominant leukocyte type infiltrating renal tissue, can be detected in early phases of progressive renal disease. In rats subjected to 5/6 renal ablation (Nx), macrophage infiltration starts already 1 week after ablation and increases progressively with time [46]. Several studies have shown that macrophages play a central role in mediating the development of renal injury in animal models [46] as well as in human diseases [3]. Macrophages can mediate tissue injury through different pathways. Besides the ability of producing proteolytic enzymes, reactive oxygen species and vasoactive substances, macrophages synthesize cytokines and fibrogenic growth factors responsible for a sustained inflammatory milieu. The maintenance of these cells in later phases of renal disease suggest that macrophages are also involved in the chronic inflammatory process leading to the development of fibrotic changes.
Evidence of the activity of the cellular component in progressive renal diseases is reflected by the ongoing state of proliferation. Most of the renal cells display proliferative activity, as demonstrated by PCNA expression, and are localized within glomeruli and in areas of tubulointerstitial lesions. In the remnant kidney model we, and others, observed a peak in renal cell proliferation at 1 week after renal ablation. The proliferative activity is a phenomen occurring not only in interstitial cells but also tubular epithelial cells pointing to a possible participation of tubular cells in the pathogenesis of renal interstitial fibrosis [4,7].
In fact, tubular cells may have an active participation in this process by controlling the cellular trafficking to the interstitium through the expression of MCP-1, osteopontin and RANTES, and adhesion molecules. In addition, tubular cells can further influence the development of interstitial inflammation due to their ability to express MHC antigens and other molecules which will enable these cells to function as antigen presenting cells and facilitate interaction with T cells [8]. Finally, tubular cells themselves can produce several inflammatory cytokines and even extracellular matrix components, such as collagen I and III, laminin, and fibronectin [9]. Thus, tubular cells may also influence the process of tissue fibrosis and scarring.
Another possible mechanism of activating tubular cells could be the massive protein reabsorption by proximal tubular cells. The nephrotoxic effect of proteinuria could result from endocytosis of protein by tubular epithelial cells with consequent cell injury [10]. In favour of this theory, one may quote the fact that reduction of urinary protein excretion in clinical trials reduces the decline in the glomerular filtration rate and ameliorates histopathologic changes in the interstitium [10].
The fibrogenic process in progressive renal disease
Fibrogenesis is a response to tissue injury and to an inflammatory process. In general, after tissue injury and the establishment of an inflammatory reaction, subsequent fibrosis occurs but this response is oriented to an attempt towards resolution, with regression of fibrous tissue. In progressive renal diseases, however, this repair response does not shut down. Rather, the process is perpetuated and is characterized by a persistent, chronic inflammatory process. Progressive fibrosis then leads to tissue scarring with destruction of organ architecture.
The fibrogenic response relies basically on fibroblasts, which are considered to be the effector cells in fibrogenesis. Fibroblasts synthesize a variety of extracellular matrix components [11]. The overproduction of these proteins during fibrogenesis leads to excessive collagen accumulation and fibrosis. The production of extracellular matrix proteins is regulated and induced by growth factors derived from macrophages and from tubular cells. One of the most relevant mediators in this process is the TGF-ß.
The amount of extracellular matrix in the interstitium reflects the balance between the production and degradation by proteases. TGF-ß contributes to fibrogenesis by acting through both pathways [12]. TGF-ß directly enhances the synthesis of all major matrix proteins, such as fibronectin, proteoglycans, and collagens. On the other hand, TGF-ß inhibits matrix degradation by enhancing the production of plasminogen activator inhibitors (PAI) and enhancing the activity of tissue inhibitors of metalloproteinases (TIMP). Additionally, TGF-ß inhibits T-cell proliferation, and this biological effect may be of relevance in limiting the acute inflammatory response.
The process of scar tissue remodelling includes tissue retraction [13]. This retractile feature is the result of the action of retractile microfilaments such as -smooth muscle actin (
-SMA) and other cytoskeletal proteins. In this process, fibroblasts change their phenotype differentiating into myofibroblasts, which can be recognized by their expression of
-SMA.
The origin of fibroblasts and myofibroblasts is still not clear: they can be derived from resident interstitial fibroblasts or from perivascular cells. They can also be attracted into the interstitium by the action of locally produced cytokines, particularly by PDGF [4]. Alternatively, interstitial fibroblasts and myofibroblasts could be derived from tubular cells through a process of transdifferentiation [7]. The phenotypic transformation of fibroblasts and myofibroblasts is regulated by fibrogenic cytokines IL-l, TNF-, PDGF, TGF-ß, FGF, among others [13]. These fibrogenic cytokines also have mitogenic properties for these cells. Proliferation of fibroblasts and myofibroblasts is considered an active form of the fibrogenic response [13].
Myofibroblasts can be identified by their expression of -SMA and have been demonstrated in several experimental and clinical progressive renal diseases [6,14]. The number of myofibroblasts present in renal tissue correlates with the prognosis of the disease progression preceding the development of renal scarring [4,6].
Finally, in the process of renal disease progression another local mediator, angiotensin II (Ang-II), deserves consideration. Besides its haemodynamic effects, Ang-II exerts actions on cells that contribute to inflammation and tissue fibrosis which contribute to the pathogenesis of renal disease. Ang-II acts as a growth factor enhancing the proliferation of myofibroblasts and mesangial cells, and stimulating extracellular matrix synthesis [11,15,16]. In addition, Ang-II has potent pro-inflammatory actions with direct effects on macrophages and T-cell functions through AT1 receptors present on these cells. Ang-II promotes T-cell proliferation [17]. It also stimulates the production of inflammatory mediators and the expression of chemokines and adhesion molecules [18]. Infusion of Ang-II to normal rats induces macrophage infiltration and stimulates interstitial fibroblasts to proliferate and to increase the synthesis of matrix proteins [19]. This effect is presumably the result of up-regulation of the TGF-ß gene by Ang-II, stimulating the production of TGF-by renal tubular cells, fibroblasts and other cells [16].
In this context, the intra-renal production of Ang-II may be of relevance. Previous reports have shown that some renal cells such tubular epithelial cells, macrophages, and fibroblasts have the machinery for the synthesis of Ang-II [11,20]. Using immunohistochemistry, we have recently demonstrated that in the remnant kidney model Ang-II is expressed not only in juxtaglomerular arterioles, but also in interstitial cells [21]. The number of Ang-II positive interstitial cells increased progressively after renal ablation. These results suggest the existence of a local interstitial intra-renal RAS, which may play an important role in the inflammatory process and progressive renal injury.
Possible therapeutic interventions
Pharmacological blockade of the RAS
Several mechanisms that are involved in the progression of renal diseases have been identified but so far no specific treatment is available that can efficiently block or even revert progression of renal disease.
Hypertension is an important factor in chronic renal disease and is associated with a more rapid rate of progression [22]. Therefore, therapeutic measures aiming to lower high blood pressure in progressive renal diseases have been widely used. However, pharmacological blockade of the RAS is non-effective in retarding the progression of renal disease as compared with equipotent hypotensive drugs, such as beta-blockers [23,24].
Renoprotection by suppressing the RAS by angiotensin I converting enzyme inhibitors or Ang-II receptor antagonists has been extensively demonstrated in animal studies and confirmed by controlled prospective trials in patients with renal disease [25,26]. The renoprotection of RAS blockers is explained, at least in part, by reduction of intra-glomerular capillary pressure [27]. Evidence in the literature suggests that the renoprotective effect of RAS blockers is also related to their anti-proteinuric effect [28]. Limiting protein filtration may be of critical relevance in preventing tubular cell activation and consequently tubulointerstitial injury [10].
Another mechanism by which pharmacological antagonists of RAS may have a beneficial effect is a blockade of non-haemodynamic actions of Ang-II. As already commented, Ang-II has potent pro-inflammatory actions. The blockade of Ang-II activity significantly inhibits macrophage infiltration, myofibroblast proliferation, and also diminishes the expression of PDGF, TGF-ß [29], and the appearance of interstitial fibrosis in experimental models renal disease thus, preventing its progression.
A role for anti-inflammatory interventions?
It is of note that both clinical and experimental studies failed to show total arrest or reversal of progression of renal disease, indicating that inhibition of the RAS afforded only incomplete control of the mechanisms involved in the progression of renal disease. Therefore, more efficient alternative therapeutic interventions are necessary. Approaches aiming more specifically at blocking the inflammatory process are likely to ameliorate progressive renal injury. Indeed, steroids, considered important but non-specific immunosuppressive drugs have been widely used in clinical practice. Although steroids have been recommended for the treatment of a variety of immune-mediated glomerular diseases, particularly proteinuric forms of renal diseases, it has limited effects in reversing progressive renal diseases. Non-steroidal anti-inflammatory drugs may also have beneficial effects against the inflammatory component. We showed that chronic treatment with a non-steroidal anti-inflammatory drug, nitroflurbiprofen, retards renal injury in the 5/6 ablation model [30]. Protective effects were also reported by using COX-2 inhibitor in the same model [31].
As macrophages have been recognized as an important component in the inflammatory process, efforts to target these cells have been proposed. Macrophage depletion by anti-macrophage serum, by systemic X-irradiation, by essential fatty acid-deficient diets or by gamma lactone protected against proteinuria and induced resolution of the renal disease [32,33].
With the same rationale in mind, calcineurin inhibitors such as cyclosporin have been used, to block lymphocyte activation. Cyclosporin has been used particularly in cases of steroid-resistance. It is currently considered an appropriate alternative in the treatment of refractory nephrotic syndrome, including minimal change disease, membranous nephropathy, and lupus nephritis [34,35].
What is the role of mycophenolate mofetil?
However, none of these treatments have so far been efficient in blocking the progression of interstitial fibrosis. Another therapeutic approach to inhibit the inflammatory phenomena is the use of mycophenolate mofetil (MMF), which is currently widely used in clinical transplantation protocols. MMF, an anti-proliferative agent, efficiently limits proliferative activity of lymphocytes and consequently, macrophage accumulation and proliferation. Additional benefit may also derive from limitation of tubular cell proliferation and down-regulation of adhesion molecules, all features considered to be key to a protective effect. We showed in the 5/6-ablation model that early treatment with MMF attenuated renal inflammation, glomerulosclerosis and interstitial expansion [5] and also diminished the number of Ang-II positive interstitial cells [21] suggesting that MMF favourably affects progressive renal diseases of non-immune origin. The amelioration of renal injury in the renal ablation model promoted by MMF was also observed by other groups and also in other models of renal disease [3638]. In human renal diseases, there are also reports on the successful MMF treatment of vasculitis, lupus nephritis, and glomerular diseases [39,40]. However, further prospective controlled randomized clinical trials are needed to demonstrate the usefulness of this drug in the treatment of progression in human chronic renal disease.
The association of MMF and RAS inhibition may afford a more potent renoprotective effect [41,42]. In Nx rats, combined losartan/MMF therapy was associated with reversal of systemic hypertension and albuminuria, as well as arrest of macrophage infiltration, glomerulosclerosis, and interstitial injury. Combined therapy, acting at distinct steps of the cascade leading to end-stage renal disease, had an additive renoprotective effect compared with the respective monotherapies. Moreover, this association was even effective when instituted in 5/6 nephrectomized rats 1 month after ablation, at a time when rats already exhibited substantial renal injury. This situation resembles more closely the conditions observed in clinical practice [42]. In the same line of investigation, simultaneous treatment of Nx rats with tacrolimus and candesartan afforded more effective renal protection than either drug alone [43].
Although the data on MMF are encouraging indeed it would be desirable to investigate further pharmacological agents that are capable of targeting other components of the inflammatory process.
Notes
Correspondence and offprint requests to: Irene L. Noronha, Renal Pathophysiology Laboratory, Renal Division, Department of Clinical Medicine, Faculty of Medicine, University of São Paulo, São Paulo, Brazil. Email: irenenor{at}usp.br
References