In this study we have investigated the role of chemokines in the development of glomerular and interstitial inflammation and progression to renal fibrosis in a murine model of crescentic glomerulonephritis. Glomerular and interstitial hypercellularity are the earliest abnormalities in this model of accelerated nephrotoxic serum nephritis (Fig. ), and they occur concomitantly with the appearance of severe proteinuria and transient acute renal failure (Fig. ). The inflammatory infiltrate in glomeruli and interstitium is composed of T-lymphocytes, mononuclear phagocytes and neutrophils (Fig. ), much as has been described in other animal models of crescentic glomerulonephritis (34
). Within one week from the onset of disease, glomerular crescents begin to form and there is perivascular and periglomerular type I collagen gene induction (Fig. ), signifying the onset of renal fibrosis. PMNs, mononuclear phagocytes and lymphocytes may contribute to acute renal cell injury and progression of glomerular disease by a variety of different mechanisms including inducing the secretion of pro-inflammatory mediators such as TNFα and IL-1, which may then induce chemokine expression by resident renal cells. The interaction between infiltrating inflammatory cells and intrinsic renal cells may lead to cell proliferation and matrix expansion which would ultimately result in glomerulosclerosis and interstitial fibrosis.
To determine the role of chemokines in eliciting the migration of inflammatory cells to the kidney in this model, we first examined their expression during the evolution of the lesion and then established the functional role of likely candidates with blocking agents. RANTES and MCP-1 are strongly expressed after disease induction (Fig. ) at a time that coincides with the influx of T-lymphocytes and mononuclear phagocytes, and that follows the earlier appearance of neutrophils. MIP1α was found to be constitutively expressed in the kidney and was not upregulated during crescentic nephritis. We did not see any expression of TCA3 during crescentic nephritis, but this may reflect the restricted secretion profile of this particular chemokine (23
). MCP-1 and RANTES are both critical mediators in the evolution of inflammatory reactions and are known to be secreted by isolated resident renal cells in vitro (11
) as well as by infiltrating inflammatory cells. Expression of both RANTES and MCP-1 has been documented in several human nephritides (35
) as well as during experimental anti-GBM GN (14
) but these studies are limited to examining glomerular changes occurring during a relatively short time period and do not provide evidence that chemokines are involved in the development of inflammation and disease progression. In our study however, administration of a RANTES antagonist resulted in a decrease in the accumulation of T cells and monocytes/macrophages concomitant with a partial attenuation of proteinuria (Fig. ). MetRANTES is a functional antagonist of RANTES and blocks calcium mobilization as well as chemotaxis of T cells and monocytes (22
). Interestingly, MetRANTES antagonizes RANTES and MIP-1α with equal potency in chemotaxis assays but not calcium mobilization. It is thought to act as a competitive inhibitor on the shared MIP-1α/ RANTES receptor. We did not find increased expression of MIP-1α in our model of crescentic GN and therefore assume that Met-RANTES is acting primarily to antagonize RANTES, thereby partially blocking the in vivo migration of T cells and mononuclear phagocytes. We have also shown that inhibition of MCP-1 by administration of neutralizing antibodies results in decreased accumulation of mononuclear phagocytes and T-lymphocytes, concomitant with a partial attenuation of proteinuria (Fig. ). Thus, we have shown that both MCP-1 and RANTES are critically involved in the primary inflammatory phase of crescentic GN.
Progression of disease in mice with crescentic GN was marked by the development of glomerular crescents and the deposition of type I collagen within the interstitium. We have examined the role that MCP-1 and RANTES play in the progression to this later stage of disease. Although administration of a RANTES antagonist resulted in a decrease in the accumulation of T cells and monocytes/macrophages, there was little effect on glomerular crescent formation or deposition of interstitial type I collagen. Previous studies have suggested that activated periglomerular T cells may be involved in the disruption of Bowman's capsule (36
), and CD8+
T cell depletion has been found to inhibit crescent formation, (37
), but we have shown that RANTES does not appear to be involved in this process. Conversely, neutralizing antibodies to MCP-1 resulted in a striking decrease in both glomerular crescent formation and collagen I deposition. It is interesting that although macrophage accumulation was only inhibited by 50% by antibody administration, this decrease is sufficient to cause such an impressive effect on markers of fibrosis. Since monocyte/macrophage accumulation was not completely inhibited by MCP-1 neutralization, it seems likely that other macrophage-specific chemokines may contribute to this migration. It is not inconceivable that MCP-1 not only affects migration and extravasation of mononuclear phagocytes but mediates direct effects upon the resident renal cells, which in turn affects the pathway to fibrosis. MCP-1 may participate in this fibrotic process directly, by inducing the secretion of extracellular matrix components, or indirectly by instigating the secretion of other pro-fibrotic mediators. It is also possible that MCP-1 alters the phenotype of resident renal cells leading to parietal glomerular epithelial cell proliferation, collagen I production by interstitial fibroblasts, or even tubular epithelial cell transmodulation (38
). Whether this is a direct effect of MCP-1, or is mediated by other fibrogenic agents such as TGF-β, PDGF, or FGF, has yet to be determined. We have preliminary data demonstrating the expression of TGF-β mRNA in murine crescentic glomerulonephritis (data not shown), however despite its well-established fibrogenic properties (40
), the functional role of this cytokine in post-inflammatory interstitial fibrosis of the kidney has yet to be documented. It is noteworthy that the mice in our model are heavily proteinuric, which some have suggested may be a sufficient stimulus for renal tubular chemokine production and consequent interstitial inflammation and fibrosis (41). On the other hand, proteinuria in the absence of accompanying glomerular inflammation does not appear to stimulate MCP-1 expression (41).
This work has centered on characterizing a murine model of crescentic nephritis and determining the role of the β chemokines MCP-1 and RANTES in the renal inflammation, crescent formation and in the development of the ensuing interstitial fibrosis. We can conclude that both chemokines play a role in the influx of inflammatory cells to the glomeruli and interstitium and in the initial development of renal dysfunction. Moreover, MCP-1 seems to play a critical role in the development of the characteristic glomerular crescents and in the deposition of type I collagen. It is likely that this chemokine mediates direct effects upon intrinsic renal cells which ultimately leads to the formation of crescents and secretion of matrix components.