RANTES and monocyte chemoattractant protein-1 (MCP-1) play an important role in the inflammatory phase of crescentic nephritis, but only MCP-1 is involved in crescent formation and interstitial fibrosis

Abstract

The involvement of chemokines in inflammation is well established, but their functional role in disease progression, and particularly in the development of fibrosis, is not yet understood. To investigate the functional role that the chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES play in inflammation and the progression to fibrosis during crescentic nephritis we have developed and characterized a murine model for this syndrome. Significant increases in T-lymphocytes and macrophages were observed within glomeruli and interstitium, paralleled by an induction of mRNA expression of MCP-1 and RANTES, early after disease initiation. Blocking the function of MCP-1 or RANTES resulted in significant decreases in proteinuria as well as in numbers of infiltrating leukocytes, indicating that both MCP-1 and RANTES (regulated upon activation in normal T cells expressed and secreted) play an important role in the inflammatory phase of crescentic nephritis. In addition, neutralization of MCP-1 resulted in a dramatic decrease in both glomerular crescent formation and deposition of type I collagen. These results highlight a novel role for MCP-1 in crescent formation and development of interstitial fibrosis, and indicate that in addition to recruiting inflammatory cells this chemokine is critically involved in irreversible tissue damage.

Figure 1

Administration of nephrotoxic serum to pre-immunized mice results in a severe proliferative and necrotizing GN. Hematoxylin and eosin staining of kidney sections from mice given either NSS (A) or NTS (B–D). NTS mice showed glomerular and interstitial infiltrates (B); glomerular crescent formation (C and D). Arrows highlight crescentic glomeruli. Original magnification: (A, B, and D) ×40; (C) ×20.

Figure 2

Nephrotoxic serum results in a decline in renal function. Renal dysfunction in mice as shown by (A) proteinuria and (B) blood urea nitrogen. Each point represents the mean values from either NSS (open circles, n = 6) or NTS (closed circles, n = 15) treated mice, and bars show the standard error of the mean.

Figure 3

Nephrotoxic serum induces expression of type I collagen mRNA and protein. Deposition of type I collagen in renal tissue isolated from NSS (A and B) or NTS (C and D) mice on day 7 of crescentic GN. Plates show (A and C) a 4-μm paraffin section hybridized with a ³⁵ScDNA probe encoding a region of the at α1 type I collagen gene and (B and D) a 4-μm frozen section stained for mouse type I collagen using indirect immunofluorescence. G, glomerulus; *, lumen of tubule; V, venule. Original magnification ×40.

Figure 4

Nephrotoxic serum induces an increase in numbers of leukocytes within renal tissue. Sections were prepared from NSS (open circles) or NTS (closed circles) mice at various times after disease induction and stained with antibodies specific for T-lymphocytes (a), mononuclear phagocytes (b), and neutrophils (c). Positively stained cells were counted in 15 high power fields per section (total area = 0.5mm²). Each dot represents one individual mouse analyzed (between 3 and 10 mice per group) with bars depicting means for each group.

Figure 5

Kidneys from mice with crescentic nepritis show expression of inflammatory chemokines. Northern blot analysis was performed using total RNA (20 μg) extracted from kidneys isolated from NSS or NTS mice throughout disease. Expression of two representative mice at each time point is included. Expression of β-actin was used to determine the quality and quantity of RNA.

Figure 6

MetRANTES reduces proteinuria and mononuclear cell infiltration. The effects of the RANTES antagonist MetRANTES on development of (A) proteinuria, (B) leukocyte infiltration, (C) glomerular crescent formation, and (D) deposition of type I collagen is shown. Groups of NSS (open circles, n = 3) and NTS (closed circles, n = 6) mice given 16 μg/day of MetRANTES or PBS were scarified on day 7 of disease. For A, C, and D, each point represents one mouse and bars indicate the mean for each group. For B, numbers of T-lymphocytes (T), mononuclear phagocytes (M) and granulocytes (G) were enumerated in immunohistochemically stained sections and bars represent the standard error of the mean for each group. Positively stained cells were counted within the glomeruli (open bars) and interstitium (shaded bars) of 15 randomly chosen, high power fields within each section. For C, one hundred glomeruli were counted in a hematoxylin and eosin-stained section from each mouse, and the percentage of glomeruli containing cellular crescents was calculated. In D, kidney sections were stained with an antibody against type I collagen, which was detected with a fluoresceinated secondary antibody. Distribution of immunofluorescence was measured in 10 random fields from each section using NIH image 1.56. For each panel the significance of differences between treated and untreated groups of NTS mice were determined by a Wilcoxon Mann-Whitney test.

Figure 7

Antibodies to MCP-1 reduce proteinuria and mononuclear cell infiltration, as well as glomerular crescent formation and collagen deposition. The effects of neutralizing antibodies to MCP-1/JE on (A) proteinuria, (B) leukocyte infiltration, (C) glomerular crescent formation, and (D) deposition of type I collagen are shown. Groups of NSS (open circles, n = 4) and NTS (closed circles, n = 10) were treated daily with 5 μg/d hamster anti–mouse MCP-1/JE or hamster immunoglobulin and were then killed on day 7 of disease. For A, C, and D, each point represents one mouse and bars indicate the mean for each group. For B, numbers of T-lymphocytes (T), mononuclear phagocytes (M), and granulocytes (G) were enumerated in immunohistochemically stained sections and bars represent the standard error of the mean for each group. Positively stained cells were counted within the glomeruli (open bars) and interstitium (shaded bars) of 15 randomly chosen, high power fields within each section. For C, 100 glomeruli were counted in a hematoxylin and eosinstained section from each mouse, and the percentage of glomeruli containing cellular crescents was calculated. In D, kidney sections were stained with an antibody against type I collagen that was detected with a fluoresceinated secondary antibody. Distribution of immunofluorescence was measured in 10 random fields from each section using NIH image 1.56. For each panel the significance of differences between treated and untreated groups of NTS mice were determined by a Wilcoxon MannWhitney test.