PMC

Changes in urinary protein excretion after the administration of anti-GBM AS. Urinary protein excretion was measured in the mice sacrificed at day 126. The open circles and closed circles represent the AT1a^+/+ and AT1a^–/– mice, respectively. The open squares with a dotted line and the closed squares with a dotted line represent the physiological protein excretion of the AT1a^+/+ and AT1a^–/– mice, respectively. Data are mean ± SE. Statistical significance was evaluated by comparing the data of the AT1a^–/– mice with those of the AT1a^+/+ mice at each time point. **P < 0.01.

Immunohistochemical findings in the kidney after the administration of anti-GBM AS in the AT1a^+/+ (a, c, and e) and AT1a^–/– mice (b, d, and f). Linear binding of rabbit IgG to the GBM at 6 h (a and b). Fine granular deposits of C3 along the capillary wall, with additional deposition in the mesangium at 6 h (c and d). Linear binding of mouse IgG, identical to rabbit IgG, in the glomeruli at 7 days (e and f). ×400.

Genotype analysis of the renin genes. After EcoRI restriction enzyme digestion, the genomic DNA was analyzed by Southern blot analysis. Lane 1, TT2 ES cells used for the generation of theAT1a ^–/– mice; Lane 2, the AT1a ^–/– mice; Lane 3, C57Bl/6 mice. The Ren-2 and Ren-1D genes were detected as 4.4-, 9.2-kb fragments and 3.9-, 8.8-kb fragments respectively.. The Ren-1C gene was detected as 3.9- and 8.8-kb fragments. The AT1a^–/– mice have the Ren-1C genotype identical to that of C57Bl/6 mice. AT1a^–/– mice, angiotensin II type 1a receptor–deficient homozygous mice.

Histological examination of the chronic renal injuries. (a) Light microscopy of the kidney at day 126 after the administration of anti-GBM AS. The kidney sections were stained using the Azan-Mallory method. The overproduction of extracellular matrix proteins led to severe glomerulosclerosis and interstitial fibrosis in the AT1a^+/+ mice(top). These histological changes were markedly reduced in the AT1a^–/– mice (bottom) ×200. (b) The percent of glomerular (left) and interstitial(right) area occupied by extracellular matrix was semiquantitated as described in Methods. The closed bars represent the AT1a^+/+ mice, and the hatched bars represent the AT1a^–/– mice. Data are mean ± SE. **P < 0.01 compared with the AT1a^+/+ mice.

Expression of MCP-1. (a) Gene expression of MCP-1 during the first 14 days after the anti-GBM AS administration was examined by Northern blot analysis. Total RNA from mice at day 0 (control), 6 h, and days 1, 7, and 14 was separated on an agarose gel and probed for MCP-1. The blots were stripped and rehybridized with a cDNA probe for the 28S rRNA. Two representative results of each animal group were shown. (b) The gene expression of MCP-1 was estimated as described in Methods. The closed bars represent the AT1a^+/+ mice, and the hatched bars represent the AT1a^–/– mice. Data are mean ± SE in arbitrary units. *P < 0.05 compared with the AT1a^+/+ mice. (c) Immunoperoxidase staining for MCP-1 at day 7 after anti-GBM AS administration. In the AT1a^+/+ mice, MCP-1 expression was propagated in the glomeruli. In contrast, MCP-1 expression was reduced in the AT1a^–/– mice. ×200. MCP-1, monocyte chemoattractant protein-1.

Expressions of TGF-β1 and collagen type I. (a) Northern blot analysis was performed using specific probes for TGF-β1 and α2(I). The blots were stripped and rehybridized with a cDNA probe for the 28S rRNA. A representative result of each animal group was shown. (b) The gene expressions of TGF-β1 (top) and collagen type I (bottom) were estimated as described in Methods. The results are presented as the fold increase compared with the values obtained before the administration of anti-GBM AS (day 0). The open circles represent the AT1a^+/+ mice, and the closed circles represent the AT1a^–/– mice. *P < 0.05, **P < 0.01 compared with day 0; ^‡P < 0.05, ^‡‡P < 0.01 compared with the AT1a^+/+ mice at each time point. (c) Immunoperoxidase staining for TGF-β1 (top left and top right) and collagen type I (bottom left and bottom right) at day 126 after the anti-GBM AS administration. In the AT1a^+/+ mice, the expression of TGF-β1 was propagated in the glomeruli, (top left)and the expression of collagen type I was propagated in the interstitium(bottom left). In the AT1a^–/– mice, reduced staining of TGF-β1 (top right) and collagen type I (bottom right)was observed. TGF-β1, ×200; collagen type I, ×100. TGF-β1, transforming growth factor-β1.

Activation of the RAS after administration of anti-GBM AS. Mice were sacrificed at 6 h and days 1, 7, and 14 after the administration of anti-GBM AS. (a) The amount of active renin in the plasma was estimated by RIA as described in the Methods. (b) The expression of the angiotensinogen mRNA in the liver was examined by Northern blot analysis. Open circles represent the AT1a^+/+ mice, and closed circles represent the AT1a^–/– mice. (c) Renal expressions of AT1a mRNA in the AT1a^+/+ mice and lacZ mRNA in the AT1a^–/– mice. Open circles and closed circles represent the gene expressions of AT1a and lacZ, respectively. Results at each time point in b and c were given as the fold increase compared with those observed before the induction of nephritis (day 0). (d) Changes in the MBP. Open circles represent the AT1a^+/+ mice, and closed circles represent the AT1a^–/– mice. Data are mean ± SE. Statistical significance was evaluated by comparing the data at each time point with those obtained before the administration of anti-GBM AS (day 0). *P < 0.05, **P < 0.01. AI, angiotension I; AS, antiserum; AT1a, angiotensin II type 1a receptor; AT1a^+/+ mice, AT1a wild-type mice; GBM, glomerular basement membrane; MBP, mean blood pressure; RAS, renin–angiotensin system.