Immunolocalization of Kim-1, RPA-1, and RPA-2 in kidney of gentamicin-, mercury-, or chromium-treated rats: relationship to renal distributions of iNOS and nitrotyrosine

Abstract

Immunohistochemical studies for kidney injury molecule-1 (Kim-1), renal papillary antigen-1 (RPA-1), and renal papillary antigen-2 (RPA-2) were conducted to explore their relationship to inducible nitric oxide synthase (iNOS) and nitrotyrosine expression. Male Sprague-Dawley rats were exposed to gentamicin (100 mg/kg/day Gen, sc, for 3 days), mercury (0.25 mg Hg/kg, iv, single dose), or chromium (5 mg Cr/kg, sc, single dose) and kidney tissue was examined 24 hours or 72 hours after the last dose of the nephrotoxicant. Another group of kidneys was evaluated 24 hours after rats were administered 3 daily doses (50, 100, 150, 200, or 300 mg/kg/day) of Gen. Gen- and Cr-treated rats exhibited increased immunoreactivity of Kim-1, RPA-1, and RPA-2 largely in the S1/S2 segments and to a lesser extent in the S3 segments of the proximal tubule of the kidney, whereas Hg-treated rats showed increased immunoreactivity of Kim-1, RPA-1, and RPA-2 in the S3 segments. Up-regulation of Kim-1, RPA-1, and RPA-2 expression correlated with injured tubular epithelial cells and also correlated with immunoreactivity of iNOS and nitrotyrosine. It is possible that iNOS activation with nitrotyrosine production in injured nephron segments may be involved in the induction of Kim-1, RPA-1, and RPA-2 following exposure to nephrotoxicants.

FIGURE 1

Representative photomicrographs of kidney injury molecule-1 (Kim-1) immunoreactivity in kidneys of rats treated with nephrotoxicants. No Kim-1 expression was found in the cytoplasm of epithelial cells of all segments in the cortex in rats treated with dH₂0 vehicle (A). In the time course study with gentamicin (Gen), Kim-1 expression was detected on the surface of epithelial cells in the S1/S2 segments in rats 24 hours after the 3rd daily injection of 100 mg Gen/kg, sc (B). Kim-1 expression was also seen in the cytoplasm of necrotic and dying epithelial cells in the S1/S2 segments in rats 72 hours after the 3rd daily injection of Gen 100 mg/kg, sc (C). Kim-1 was observed in the cytoplasm of desquamated necrotic cells in the lumen of these segments 24 hours after the 3rd injection of Gen 300 mg/kg (D). In the time course studies with mercury (Hg) or chromium (Cr), Kim-1 expression was detected in rats treated with Hg 0.25 mg/kg, iv, after 24 hours (E) and 72 hours (F) or Cr 5 mg/kg after 24 hours (G) and 72 hours (H). Original magnification of all figures: ×400.

FIGURE 2

Representative low-magnification photomicrographs showing a higher level of kidney injury molecule-1, renal papillary antigen-1 (RPA-1), and renal papillary antigen-2 (RPA-2) expression in the S1/S2 segments of the nephron when compared with S3 segments in rats 72 hours after the 3rd daily dose of gentamicin 100 mg/kg, sc. Note: RPA-1 expression in collecting ducts and RPA-2 expression in loop of Henle are also visible but are comparable to their respective counterparts in control rats. Original magnification of all figures: ×50.

FIGURE 3

Representative photomicrographs of renal papillary antigen-1 (RPA-1) immunoreactivity in kidneys of rats treated with nephrotoxicants. RPA-1 expression was located predominantly in the medullary collecting duct (CD) epithelial cells (A) and sporadically in the cortical CD epithelial cells (B) in rats treated with saline, iv, for 24 hours. Treatment of rats with gentamicin (Gen) 50 mg/kg, sc, daily for 3 days did not change RPA-1 expression in the medullary CD epithelial cells (C), but RPA-1 appeared in proximal tubular epithelial cells (D). Treatment of rats with high doses of Gen, 300 mg, kg sc, daily for 3 days did not induce significant changes in medullary RPA-1 expression (E), but did result in enhanced S1/S2 segment expression (F). Rats treated with mercury 0.25 mg/kg, iv, for 24 hours expressed RPA-1 in the S3 segments (G). RPA-1 expression was noted in the S1/S2 segments in rats treated with chromium 5 mg/kg, sc, for 24 hours (H). Original magnification of all figures: ×400, except figures (A, C, E): ×50.

FIGURE 4

Representative photomicrographs of renal papillary antigen-2 (RPA-2) immunoreactivity in kidneys of rats treated with nephrotoxicants. RPA-2 expression was located predominantly in the epithelial cells of the descending and ascending thin loop of Henle (LH) segments in the medulla (A) in control rats. The immunostaining for RPA-2 in control rats showed homogenous reaction within the cytoplasm of LH cells and intense reaction on their surfaces (B). Note the epithelial cells of medullary collecting ducts in the vicinity of RPA-2-expressing tubules were completely negative. RPA-2 expression was sporadic in the epithelial cells in the ascending thick LH segments of the cortex in control rats (C). Treatment of rats with gentamicin 100 mg/kg, sc, for 3 days did not change RPA-2 expression in the epithelial cells of the descending and ascending thin LH segments in the medulla (D) but resulted in new expression of RPA-2 in proximal tubular epithelial cells 24 hours (E) and 72 hours (F) after the last dose. There was no RPA-2 expression in the S1/S2 segments in mercury-treated rats (G). In contrast, RPA-2 expression was noted in the S1/S2 segments in chromium-treated rats (H). Original magnification of all figures: ×400, except figures (A, D, G), ×50.

FIGURE 5

Representative photomicrographs demonstrating immunostaining for apoptosis (A, D, G, J), expression of inducible nitric oxide synthase (iNOS) (B, E, H, K), and expression of nitrotyrosine (C, F, I, L). No immunostaining for apoptosis, iNOS, or nitrotyrosine was observed in saline-treated rats (A-C). Gen- (D-F) and Cr- (J-L) treated rats exhibited immunostaining for apoptosis, iNOS, and nitrotyrosine largely in the S1/S2 segments and to a lesser degree in the S3 segments, whereas Hg- (G-I) treated rats exhibited increased immunostaining for apoptosis, iNOS, and nitrotyrosine almost exclusively in the S3 segments. Apoptosis is indicated by blue color of nuclei. Original magnification of all figures: ×400.