Possible involvement of myofibroblasts in cellular recovery of uranyl acetate-induced acute renal failure in rats

Abstract

Cellular recovery in acute renal failure is a form of wound healing. Fibroblast-like cells or myofibroblasts are involved in wound healing. We examined the serial changes in tubular damage and origin and kinetics of regenerating cells in uranyl acetate-induced acute renal failure, with a special emphasis on interstitial myofibroblasts. Acute renal failure was induced in rats by intravenous injection of uranyl acetate (5 mg/kg). All rats received bromodeoxyuridine intraperitoneally 1 hour before sacrifice. Serial changes in the distribution of tubular necrosis and bromodeoxyuridine-incorporated or vimentin-positive regenerating cells, and their spatial and temporal relation to alpha-smooth muscle actin-positive myofibroblasts as well as ED 1-positive monocytes/macrophages were examined. Necrotic tubules initially appeared around the corticomedullary junction after uranyl acetate injection, then spread both downstream and upstream of proximal tubules. Peritubular alpha-smooth muscle actin-positive myofibroblasts appeared and extended along the denuded tubular basement membrane, establishing network formation throughout the cortex and the outer stripe of outer medulla at days 4 to 5. Tubular regeneration originated in nonlethally injured cells in the distal end of S3 segments, which was confirmed by lectin and immunohistochemical staining using markers for tubular segment. Subsequently, upstream proliferation was noted along the tubular basement membrane firmly attached by myofibroblasts. During cellular recovery, no entry of myofibroblasts into the tubular lumen across the tubular basement membrane was noted and only a few myofibroblasts showed bromodeoxyuridine positivity. The fractional area of alpha-smooth muscle actin-positive interstitium reached a peak level at day 7 in the cortex and outer stripe of outer medulla, then gradually disappeared by day 15 and remained only around dilated tubules and in the expanded interstitium at day 21. ED 1-positive monocytes/macrophages were transiently infiltrated mainly into the region of injury. They did not show specific association with initially necrotic tubules, but some of them located in close proximity to regenerating tubules. Nonlethally injured cells at the distal end of proximal tubules are likely to be the main source of tubular regeneration, and the transient appearance of interstitial myofibroblasts attached to the tubular basement membrane immediately after tubular necrosis might play a role in promoting cellular recovery in possible association with monocytes/macrophages in uranyl acetate-induced acute renal failure.

Figure 1.

Serial changes in serum creatinine (Scr) after induction of UA-induced ARF. Data represent the mean ± SEM values of five animals. *, P < 0.05 versus before induction of ARF; #, P < 0.001 versus before induction of ARF.

Figure 2.

Photomicrographs of periodic acid-Schiff-stained sections, showing PT injury in OSOM before induction of ARF (A); day 2 (B); day 3 (C); day 5 (D); day 7 (E and H); and day 9 (F and I). G: Enlarged photomicrograph of the inset in B; arrows, border between CO and OSOM; arrowheads, border between OSOM and ISOM. PT necrosis initially occurred around the corticomedullary junction where denuded TBM was evident at day 2 (B and G), then spread upstream and downstream of the nephron, reaching a peak level around day 5 (D). At day 7 (E) the TBM was almost covered with flattened, regenerated tubular cells (H, arrowheads). Hyperplastic tubules (I, arrow) were evident at day 9. Original magnifications, ×100 (A–F); ×300 (G); ×650 (H and I).

Figure 3.

Morphometric analysis of serial changes in the distribution of PT necrosis in CO and OSOM. S, surface of the kidney; B1, border between CO and OSOM; B2, border between OSOM and ISOM. Data represent the mean values of three or four animals.

Figure 4.

Serial changes in the distribution of BrdU-positive cells (black nuclei) in OSOM before induction of ARF (A), day 2 (B), day 3 (C), day 4 (D), day 5 (E), day 7 (F), and day 9 (G). Arrows, border between CO and OSOM; arrowheads, border between OSOM and ISOM. Original magnifications, ×100 (A–G).

Figure 5.

Serial changes in the frequency distribution of tubular BrdU-positive cells in CO, OSOM, and ISOM. S, surface of the kidney; B1, border between CO and OSOM; B2, border between OSOM and ISOM; B3, border between ISOM and inner medulla. Data represent the mean values of three or four animals.

Figure 6.

Double-staining of AQP-1 (pink color) and BrdU (brown color) (A), and THP (pink color) and BrdU (brown color) (B), or staining of vimentin (brown color) (C) around the border between OSOM and ISOM in consecutive sections at day 2. Arrow, weak AQP-1 expression in PT; arrowheads, border between OSOM and ISOM; 1 and 2, identical PTs; 3, identical collecting duct. Original magnifications, ×350 (A–C).

Figure 7.

Morphometric analysis of the number of BrdU-positive tubular or interstitial cells in CO, OSOM, and ISOM. Each bar represents the mean ± SEM of four or five animals. *, P < 0.05 versus before induction of ARF; #, P < 0.001 versus before induction of ARF; +, P < 0.05; ++, P < 0.001; N, no significant difference.

Figure 8.

Serial changes in the distribution of cells expressing vimentin (black color) before induction of ARF (A), day 2 (B, H, and I), day 3 (C), day 4 (D), day 5 (E), day 7 (F), day 9 (G and J). Arrows, border between CO and OSOM; arrowheads, border between OSOM and ISOM. Vimentin-positive spindle-like cells with large oval nuclei (arrowheads) could be seen at day 2 around the border between OSOM and ISOM (H); interstitial vimentin expression (arrows) was increasingly seen at day 2 (I); and vimentin expression (arrow) was also found in hyperproliferative PT (J). Original magnifications, ×100 (A–G); ×700 (H–J).

Figure 9.

Location of PHA-E binding sites in normal rats (A) and in experimental rats at days 5 (B) and 7 (C). Staining of brush-border was quite strong (arrows) in PTs of normal rats (A), but totally negative in regenerating PTs at day 5 (B). A faint staining could be seen on the reappeared brush-border (arrowheads) at day 7 (C). Original magnifications, ×750 (A–C).

Figure 10.

Serial changes in the distribution of cells expressing α-SMA (black color) before induction of ARF (A), day 2 (B), day 3 (C), day 5 (D), day 7 (E and G), day 9 (F), day 15 (H), and day 21 (I). G: Expanded interstitium around hyperplastic tubules showed α-SMA-positive staining (arrowheads). H: At day 15, peritubular α-SMA staining became faint (arrows), however, prominent positive stainings were noted in the periglomerular region (arrowheads). I: At day 21 α-SMA-positive cells were seen only around the very dilated tubules (arrows) and in small areas of focally expanded interstitium (arrowhead). Original magnifications, ×100 (A–F); ×300 (G–I).

Figure 11.

Expression of α-SMA (A), vimentin (B), and ED 1-positive monocytes/macrophages (C) in OSOM in consecutive sections at day 5. α-SMA staining was seen only in peritubular interstitial cells (myofibroblasts, thick arrows) (A). In contrast, vimentin staining was found in both regenerating tubules (arrowheads) and peritubular interstitial cells (thick arrows) (B). ED 1-positive monocytes/macrophages (thin arrows) could be seen in peritubular regions, some located in close proximity to regenerating tubules (C). 1 and 2, identical PTs. Original magnifications, ×500 (A–C).

Figure 12.

Immunoelectron micrographs for α -SMA. A: Interstitial cell processes labeled by gold particles (arrowheads), indicating α-SMA attached firmly to the denuded TBM (arrows) at day 5. B: Enlarged micrograph of A. M, Myofibroblast; P, necrotic PT cell. Scale bars: A, 2.0 μm; B, 0.5 μm.

Figure 13.

Double-immunostaining of α-SMA (pink color) and BrdU (brown color) in OSOM at day 5. A proliferating (α-SMA-negative/BrdU-positive) interstitial cell is indicated by the arrow in A and at higher magnification in B, whereas nonproliferative (α-SMA-positive/BrdU-negative) myofibroblast is indicated by the arrowhead in A and higher magnification in C. Original magnifications, ×350 (A); ×1,500 (B and C).

Figure 14.

Morphometric analysis of α-SMA fractional area in CO, OSOM, and ISOM. α-SMA fractional area significantly increased from day 4 in all three layers, and reached peak values at days 7, 7, or 9, respectively. Each bar represents the mean ± SEM of four or five animals. *, P < 0.05 versus before induction of ARF; #, P < 0.001 versus before induction of ARF; +, P < 0.05; ++, P < 0.001; N, no significant difference.

Figure 15.

Morphometric analysis of the number of ED 1-positive interstitial monocytes/macrophages in CO, OSOM, and ISOM. Each bar represents the mean ± SEM of four or five animals. *, P < 0.05 versus before induction of ARF; #, P < 0.001 versus before induction of ARF; +, P < 0.05; ++, P < 0.001; N, no significant difference.