Mycophenolate mofetil inhibits regenerative repair in uranyl acetate-induced acute renal failure by reduced interstitial cellular response

Abstract

We recently reported that transient appearance of interstitial myofibroblasts and infiltrating macrophages might play a role in cellular recovery in uranyl acetate (UA)-induced acute renal failure (ARF). Here we tested the effects of mycophenolate mofetil (MMF), which attenuates infiltration of lymphocytes, macrophages, and myofibroblasts, but does not suppress epithelial regeneration, on renal tissue repair. Rats treated with MMF (20 mg/kg/day) or vehicle were sacrificed at 2, 5, and 7 days after induction of ARF by injection of 5 mg/kg UA. Renal tissues were immunostained for bromodeoxyuridine (BrdU) and Ki67, alpha-smooth muscle actin (alpha-SMA), ED1, and CD43. The expression levels of alpha-SMA mRNA were examined by reverse transcription-polymerase chain reaction. Body weight loss or serum albumin levels were similar in MMF and vehicle rats during the experiment. In vehicle group, serum creatinine (Scr) significantly increased after day 5, but proximal tubular (PT) damage score increased as early as day 2 after UA injection. BrdU- or Ki67-positive regenerating tubular cells, ED1-positive macrophages and alpha-SMA-positive myofibroblasts significantly increased in the interstitium after day 5. In MMF-treated rats, Scr and PT damage score significantly increased at day 7 and the number of regenerating PT were significantly reduced compared with vehicle-treated rats at days 5 and 7. The numbers of macrophages and myofibroblasts and the expression of alpha-SMA mRNA were significantly lower in MMF than in vehicle rats at day 5, indicating that reduced interstitial cellular response is linked to the inhibition of regenerative repair. CD43-positive lymphocytes were significantly reduced in MMF group than in vehicle group at day 7, suggesting that lymphocyte infiltration does not seem to contribute to early regenerative response of proximal tubules. The transient appearance of myofibroblasts and macrophages in the interstitium may promote regenerative repair in UA-induced ARF in rats.

Figure 1.

Body weight loss (A) and serum albumin (B) in MMF- and vehicle-treated rats. Data represent the mean ± SEM values of six animals. *, P < 0.05 versus normal control (con); **, P < 0.001 versus normal control; N, no significant difference.

Figure 2.

Scr in MMF- and vehicle-treated rats. Data represent the mean ± SEM values of six rats. **, P < 0.001 versus normal control (con); N, no significant difference; ++, P < 0.001.

Figure 3.

Photomicrographs of periodic acid-Schiff-stained sections in the OSOM in vehicle-treated (A, C) and MMF-treated (B, D) rats at days 5 (A, B) and 7 (C, D) after induction of ARF. Damaged proximal tubules with bare TBM (asterisks) were almost maximally distributed in OSOM at day 5 in both groups. At day 7, damaged proximal tubules with bare TBM were still predominant in MMF-treated rats, whereas regenerating cells (arrows) covered most of the TBM and hyperproliferative proximal tubules (arrowhead) could be found in vehicle-treated rats. Original magnifications, ×300.

Figure 4.

The PT damage score in OSOM. Data represent the mean ± SEM values of six rats. **, P < 0.001 versus normal control (con); N, no significant difference; ++, P < 0.001.

Figure 5.

Photomicrographs of immunostaining for BrdU (A, B) and Ki67 (C–F) in OSOM in vehicle-treated (A, C, and E) and MMF-treated (B, D, and F) rats at days 5 (A–D) and 7 (E, F) after induction of ARF. Arrows, immunoreactive tubular cells; arrowheads, immunoreactive interstitial cells. Original magnifications, ×320.

Figure 6.

A: The number of BrdU-positive cells in proximal tubules (PT) per field in the OSOM. B: The number of Ki67-positive cells in proximal tubules (PT) or interstitium per field in the OSOM. Data represents the mean ± SEM values of six rats. *, P < 0.05 versus normal control (con); **, P < 0.001 versus normal control; N, no significant difference; ++, P < 0.001.

Figure 7.

Photomicrographs of immunostaining for α-SMA (A, B), ED1 (C, D), and CD43 (E, F) in the OSOM in vehicle-treated (A, C, and E) and MMF-treated (B, D, and F) rats at days 5 (A–D) and 7 (E, F). Arrows, α-SMA-positive myofibroblasts (A, B), ED1-positive macrophages (C, D), or CD43-positive lymphocytes (E, F). Original magnifications, ×300.

Figure 8.

A: Morphometric analysis of fractional area of α-SMA in OSOM in MMF-treated and vehicle-treated rats. B: Ratio of α-SMA to GAPDH mRNA in the kidney in MMF-treated and vehicle-treated rats. Data represent the mean ± SEM values of six rats. *, P < 0.05 versus normal control (con); **, P < 0.001 versus normal control; N, no significant difference; ++, P < 0.001.

Figure 9.

Correlation of percent fractional area of α-SMA in OSOM to Scr and score of PT damage in vehicle and MMF groups. A: Percent fractional area of α-SMA versus Scr; r = 0.81 in vehicle group, P < 0.01; r = 0.80 in MMF group, P < 0.01. B: Percent fractional area of α-SMA versus score of PT damage; r = 0.73 in vehicle group, P < 0.01; r = 0.65 in MMF group, P < 0.05.

Figure 10.

Morphometric analysis of the number of interstitial ED1-positive monocytes/macrophages (A) and CD43-positive lymphocytes (B) in OSOM in MMF-treated and vehicle-treated rats. Data represent the mean ± SEM values of six rats. **, P < 0.001 versus normal control (con); N, no significant difference; +, P < 0.05; ++, P < 0.001.