CDK inhibitor p21 is prosurvival in adriamycin-induced podocyte injury, in vitro and in vivo

Abstract

In response to injury, the highly specialized and terminally differentiated glomerular visceral epithelial cell, or podocyte, may undergo several cell fates, including dedifferentiation and proliferation, persistent cell cycle arrest, hypertrophy, apoptosis, or necrosis. Common to these potential outcomes of injury is their ultimate regulation at the level of the cell cycle. There is now a large body of literature confirming the importance of cell cycle regulatory proteins in the cellular response to injury. Although CDK inhibitor p21 levels increase in podocytes following injury, the role of p21 is unclear in focal segmental glomerulosclerosis (FSGS), in part because its function depends heavily on the cytotoxic stimulus and the cellular context. Adriamycin (ADR) is a podocyte toxin used to induce experimental FSGS. The purpose of this study was to define the role of p21 in ADR-induced podocyte injury. BALB/c mice, a strain carrying the recessive ADR susceptibility gene, were backcrossed against c57B6 p21-/- mice to yield a 12th generation BALB/c p21-/- strain. Experimental FSGS was induced by injection of ADR 12 mg/kg × 2 doses (n = 8/group), with mice killed at 1, 2, 8, and 11 wk. Diseased p21-/- mice demonstrated worse albuminuria, more widespread glomerulosclerosis, and higher blood urea nitrogen compared with diseased p21+/+ mice. In diseased p21-/- mice vs. p21+/+ mice, apoptosis [measured by TdT-mediated dUTP nick end labeling (TUNEL) assay] was increased, and podocyte number (measured by WT-1 immunostaining) was decreased. To validate these findings in vitro, we utilized differentiated mouse podocytes, p21-/- and p21+/+, exposed to 0.125 μg/ml ADR. Apoptosis, measured by Hoechst 33342 staining and TUNEL assay, was greater in cultured p21-/- podocytes compared with p21+/+ podocytes. Reconstitution of p21 via retroviral transfection rescued the p21-/- podocytes from apoptosis. We conclude that p21 is prosurvival in the podocyte's response to ADR-induced injury. Ongoing studies are defining the mechanisms of this protective effect as it relates to DNA damage and apoptosis.

Valproic Acid Attenuates Proteinuria and Kidney Injury

“…Furthermore, we confirm that ADR-induced damaged DNA leads to apoptosis, as was recently described by Marshall et al 31 HDAC inhibition by VPA in the ADR nephropathy model results in less glomerular apoptosis. Less apoptosis was also shown for both VPA and SAHA (Suberoylanilide Hydroxamic Acid) in the hemorrhagic shock model.…”

“…31,39,40 HDAC inhibitors are found to inhibit EMT of renal proximal tubular epithelial cells. 12,13,15,18 -21 Specifically, VPA is found to prevent EMT in NRK-52E cells, along with the selective class I HDAC inhibitor SK-7041, which indicates that class I HDACs play a pivotal role in EMT.…”

“…Cell depletion by apoptosis is a typical phenomenon in renal disorders, as is seen in the ADR nephropathy model by Marshall et al 31 The TUNEL assay was performed to determine whether VPA could modulate apoptosis in the ADR model ( Figure 6A and Supplemental Figure S1). The number of TUNEL-positive cells in 100 glomeruli in the ADR-10d group was higher than those at the end of the experiment.…”

Role of smooth muscle protein SM22α in glomerular epithelial cell injury

“…The adriamycin (ADR) nephropathy model, proven to be a robust experimental analog of human FSGS, was chosen to examine more closely the expression of SM22␣ in the initiation and propagation of FSGS in mice. ADR nephropathy was induced in male BALB/c mice, a strain carrying the recessive ADR susceptibility gene, aged 12 wk, by tail vein injection of ADR (12 mg/kg body wt ϫ 2 doses; n ϭ 8/group) as previously described (35). Control animals were injected with an equal volume of vehicle only (0.9% NaCl).…”

“…6). To assess semiquantitatively the severity of glomerular injury, a glomerular disease severity score (GDS score) was calculated by examining histopathological sections stained with periodic acid-Schiff, as a modification of what has been described previously (35), Fig. 5.…”