Podocyte hypertrophy precedes apoptosis under experimental diabetic conditions

Abstract

Background: Diabetic nephropathy, the leading cause of end-stage renal disease worldwide, is characterized pathologically by glomerular hypertrophy and clinically by proteinuria. Apoptosis has also been implicated in the pathogenesis of diabetic nephropathy. To date, however, the sequence of hypertrophy and apoptosis especially of podocytes, two hallmarks in diabetic glomeruli, is still a matter of debate. The aim of this study was to elucidate the consequences of inhibiting hypertrophy on apoptosis and of blocking apoptosis on hypertrophy in podocytes under diabetic conditions.

Methods: In vitro, After confirming differentiation of immortalized mouse podocytes and serum restriction for 24 hr, the medium was changed to serum-free RPMI medium containing 5.6 mM glucose (NG), NG+24.4 mM

mannitol (NG+M), or 30 mM glucose (HG) with or without 10 μM PKI 166, an epidermal growth factor receptor (EGFR) inhibitor or 10-7 M zAsp-DCB (N-aspartyl-2,6-dichlorobenzoyloxy-methylketone), a pancaspase inhibitor.

In vivo, sixty-four male Sprague-Dawley rats, weighing 250-280 g, were used. Thirty-two rats were injected intraperitoneally with diluent [control (C)] and the other 32 rats with 65 mg/kg streptozotocin (STZ) [diabetes mellitus (DM)]. After confirming DM, 8 rats each from the C and DM groups were treated with 100 mg/kg/day of PKI 166 by gavage for 3 months. Another 8 rats from each group were treated with zAsp-DCB (2 mg/day) via subcutaneously implanted osmotic minipumps for 3 months. 24-hr urinary albumin excretion was checked at the time of sacrifice.

Western blotting for p27, p21, phospho-4EBP1, phospho-p70S6 kinase, Bax, Bcl-2, active fragments of caspase-3, cleaved poly(ADP-ribose)polymerase (PARP), or β-actin were performed with cell lysates and sieved glomeruli. Podocyte hypertrophy was assessed by measurement of cellular protein/cell number and by flow cytometry. Hoechst 33342 staining and TUNEL assay were performed to identify apoptosis.

Results: Compared to NG cells and C glomeruli, there were significant increases in p27, p21, phospho-4EBP1, and phospho-p70S6K protein expression in cultured podocytes exposed to HG medium and DM glomeruli, respectively, and these increases were significantly abrogated by PKI 166, but not by zAsp-DCB treatment. In addition, both cellular protein/cell number and relative cell size and glomerular volume were significantly greater in podocyte cultured under HG conditions and DM glomeruli, respectively, and these changes were significantly ameliorated by PKI 166 treatment, but not by zAsp-DCB. Meanwhile, the increases in the ratio of Bax/Bcl-2 and the protein expression of active fragments of caspase-3 and cleaved PARP in HG-stimulated podocytes and DM glomeruli were significantly attenuated by

not only zAsp-DCB but also PKI 166 treatment. The increase in apoptotic cells assessed by Hoechst 33342 staining and TUNEL assay in podocytes exposed to HG medium and DM glomeruli, respectively, was also significantly abrogated by PKI 166 as well as zAsp-DCB. Moreover, both EGFR inhibitor and pancaspase inhibitor significantly reduced albuminuria in DM rats.

Conclusion: In conclusion, inhibition of podocyte hypertrophy results in less apoptosis, whereas blocking apoptosis has no effect on podocyte hypertrophy under diabetic conditions, suggesting that podocyte hypertrophy precedes apoptosis. Based on these findings, targeting podocyte hypertrophy may be more effective in the treatment of diabetic nephropathy.