Laboratory Investigation

Kidney International (1996) 50, 1150–1157; doi:10.1038/ki.1996.422

Modulation of hypoxia-induced calpain activity in rat renal proximal tubules

Charles L Edelstein, Muhammad M Yaqoob, Ahmed M Alkhunaizi, Patricia E Gengaro, Raphael A Nemenoff, Kevin K W Wang and Robert W Schrier

Department of Medicine, University of Colorado School of Medicine, Denver, Colorado, and Department of Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, Michigan, USA

Correspondence: Robert W Schrier MD, C281, University of Colorado School of Medicine, 4200 E. 9th Ave, Denver, CO 80262, USA.

Received 3 November 1995; Revised 16 May 1996; Accepted 16 May 1996.

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Abstract

Modulation of hypoxia-induced calpain activity in rat renal proximal tubules. The effect of the newly developed, nonpeptide, calpain inhibitor, PD 150606, on hypoxia and ionomycin-induced increases in calpain activity in rat proximal tubules (PT) was determined. PD 150606 inhibited both hypoxia and ionomycin-induced calpain activity as determined by the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl coumarin (N-succinyl-Leu-Leu-Val-Tyr-AMC). This decrease in calpain activity was accompanied by dose-dependent cytoprotection against hypoxia and ionomycin-induced cell membrane damage. PD150606 had no effect on cathepsin B and L activity in PT as measured by the fluorescent substrate, benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methyl coumarin (Z-Phe-Arg-AMC). The effects of low intracellular pH (pHi) or low free cytosolic calcium [Ca2+]i on this hypoxia-induced calpain activity were also determined. Both low pHi and low [Ca2+]i attenuated the hypoxia-induced increase in calpain activity. This attenuation of calpain activity was observed early before hypoxia-induced membrane damage and was associated with marked reduction in the typical pattern of hypoxia-induced cell membrane damage observed in this model. To identify the isoform of calpain activated in rat proximal tubules, normoxic, hypoxic and ionomycin treated tubules were fractionated by MONO-Q anion exchange chromatography and the fractions were assayed for calpain activity. A single peak of calpain activity characteristic of mu-calpain was found. The calcium dependency of the calpain activity was in the nanomolar range, further confirming that the activity was the low Ca2+-sensitive mu-calpain. The present study suggests that in rat proximal tubules: (1) PD 150606 is a specific inhibitor of calpain and not cathepsins B and L; (2) the cytoprotective effects of low pHi and low [Ca2+]i are mediated, at least in part, by inhibition of calpain activity; and (3) the predominant active form of calpain is the isoenzyme mu-calpain.

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