MODULATION OF HYPOXIA-INDUCED CALPAIN ACTIVITY IN RAT RENAL PROXIMAL TUBULES

The effect of the newly developed, nonpeptide, calpain inhibitor, PD 1 50606, on hypoxia and ionomycin-induced increases in calpain activity in rat proximal tubules (PT) was determined. PD150606 inhibited both h ypoxia and ionomycin-induced calpain activity as determinrd by the flu orescent substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl coumari n (N-succinyl-Leu-Leu-Val-Tyr-AMC). This decrease in calpain activity was accompanied by dose-dependent cytoprotection against hypoxia and i onomycin-induced cell membrane damage. PD150606 had no effect on cathe psin B and L activity in PT as measured by the fluorescent substrate, -phenylalanyl-L-arginine-7-amido-4-methyl-coumarin (Z-Phe-Arg-AMC). Th e effects of low intracellular pH (pH(i)) or low free cytosolic calciu m [Ca2+](i) on this hypoxia-induced calpain activity were also determi ned. Both low pH(i) and low [Ca2+](i) attenuated the hypoxia-induced i ncrease in calpain activity. This attenuation of calpain activity was observed early before hypoxia-induced membrane damage and was associat ed with marked reduction in the typical pattern of hypoxia-induced cel l membrane damage observed in this model. To identify the isoform of c alpain activated in rat proximal tubules, normoxic. hypoxic and ionomy cin treated tubules were fractionated by MONO-Q anion exchange chromat ography and the fractions were assayed for calpain activity. A single peak of calpain activity characteristic of mu-calpain was found. The c alcium dependency of the calpain activity was in the nanomolar range, further confirming that the activity was the low Ca2+-sensitive mu-cal pain. The present study suggest that in rat proximal tubules: (1) PD 1 50606 is a specific inhibitor of calpain and not cathepsins B and L; ( 2) the cytoprotective effects of low pH(i) and low [Ca2+](i) are media ted, at least in part, by inhibition of calpain activity; and (3) the predominant active form of calpain is the isoenzyme mu-calpain.