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. PD150606 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 Ca(2+)-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.
Edelstein
C L
CL
Department of Medicine, University of Colorado School of Medicine, Denver, USA.
Yaqoob
M M
MM
Alkhunaizi
A M
AM
Gengaro
P E
PE
Nemenoff
R A
RA
Wang
K K
KK
Schrier
R W
RW
D000179:Acrylates / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D002118:Calcium / Q000502:physiology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002403:Cathepsins / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D000860:Hypoxia / Q000378:metabolism*; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
D000179:Acrylates; C100442:PD 150606; D007770:L-Lactate Dehydrogenase; D002403:Cathepsins; D002154:Calpain; D002118:Calcium