title: Calpain is activated in experimental uremia: is calpain a mediator of uremia-induced myocardial injury?
abstract: BACKGROUND
  The cysteine proteases calpain and caspase-3 are known mediators of cell death. The aim of this study was to assess their contribution to the tissue damage found in experimental uremia.
  
  
  METHODS
  Calpain and caspase-3 activities were measured in the hearts of rats that were sham-operated (control), sham-operated and spontaneously hypertensive (SHR), and those rendered uremic by 5/6 nephrectomy (uremic). In an in vitro study, heart myoblasts (Girardi) were incubated with human serum from healthy subjects (control serum conditioned media, CSCM) or uremic patients (uremic serum conditioned media, USCM), in the presence and absence of calpain and caspase-3 inhibitors. After 48 hours the activity of calpain and caspase-3 was measured, and cell injury determined by DNA fragmentation (ELISA) and lactate dehydrogenase (LDH) release. An in situ assay was designed to study how USCM affects calpain activity over time.
  
  
  RESULTS
  In the in vivo study, mean calpain activities were almost identical in the control and SHR groups, but calpain and caspase-3 activities were much elevated in the uremic group (P < 0.01 and 0.001 respectively vs. control). The SHR group had significantly higher mean arterial blood pressure (P < 0.001 vs. control, 0.01 vs. uremic). In the in vitro study calpain activity and DNA fragmentation were markedly higher in USCM treated cells compared to CSCM (both P<0.05). Both were reduced in USCM cells containing calpain inhibitors (E64d, calpastatin, or PD 150606). LDH release was raised also in USCM treated cultures (P < 0.05), which only the E64d treatment could significantly reduce (P < 0.02). Caspase-3 activities were similar in USCM and CSCM groups. The in situ assay showed significant increases in calpain activity in USCM treated cells compared to CSCM after just 3.5 hours (P<0.01).
  
  
  CONCLUSIONS
  In vivo results suggest that the increases in calpain and caspase-3 activity in uremic rat hearts were primarily due to uremia and not to hypertension. In vitro data demonstrate that uremia-induced cell injury can be attenuated by calpain inhibition. Therefore, it is likely that calpain is a mediator of uremia-induced myocardial injury.
authors.affiliation: Department of Experimental Medicine and Nephrology, St Bartholomew's, and Royal London School of Medicine and Dentistry, Queen Mary, University of London, London, England, United Kingdom. s.m.harwood@qmul.ac.uk
authors.forename: Steven M
authors.initials: SM
authors.lastname: Harwood
authors.forename: David A
authors.initials: DA
authors.lastname: Allen
authors.forename: Alistair M S
authors.initials: AM
authors.lastname: Chesser
authors.forename: David I
authors.initials: DI
authors.lastname: New
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Raftery
authors.forename: Muhammad M
authors.initials: MM
authors.lastname: Yaqoob
mesh_terms: D000179:Acrylates / Q000494:pharmacology; D000818:Animals; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004195:Disease Models, Animal; D006801:Humans; D017379:Hypertrophy, Left Ventricular / Q000378:metabolism*; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008297:Male; D009392:Nephrectomy; D009842:Oligopeptides / Q000494:pharmacology; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D014511:Uremia / Q000378:metabolism*
chemical_list: D000179:Acrylates; D002135:Calcium-Binding Proteins; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; C100442:PD 150606; C108637:aspartyl-glutamyl-valyl-aspartal; C033668:calpastatin; C505407:CASP3 protein, human; D002154:Calpain; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D007930:Leucine; C108192:aloxistatin