Cloning and expression of rat caspase-6 and its localization in renal ischemia/reperfusion injury

Amar B. Singh, Varsha Kaushal, Judit K. Megyesi, Sudhir V. Shah, Gur P. Kaushal

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Background. Caspase-6 is an important member of the executioner caspases in the caspase family of cell death proteases. The executioner caspases are the major active caspases detected in apoptotic cells and are generally considered to mediate the execution of apoptosis by cleaving and inactivating intracellular proteins. However, the complete characterization of mRNA and protein of caspase-6 in rat and its expression in normal kidney and in disease state has not been previously elucidated. Methods. A rat kidney cortex λgt10 cDNA library was screened to isolate the full-length caspase-6 cDNA. The recombinant caspase-6 protein was characterized by expression in bacteria and by transient transfection in mammalian cells. The expression in various tissues was analyzed by Northern blot, and localization in normal and ischemic kidney was performed by immunohistochemistry. Results. The predicted amino acid sequence of rat caspase-6 contains 277 amino acids, with two potential glycosylation sites, an integrin binding site (KGD), the caspase active site pentapeptide QACRG and the caspase family signature, HX2-4(S,C) X4(L,I,V,M,F)2(S,T)HG (HVDADCFVCVFLSHG). Rat caspase-6 is unique among known caspases by possessing a relatively long 5′ untranslational region. Among various tissues tested, caspase-6 was expressed in varying levels in kidney, liver, spleen, heart, muscle, testis, and lung. Bacterial expression of recombinant rat caspase-6 resulted in production of both of the proform and active form of the enzyme suggesting autoactivation. Transient overexpression of rat caspase-6 in COS-1 cells induced DNA fragmentation, a hallmark of apoptosis. We also examined the localization and expression of caspase-6 by immunohistochemistry in kidneys subjected to 40 minutes of ischemia followed by 24 hours of reperfusion injury. Normal kidney showed mostly cytoplasmic and some nuclear staining of the tubules. Kidneys 24 hours after 40 minutes of ischemia showed more intense and diffused cytoplasmic staining with prominent nuclear staining, indicating increased expression and translocation from the cytoplasm to the nuclei. The staining in glomeruli was negative in both normal and ischemic kidney. Conclusions. These studies demonstrate cloning, expression and characterization of the full-length rat caspase-6 and its localization in normal kidneys and kidneys subjected to ischemia/reperfusion injury. Since caspase-6 is involved in the degradation of nuclear matrix proteins and in activation of caspase-3, it may play an important role during renal ischemic injury.

Original languageEnglish (US)
Pages (from-to)106-115
Number of pages10
JournalKidney International
Volume62
Issue number1
DOIs
StatePublished - Jan 1 2002

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Caspase 6
Reperfusion Injury
Organism Cloning
Kidney
Caspases
Staining and Labeling
Ischemia
Immunohistochemistry
Nuclear Matrix-Associated Proteins
Apoptosis
Kidney Cortex
COS Cells
Kidney Diseases
DNA Fragmentation
Gene Library
Glycosylation
Integrins
Caspase 3
Northern Blotting
Transfection

Keywords

  • Acute renal failure
  • Apoptosis mediators
  • Cell death
  • Executioner caspases
  • Rat kidney
  • Renal injury

ASJC Scopus subject areas

  • Nephrology

Cite this

Cloning and expression of rat caspase-6 and its localization in renal ischemia/reperfusion injury. / Singh, Amar B.; Kaushal, Varsha; Megyesi, Judit K.; Shah, Sudhir V.; Kaushal, Gur P.

In: Kidney International, Vol. 62, No. 1, 01.01.2002, p. 106-115.

Research output: Contribution to journalArticle

Singh, Amar B. ; Kaushal, Varsha ; Megyesi, Judit K. ; Shah, Sudhir V. ; Kaushal, Gur P. / Cloning and expression of rat caspase-6 and its localization in renal ischemia/reperfusion injury. In: Kidney International. 2002 ; Vol. 62, No. 1. pp. 106-115.
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AU - Shah, Sudhir V.

AU - Kaushal, Gur P.

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N2 - Background. Caspase-6 is an important member of the executioner caspases in the caspase family of cell death proteases. The executioner caspases are the major active caspases detected in apoptotic cells and are generally considered to mediate the execution of apoptosis by cleaving and inactivating intracellular proteins. However, the complete characterization of mRNA and protein of caspase-6 in rat and its expression in normal kidney and in disease state has not been previously elucidated. Methods. A rat kidney cortex λgt10 cDNA library was screened to isolate the full-length caspase-6 cDNA. The recombinant caspase-6 protein was characterized by expression in bacteria and by transient transfection in mammalian cells. The expression in various tissues was analyzed by Northern blot, and localization in normal and ischemic kidney was performed by immunohistochemistry. Results. The predicted amino acid sequence of rat caspase-6 contains 277 amino acids, with two potential glycosylation sites, an integrin binding site (KGD), the caspase active site pentapeptide QACRG and the caspase family signature, HX2-4(S,C) X4(L,I,V,M,F)2(S,T)HG (HVDADCFVCVFLSHG). Rat caspase-6 is unique among known caspases by possessing a relatively long 5′ untranslational region. Among various tissues tested, caspase-6 was expressed in varying levels in kidney, liver, spleen, heart, muscle, testis, and lung. Bacterial expression of recombinant rat caspase-6 resulted in production of both of the proform and active form of the enzyme suggesting autoactivation. Transient overexpression of rat caspase-6 in COS-1 cells induced DNA fragmentation, a hallmark of apoptosis. We also examined the localization and expression of caspase-6 by immunohistochemistry in kidneys subjected to 40 minutes of ischemia followed by 24 hours of reperfusion injury. Normal kidney showed mostly cytoplasmic and some nuclear staining of the tubules. Kidneys 24 hours after 40 minutes of ischemia showed more intense and diffused cytoplasmic staining with prominent nuclear staining, indicating increased expression and translocation from the cytoplasm to the nuclei. The staining in glomeruli was negative in both normal and ischemic kidney. Conclusions. These studies demonstrate cloning, expression and characterization of the full-length rat caspase-6 and its localization in normal kidneys and kidneys subjected to ischemia/reperfusion injury. Since caspase-6 is involved in the degradation of nuclear matrix proteins and in activation of caspase-3, it may play an important role during renal ischemic injury.

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KW - Acute renal failure

KW - Apoptosis mediators

KW - Cell death

KW - Executioner caspases

KW - Rat kidney

KW - Renal injury

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