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The role of cysteine proteases in hypoxia-induced renal proximal tubular injury
Thesis (PhD)--Stellenbosch University, 1998.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2012
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| _version_ | 1867614136385404928 |
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| access_status_str | Open Access |
| author | Edelstein, Charles Louis |
| author2 | Taljaard, J. J. F. |
| author_browse | Edelstein, Charles Louis Taljaard, J. J. F. |
| author_facet | Taljaard, J. J. F. Edelstein, Charles Louis |
| author_sort | Edelstein, Charles Louis |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 1998. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/56026 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:47:14.760Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2012 |
| publishDateRange | 2012 |
| publishDateSort | 2012 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/56026 The role of cysteine proteases in hypoxia-induced renal proximal tubular injury Edelstein, Charles Louis Taljaard, J. J. F. Joubert, J. R. Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of General Internal Medicine. Calpain Cysteine proteinases Kidney tubules -- Diseases Dissertations -- Medicine Thesis (PhD)--Stellenbosch University, 1998. It has been previously found in suspensions of proximal tubules (PT) that hypoxia is associated with a significant rise in cytosolic free calcium [Ca²⁺], which anteceded evidence of membrane damage as measured by propidium iodide uptake. Thus, the role of the Ca²⁺-dependent cysteine protease, calpain in the pathophysiology of PT injury and dysfunction was investigated. An assay for calpain activity in suspensions of PT using fluorescent substrates was developed. The increase in [Ca²⁺] was found to activate calpain, an effect which preceded cell membrane damage and occurred in the presence of the cytoprotective agent, glycine. Inhibition of hypoxic- and the Ca²⁺-ionophore, ionomycin-induced increases in calpain activity with the chemically-dissimilar cysteine protease inhibitors, E-64-d, CBZ and PD150606 elicited cytoprotection against PT cell membrane damage. PD150606 selectively inhibited calpain but not the other cysteine proteases, cathepsins B and L in PT. Both low intracellular pH (pHi) and low [Ca²⁺] 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. Normoxic, hypoxic and ionomycin treated tubules, fractionated by MONO-Q anion exchange chromatography demonstrated a single peak of calpain activity characteristic of the μ-calpain isoform. The calcium dependency of the calpain activity was in the nanomolar range further confirming that the activity was due to the low Ca²⁺ sensitive, μ-calpain. Northern analysis using cDNA probes to μ-calpain and m-calpain indicated the presence of μ-calpain mRNA but not m-calpain mRNA in both normoxic and hypoxic tubules suggesting that μ-calpain is the isoform present in rat PT. As cytoskeletal proteins are known substrates for calpain, we investigated the cytoskeletal targets of calpain during sublethal hypoxia. On immunoblot analysis, the majority of actin and calpain-mediated spectrin breakdown products was in the Triton X-100 insoluble fraction during normoxia and increased in the Triton soluble fraction during hypoxia. In contrast, the majority of NaK-ATPase was in the TS fraction during normoxia and did not change during hypoxia. However, immunoblots of particulate and soluble fractions demonstrated an increase in soluble spectrin breakdown products and NaK-ATPase in the cytosolic fraction during hypoxic injury compared to normoxic controls, suggesting that during hypoxia NaK-ATPase translocated to either the cytoplasm, or a vesicle that was too light to precipitate out in the membrane fraction. Furthermore, the increase in soluble NaK-ATPase was attenuated by the calpain inhibitor, PD150606. In summary, the low Ca²⁺-sensitive isoform, μ-calpain is a mediator of hypoxic and ionomycin-induced PT injury, even in the presence of the cytoprotective agent glycine. The cytoprotective effect of low pHi and low [Ca²⁺] is mediated, at least in part, through inhibition of calpain activity. During hypoxia, there is a calpain-mediated breakdown of spectrin and translocation of NaK-ATPase from the membrane to the cytoplasm. Doctoral 2012-08-27T11:37:21Z 2012-08-27T11:37:21Z 1998 Thesis http://hdl.handle.net/10019.1/56026 en Stellenbosch University application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Calpain Cysteine proteinases Kidney tubules -- Diseases Dissertations -- Medicine Edelstein, Charles Louis The role of cysteine proteases in hypoxia-induced renal proximal tubular injury |
| title | The role of cysteine proteases in hypoxia-induced renal proximal tubular injury |
| title_full | The role of cysteine proteases in hypoxia-induced renal proximal tubular injury |
| title_fullStr | The role of cysteine proteases in hypoxia-induced renal proximal tubular injury |
| title_full_unstemmed | The role of cysteine proteases in hypoxia-induced renal proximal tubular injury |
| title_short | The role of cysteine proteases in hypoxia-induced renal proximal tubular injury |
| title_sort | role of cysteine proteases in hypoxia induced renal proximal tubular injury |
| topic | Calpain Cysteine proteinases Kidney tubules -- Diseases Dissertations -- Medicine |
| url | http://hdl.handle.net/10019.1/56026 |
| work_keys_str_mv | AT edelsteincharleslouis theroleofcysteineproteasesinhypoxiainducedrenalproximaltubularinjury AT edelsteincharleslouis roleofcysteineproteasesinhypoxiainducedrenalproximaltubularinjury |