Analgesic nephropathy : mechanistic studies using human renal medullary interstitial cells
The aim of this research was to develop a culture of human renal medullary interstitial cells to study the molecular mechanism underlying toxicity of analgesics and NSAIDs in vitro. The method described in this thesis, was developed based on the culture of primary human renal medullary explants, selective detachment of interstitial cells and selective overgrowth of these cells in a serum-rich medium after dilution cloning. The cultured cell populations exhibited the morphological and functional characteristics of Type 1 medullary interstitial cells as observed in situ. Ultrastructural and immunocytochemical studies revealed the characteristic features of human renal medullary interstitial cells (hRMIC) including numerous lipid droplets, abundant endoplasmic reticulum and mitochondria and fine filaments underlying the cell membrane. The production of extracellular matrix substance was also demonstrated. Prostaglandin E2, synthesis was demonstrated in cultures of hRMIC and prostaglandin synthase-1 was localised by immunohistochemical studies to renal medullary interstitial cells both in situ and in culture. The synthesis of 5-HETE, the product of 5-lipoxygenase, was also demonstrated in cultured cells but not in situ. 2-Bromoethanamine, a haloalkyl compound used to induce renal papillary necrosis in experimental animals, exhibited an early onset of cytotoxicity leading to complete cell death within 24 hours in both a time and dose-dependent manner in hRMIC. Comparison of the ED50 value demonstrated that the PGHS containing 3T3 cells were most sensitive to the cytotoxic effect of BEA, followed by hRMIC and LLC-PK1 cells which lack eicosanoid synthesising enzymes. In vitro cytotoxicity studies with a range of analgesics demonstrated minimum toxicity following short-term exposure. However, prolonged treatment for up to 7 days augmented the cytotoxic potential of all analgesics. The rank order of toxicity observed in hRMIC was phenylbutazone>mefenamic acid>aspirin>paracetamol.