No abstract

The degree of albuminuria is a well-known adverse prognostic indicator in human glomerular diseases. However, the mechanisms by which albuminuria by itself contributes to tubulointerstitial injury and progression of renal disease remain unclear. We tested the hypothesis that apoptosis may represent one of the mechanisms by which tubule epithelial cells are damaged after albumin overload in vitro. Cultured LLC-PK(1) proximal tubule cells were incubated with varying concentrations of BSA. This resulted in a dose- and duration-dependent induction of apoptosis, as evidenced by internucleosomal DNA cleavage (DNA laddering and nick-end labeling), externalization of plasma membrane phosphatidylserine (annexin labeling), and characteristic morphological changes (cell shrinkage and nuclear condensation). Albumin overload also resulted in a dose-dependent upregulation of Fas and Fas-associated protein with death domain (FADD), and activation of caspase 8. Incubation with the caspase 8 inhibitor IETD ameliorated the albumin-induced apoptosis. Collectively, our results indicate that albumin overload induces apoptosis of cultured LLC-PK(1) cells, mediated at least in part by the Fas-FADD-caspase 8 pathway.

Nephrotic-range proteinuria is considered a poor prognostic factor. A correlation between tubulointerstitial injury and the degree of proteinuria is well established. In an attempt to explain the tubular atrophy that is observed in advanced glomerulonephritides, this study investigated apoptotic mechanisms in cultured human proximal tubule cells (HKC-8) that were exposed to endotoxin-free albumin (5, 10, and 20 mg/ml). Apoptosis was detected by Hoechst 33342; annexin staining; and assays for caspases 3, 8, and 9. The apoptotic effect of albumin was maximal at 10 mg/ml albumin, and necrosis prevailed in cells that were incubated with 20 mg/ml. Increase in caspase-9 and -3 activity was observed starting at 6 and maximally at 16 to 24 h. The proapoptotic Bcl-2 protein Bax was upregulated at 6 h, associated with translocation of cytochrome-c from mitochondria to cytosol and alteration in the mitochondrial membrane potential. Production of reactive oxygen species (ROS) was significant at 6 h but declined at 16 and 24 h. Treatment with ROS scavenger dimethylthiourea or antioxidant N-acetylcysteine did not alleviate caspase-3 production. Pan protein kinase C inhibitor bisindolylmaleimide-1 protected the cells from apoptosis. It is concluded that albumin induces apoptosis in human proximal tubule cells by stimulating mitochondrial apoptotic pathway independent of ROS production.

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