Kidney androgen-regulated protein transgenic mice show hypertension and renal alterations mediated by oxidative stress.

BACKGROUND: Kidney androgen-regulated protein (KAP), a proximal tubule androgen-regulated gene, codes for a protein of unknown function. METHODS AND RESULTS: To investigate the consequences of KAP overexpression in kidney, we produced KAP transgenic mice and performed microarray expression analyses in kidneys of control and transgenic males. Downregulation of the androgen-sensitive Cyp4A14 monooxygenase gene in KAP transgenic mice prompted us to analyze blood pressure levels, and we observed that transgenic mice were hypertensive. Inhibition of 20-hydroxyeicosatetraenoic acid synthesis by N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) reduced the increased 20-hydroxyeicosatetraenoic acid levels in urine and normalized arterial pressure in transgenic mice, as did the NADPH oxidase inhibitor apocynin. Increased oxidative stress in transgenic mice was demonstrated by (1) enhanced excretion of urinary markers of oxidative stress, 8-iso-prostaglandin F2alpha, 8-hydroxydeoxyguanosine, and thiobarbituric acid-reacting substances; (2) augmented mitochondrial DNA damage and malondialdehyde levels in kidneys; and (3) diminished catalase and glutathione peroxidase activity in transgenic kidneys. Mice exhibited renal defects that included focal segmental glomerulosclerosis, proteinuria, glycosuria, and fibrosis. CONCLUSIONS: Taken together, these results indicate that KAP expression is critical for cardiovascular-renal homeostasis maintenance and that hypertension is associated with increased oxidative stress. This is the first report showing that overexpression of an androgen-regulated, proximal tubule-specific gene induces hypertension. These observations may shed light on the molecular pathophysiology of gender differences in the prevalence and severity of hypertension and chronic renal disease.

Phosphoinositide 3-kinase inhibitors protect mouse kidney cells from cyclosporine-induced cell death.

The use of cyclosporine has been restricted by its nephrotoxic effects mediated, in part, by reactive oxygen species (ROS). Phosphoinositide 3-kinase, protein kinase B, and extracellular regulated kinase (ERK) pathways are related to survival and cell death and are activated after ROS generation. In this study, we evaluated the effects of cyclosporine on these pathways and their contribution to cyclosporine-induced toxicity. Viability of cells derived from the proximal tubule of transgenic mice was measured with Trypan Blue, ROS generation by a fluorescent probe, while ERK and phosphoinositide 3-kinase/protein kinase B activation were monitored with phospho-specific antibodies. Cyclosporine decreased cell viability and induced ROS generation and ERK and phosphoinositide 3-kinase activation. Both pathways were activated by the epidermal growth factor receptor (EGFR). Antioxidants blocked ERK activation but failed to inhibit protein kinase B phosphorylation or prevent cyclosporine toxicity. ERK inhibition did not protect from cyclosporine-induced cell death. EGFR or phosphoinositide 3-kinase inhibitors protected from cyclosporine-triggered cell death without decreasing ROS. Small interfering RNA against the catalytic subunit of phosphoinositide 3-kinase decreased protein kinase B phosphorylation but did not prevent cyclosporine-mediated cell death. Our results show that EGFR mediates the cytotoxic effects of cyclosporine through an ROS-independent mechanism. Cyclosporine-induced cell death is triggered by a non-classical phosphoinositide 3-kinase and does not require ERK activation.

The characterization and hormonal regulation of kidney androgen-regulated protein (Kap)-luciferase transgenic mice.

The androgen-dependent regulation for the gene encoding the kidney androgen regulated protein (Kap) was examined in transgenic mice expressing luciferase (luc) under the control of the murine Kap promoter. Biophotonic imaging was used to visualize luciferase expression from the kidneys and various organs that was confirmed using luminometer assays. Kap-luc expression was observed at high levels in kidneys, epididymides, testes, and seminal vesicles in male mice, and in kidneys, ovaries, and uterus in female mice. Kap-luc expression was modulated by androgen and anti-androgen treatment in both male and female mice. Male mice were treated daily with the anti-androgenic compounds, cyproterone acetate (50 and 100 mg/kg/day) and flutamide (50 and 100 mg/kg/day), or vehicle for 16 days. Endpoints evaluated included in vivo biophotonic imaging, body weights, organ weights (liver, kidney, testes, epididymides, preputial gland, and seminal vesicles), protein luciferase assays and Western blot analysis. Biophotonic imaging was used to follow Kap-luc expression from each animal throughout the experiment using a sensitive imaging system. These imaging results correlated well with Western blot analysis and traditional endpoints of body and organ weights. Following treatment with anti-androgens, the luciferase signal was found to significantly decrease in the intact male mouse using in vivo biophotonic imaging and correlated with measurements of luciferase activity in homogenized organ extracts. The decrease in epididymal and seminal vesicle weight confirmed the action of the anti-androgens. In vivo imaging documented significant changes in luciferase expression within the first few days of the experiment indicative of the anti-androgenic activity of the drugs. Testosterone treatment significantly increased the Kap-luc bioluminescent signal in female mice. This increased luciferase induction was shown to be inhibited by coadministration of cyproterone (100 mg/kg/day). Our results indicate that biophotonic imaging may provide a useful approach for noninvasively tracking the effects of endocrine disruptors in specific tissues.