Beads were washed three times with solution A (in mM: 50 Tris?HCl, pH 7.4, 100 NaCl, 5 EDTA), two times with solution B (in mM: 50 Tris?HCl, pH 7.4, 500 NaCl), and once with solution selleckchem MG132 C (50 mM Tris?HCl, pH 7.4), each time with recovery by centrifugation at 1,000 g for 1 min. Beads were then heated to 95��C in 2.5�� loading buffer, and the supernatant was subjected to SDS-PAGE and immunoblotting as above. Apical Na+/H+ exchange activity in OKP cells. Intracellular pH (pHi) was monitored with a computer-controlled spectrofluorometer (��excitation: 500 and 450 nm, ��emission: 530 nm) in cells grown on glass coverslips and loaded with the intracellularly trapped pH-sensitive dye BCECF as described previously (7, 32). Calibration of the 500-to-450 nm fluorescence ratio to pHi was performed with the K+/nigericin method.

Apical Na+/H+ exchange activity was estimated from the initial rate of the Na+-dependent pHi increase after nigericin-induced acid load, in the absence of CO2/HCO3?. Intracellular buffer capacity (��) measured with the NH4Cl pulse method was not significantly different between the different incubation conditions (not shown). Statistics. Statistical analysis was performed with Student’s t-test and ANOVA as specified in Figs. 1�C5. Results are presented graphically as means and SD. For animal experiments, n = 8 per condition unless otherwise noted. Fig. 1. Thiazolidinedione (TZD) treatment reduces plasma and renal lipid abnormalities in Zucker diabetic fatty (ZDF) rats. Eight-week-old ZDF rats and lean littermates were treated with TZD for 4 wk.

Plasma free fatty acids (FFA, A) and renal cortical triglyceride … Fig. 5. Effect of rosiglitazone on NHE3 activity in OKP cells. Apical Na+/H+ exchange activity was measured in confluent quiescent OKP cells incubated with vehicle (albumin) or 0.75 mM FFA for 24 h, with addition of rosiglitazone at the indicated concentrations. … RESULTS Effect of TZD on plasma and renal lipid abnormalities in ZDF rats. Compared with their lean littermates, ZDF rats have higher levels of FFA in the plasma and accumulate excess triglycerides in the renal cortex (6). Treatment of ZDF rats with PPAR�� agonists has been shown to reduce plasma FFA (13, 52) and lipid accumulation in the heart, skeletal muscle, and liver (22, 52), but the effect on kidney triglyceride accumulation has not been studied.

ZDF rats treated with TZD for 4 wk gained more weight than untreated ZDF rats, but their average decapsulated kidney weight was not different (Table 1). Treatment with TZD significantly decreased plasma FFA levels and renal cortical triglyceride content in ZDF rats, while having no detectable effect in their lean littermates (Fig. 1). Table 1. Plasma and urinary parameters in ZDF rats with or without TZD treatment Effect Cilengitide of TZD on urinary acidification parameters in ZDF rats.