Fig. 6.

Endogenous Kif3b expression and albumin endocytosis in mouse proximal renal tubular primary cell cultures (mPTCs). A: endogenous expression of Kif3b in mPTC cultures from Clcn5^Y/+ and Clcn5^Y/− mice. The expression of Kif3b was determined by qPCR and was found to be significantly higher in the Clcn5^Y/− mice and consistent with the observations from mouse whole kidney extracts (Fig. 5A). B: expression of human KIF3B (hKIF3B) in transfected mPTCs from Clcn5^Y/+ (n = 3) and Clcn5^Y/− (n = 3) mice was assessed by qPCR and this showed an ∼3,000-fold higher expression of the exogenous human KIF3B, compared with the endogenous expression of Kif3b [Kif3b(m)] and Kif3a. C: uptake of FITC-albumin in mPTC cultures from Clcn5^Y/+ and Clcn5^Y/− mice in mock-transfected cells, in the absence or presence of competition by excess unlabeled albumin and transferrin, or after energy depletion by deoxyglucose (DOG) and sodium azide (NaN₃), or in KIF3B-transfected cells. In Clcn5^Y/+ mPTCs, overexpression of hKIF3B decreases specific albumin endocytic uptake. In Clcn5^Y/− mPTCs, albumin uptake is fully abrogated (to the level of metabolic inhibition and albumin competition) by hKIF3B overexpression. D: RT-PCR analyses of caveolin-1 (Cav-1) and caveolin-2 (Cav-2) in HEK293 cells, OK cells, Clcn5^Y/+ and Clcn5^Y/− whole mouse kidneys, and Clcn5^Y/+ and Clcn5^Y/− mPTCs. GAPDH and a water blank were used as positive and negative controls, respectively. Means ± 1 SE and P values were calculated by the Student's unpaired, 2-tailed t-test.