engleski

Localization of PDZK1 protein in the mammalian kidney and liver

In the epithelial cells of mammalian liver (L) and kidney (K), elimination of various substances is mediated by membrane transporters, which can be regulated by PDZ (PSD95/Discs Large/ZO-1) domain-containing proteins. PDZK1 is a 4-PDZ domain-containing scaffold protein of ~70 kDa, which binds to the C-terminus of numerous membrane transporters. It has been immunochemically localized to the proximal tubule (PT) brush-border membrane (BBM) in the K of various mamammals, and to the hepatocyte sinusoidal membrane in the mouse L. However, due to absence of specific antibodies, the exact localization of this protein in the mammalian organs is still controversial. Here we used 2 polyclonal antibodies (Ab) against the NH2- (NT-Ab) and COOH- (CT-Ab) termini of the human PDZK1, generated by us, and one commercial polyclonal Ab against an internal region of the human PDZK1 (CM-Ab), and studied in rats, mice, and humans: a) cellular localization of PDZK1 protein in tissue cryosections by immunocytochemistry (IC), and b) its abundance in total cell membranes (TCM) from K and L, and in BBM from K by Western blotting (WB). Due to sequence homology of the immunizing peptide, the NT-Ab and CM-Ab reacted with rat, mouse and human, whereas the CT-Ab was human-specific. By IC, the NT-Ab stained strongly, and CM-Ab stained weakly the BBM of PT S1 & S2 in the cortex and S3 in the outer stripe of human, rat, and mouse K. The CT-Ab strongly stained the BBM of S1-S3 in humans. In the L: a) the CM-Ab stained nothing in all 3 species, b) the CT-Ab and NT-Ab stained nothing in rats and mice, whereas in humans, both Abs stained some non-identified, possibly reticuloendothelial cells ; the sinusoidal and canalicular membranes in hepatocytes remained unstained. By WB of TCM and BBM from the human K, the CT-Ab and NT-Ab labeled strongly a single protein band of ~70 kDa. In TCM from the rat and mouse K and L, the CT-Ab labeled the same, and a few smaller proteins. Whereas our data confirmed localization of PDZK1 protein in the PT BBM in various species, we could not support its localization in the hepatocyte sinusoidal membrane.

immunocytochemistry; hepatocyte; human; mouse; rat; proximal tubule

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