Nuclear pore complex oxalate binding protein p62: its expression on oxalate exposure to VERO cells.

Oxalate rich stones are the most common among the various stones. Oxalate binding protein plays a vital role in the transport of oxalate. Nuclear pore complex (NPC) contains a protein of molecular weight 62 kDa and it has maximum oxalate binding activity. The physiological significance of the presence of oxalate binding protein in the NPC is not well understood. In order to study its function, the expression of this protein during oxalate stress condition and the morphological changes on oxalate exposure to synchronized VERO cells have been determined. VERO cells were synchronized at different stages of cell cycle using cell cycle blockers and expression of the NPC p62 was assessed using enzyme linked immunosorbent assay (ELISA) technique with p62 antibody (MAb 414). Expression of NPC p62 was more pronounced in 1.0 mM oxalate concentration in mitotic phase than in S phase, suggesting cell cycle dependency. During oxalate exposure there is cell aggregation and complete degeneration of cell morphology occurs, which in turn lead to the expression of certain genes, including the NPC oxalate binding protein p62. Thus, oxalate induces degeneration of cells (may be due to the lipid peroxidation) and leads to the expression of NPC oxalate binding protein and the expression is of cell cycle dependent manner.

Nuclear pore complex oxalate binding protein p62: expression in different kidney disorders.

BACKGROUND: Urolithiasis is a multifactorial process that starts with the formation of microcrystals in the urine and terminates as mature renal calculi. The oxalate binding protein plays a vital role in the transport of oxalate. The physiological significance of the presence of oxalate binding protein in the nuclear pore complex is not well understood. METHODS: The nuclear envelope was extracted from human cadaver kidneys. 14C oxalate was labeled, nuclear pore complex proteins were extracted and loaded onto Sephadex G-200, and further purified in DEAE-Sephadex A-50 column. The radioactive protein peak was pooled, concentrated and checked for purity in SDS-PAGE. The purified protein showed cross-reactivity with the monoclonal antibody (MAb 414) and was homogeneous. Urine samples of healthy individuals with no history of kidney disease served as control. Blood and urine samples were collected from kidney and autoimmune disorder patients and checked for the expression of p62 protein by ELISA. RESULTS: Extracted and purified nuclear pore complex oxalate binding protein had a molecular weight of 62 kDa. A threefold increase in oxalate excretion was observed in hyperoxaluric patients compared to control subjects. The protein expression was found to be higher in hyperoxaluric patients vs. controls, chronic renal failure (CRF) and acute renal failure (ARF), whereas decreased expression was observed in nephrotic syndrome (NS) patients. p62 autoantibodies was observed in hyperoxaluria (HO), systemic lupus erythematosus (SLE) and primary biliary cirrhosis (PBC), whereas it was absent in controls. CONCLUSION: Increased expression of p62 may be due to membrane damage induced by oxalate stress, and may be used as a diagnostic marker. This study also confirms the presence of p62 autoantibodies in HO patients.

Expression of nuclear pore complex oxalate binding protein p62 in experimental hyperoxaluria.

Proteins are thought to play a major role in stone formation. Oxalate binding protein plays a vital role in the transport of oxalate. This study was aimed at determining whether hyperoxaluria induces the expression of nuclear pore complex oxalate binding protein p62 which has the transport function. Hyperoxaluria was induced in male Wistar rats by feeding 0.75% ethylene glycol in water. The oxalate binding activity of the nuclear pore complex protein increased markedly during experimental hyperoxaluria, whereas nuclear lamina had no binding at all. There was an alteration in the elution profile of the nuclear pore complex oxalate binding protein during the hyperoxaluric condition. The protein was purified and had a molecular weight of 62 kDa (data not shown). The purified protein showed cross-reactivity with the monoclonal antibody (MAb 414) and it showed homogeneity. The expression of this protein (p62) during the hyperoxaluric condition was determined by ELISA and a 3-fold increase was observed when compared to control rats. The increased expression is further confirmed by Western blotting and immunohistochemistry. The increase in p62 protein expression may be either due to increased expression of certain genes or degradation of the cell membrane by oxalate-induced cell injury. Thus, the present study suggests that the increased expression of this protein (p62) may be due to the oxalate induction.

Characterisation of nuclear pore complex oxalate binding protein from human kidney.

Both rat and human kidney nuclei exhibited time and pH dependent oxalate or histone-oxalate uptake which was inhibited by anion transport inhibitor, 4,4'-dithiocyanostilbene-2,2'-disulphonic acid. Sodium chloride had no effect. Nuclear membrane had oxalate binding at pH 7.4. Extraction of nuclear membrane by Triton-high salt mixture showed maximal oxalate binding activity with nuclear pore complex while nuclear lamin had no oxalate binding. The rat and human kidney nuclear pore complex showed oxalate binding of 144 and 220 pmoles/mg protein respectively. Subsequent purification of the protein on diethyl amino ethyl-Sephadex A 50 column and Sephadex G-200 column yielded 4-fold purification. The protein revealed a molecular weight of 205 kDa on SDS-PAGE. The protein was found to be saturable at 2 microM oxalate and had a Kd of 2.98 pM and a Bmax of 197 pmoles. Antibody for 205 kD was separated from primary biliary cirrhosis serum containing auto antibody against 205 kDa using affinity column chromatography. The oxalate binding activity as well as the nuclear uptake of oxalate or histone-oxalate were inhibited by its antibody.