Cytosolic calcium signalling controls a vast number of cellular functions, such as contraction, secretion, cell growth and cell division. The increase of [Ca2+]i evoked by G-protein coupled receptor activation has two closely related components, i.e. the rapid phase of inositol 1,4,5-triphosphate-mediated Ca2+ release from ER stores, and followed by Ca2+ entry through store-operated channels (SOCs) in plasma membrane. Orai and STIM proteins are recently identified store-operated channel molecules, but their expression and function in human kidney are unknown. Here, we aimed to determine the expression and distribution of Orai in normal and diabetic human kidney.
Methods: Human kidney samples were obtained from Hull and East Yorkshire NHS Trust with approval of the local ethics committee from patients undergoing a nephrectomy, or from patients with diabetes undergoing a kidney biopsy. The mRNA expression was detected by RT-PCR or real-time RT-PCR. The protein expression and localisation were detected by western blotting and immunostaining.
Results: The mRNA and protein expression of Orai1, Orai2 and Orai3 was detected in normal human kidney samples by RT-PCR and western blotting, respectively. Orai proteins were highly expressed in proximal tubules in the kidney, but very weakly expressed in the glomerulus. The expression of Orai isoforms in diabetic kidney was less abundant than that in normal kidney. In primary cultured human kidney tubular cells, Orai 1 was localised in the plasma membrane detected by fluorescence staining, while Orai 2 and 3 were intracellularly located.
Conclusions: We have demonstrated that the store-operated Orai channels existed in human kidney, and highly expressed in the proximal tubules. The expression level could be changed in some kidney diseases, such as diabetes. The finding provides the first evidence of Orai proteins in normal human kidney and diabetic nephropathy.