The sodium-bicarbonate cotransporter NBCe2 (slc4a5) expressed in human renal proximal tubules shows increased apical expression under high-salt conditions.

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake.

Exosomal transfer from human renal proximal tubule cells to distal tubule and collecting duct cells.

OBJECTIVES: Exosomes are 50-90nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have reported that human urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. This study examines exosomal transfer between cultured human renal proximal tubule cells (RPTCs) and from RPTCs to human distal tubule and collecting duct cells. DESIGN AND METHODS: For RPTC-to-RPTC exosomal transfer, we utilized 5 RPTC lines producing exosomes that were fluorescently labeled with exosomal-specific markers CD63-EGFP or CD9-RFP. Transfer between RPTCs was demonstrated by co-culturing CD63-EGFP and CD9-RFP stable clones and performing live confocal microscopy. For RPTC-to-distal segment exosomal transfer, we utilized 5 distal tubule and 3 collecting duct immortalized cell lines. RESULTS: Time-lapse videos revealed unique proximal tubule cellular uptake patterns for exosomes and eventual accumulation into the multivesicular body. Using culture supernatant containing exosomes from 3 CD9-RFP and 2 CD63-EGFP RPTC cell lines, all 5 distal tubule cell lines and all 3 collecting duct cell lines showed exosomal uptake as measured by microplate fluorometry. Furthermore, we found that RPTCs stimulated with fenoldopam (dopamine receptor agonist) had increased production of exosomes, which upon transfer to distal tubule and collecting duct cells, reduced the basal reactive oxygen species (ROS) production rates in those recipient cells. CONCLUSION: Due to the complex diversity of exosomal contents, this proximal-to-distal vesicular inter-nephron transfer may represent a previously unrecognized trans-renal communication system.