Sodium bicarbonate cotransporter NBCe2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells

Abstract

RationaleSalt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure.ObjectiveTo characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5.Methods and resultsThe expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte nuclear factor type 4A (HNF4A) to DNA was increased in hRPTCs carrying HV SLC4A5 rs7571842 but not rs10177833. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was abolished by HNF4A antagonists.ConclusionNBCe2 activity is stimulated by an increase in intracellular sodium and is hyper-responsive in hRPTCs carrying HV SLC4A5 rs7571842 through an aberrant HNF4A-mediated mechanism.

Gene Level Regulation of Na,K-ATPase in the Renal Proximal Tubule Is Controlled by Two Independent but Interacting Regulatory Mechanisms Involving Salt Inducible Kinase 1 and CREB-Regulated Transcriptional Coactivators

“…Examples include the hypotension in an RPT specific Angiotensin II receptor (AT1A) knockout [ 9 ], and the salt-sensitive hypertension in a RPT specific Knockout (KO) of aromatic L-amino acid decarboxylase [ 10 ], the enzyme responsible for renal dopamine synthesis. In addition are studies with animals with mutations in dopamine and adrenergic receptors [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ], as well as directed KOs in RPT transporters including NHE3 and NBCe2 [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ]. All of the altered genes affect Na + efflux by RPT Na,K-ATPase.…”

Inverse Salt Sensitivity of Blood Pressure Is Associated with an Increased Renin-Angiotensin System Activity

“…Details and specificity of this assay, in which ouabain was used to inhibit Na + -K + /ATPase activity, permitting the quantification of sodium accumulation in RPTCs, were previously published [ 72 ]. Briefly, the uRPTCs were cultured in 96-well glass bottom collagen-coated Matrical plates (Dot Scientific, Burton, MI, USA, MGB096-1-2LGL) at 37 °C until they reached 80% confluence.…”

Lipid rafts are required for effective renal D1dopamine receptor function

“…Immortalized hRPTCs were generated from the normal portions of renal surgical specimens from normotensive Caucasian male patients who had renal cell carcinoma 8,19‐21 . Human embryonic kidney (HEK) 293 cells (ATCC, Manassas, VA, USA) and hRPTCs were maintained at 37°C in 95% of air/5% of CO 2 in Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 (DMEM/F‐12) medium (Gibco, Grand Island, NY, USA) supplemented with 5% of penicillin‐streptomycin‐amphotericin (Corning Life Sciences, Manassas, VA) and 10% of FBS (Gibco, Grand Island, NY, USA).…”