SLC4A5 is a sodium-bicarbonate co-transporter involved with sodium homeostasis. Based on unpublished data, two SLC4A5 single nucleotide polymorphisms (SNPs rs1017783 and rs7571842) have been highly associated with an individual’s salt-sensitivity status. Since the renal proximal tubule (RPT) regulates a large percentage of renal sodium transport, we investigated whether SLC4A5 was present in this nephron segment. Using confocal immunofluorescence microscopy, we found expression of SLC4A5 in human RPT cell plasma membrane and intracellular membrane vesicles. We then examined the physiologic implications of the SLC4A5 SNPs in human RPT cells. Using immunoblotting and RT-PCR, we found no significant differences in basal SLC4A5 expression in RPT cells between individuals that are homozygous variant at both SNPs and individuals that are wild-type (WT) for both alleles. Stimulation of the dopaminergic system with 1μM fenoldopam, or the renin-angiotensin system with 10 nM angiotensin II or 10 nM angiotensin III (n=18 per treatment) over 3 and 24 hours did not significantly alter SLC4A5 protein or 24 hour mRNA expression. These data indicate that SLC4A5 is not directly regulated by either the renal dopaminergic or renin-angiotensin system. However, 24 hour stimulation with the sodium ionophore monensin (MON, 1μM) significantly increased overall mRNA expression of SLC4A5 by 182±0.098% over vehicle (VEH) (ΔCq VEH=0.283±0.035; n=18, p<0.001). There was also a significant increase in SLC4A5 mRNA in three cell lines homozygous variant for both alleles compared to three WT cell lines following MON treatment at both 3 hours (138±0.10%; ΔCq WT MON = 0.5±0.052; n=9, p<0.05) and 24 hours (161±0.11%; ΔCq WT MON = 0.39±0.066; n=9, p<0.02). Three but not 24 hour stimulation with MON also significantly increased overall expression of SLC4A5 protein (137±0.00041%; RFU VEH=0.0030±0.00022; n=18, p<0.01). MON, by allowing salt to enter a cell, may be activating an enhancer that leads to increased transcription of SLC4A5 mRNA that is more effective in homozygous variant cell lines. These novel observations demonstrate that SNPs located in a non-promoter DNA intron are associated with enhanced promoter activity that is regulated by altered intracellular sodium.
Demonstration of renin mRNA, angiotensinogen mRNA, and angiotensin converting enzyme mRNA expression in the human eye: evidence for an intraocular renin-angiotensin system.