HIV-1 Vpr suppresses expression of the thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule

Abstract

HIV-associated nephropathy (HIVAN) impairs functions of both glomeruli and tubules. Attention has been previously focused on the HIVAN glomerulopathy. Tubular injury has drawn increased attention because sodium wasting is common in hospitalized HIV/AIDS patients. We used viral protein R (Vpr)-transgenic mice to investigate the mechanisms whereby Vpr contributes to urinary sodium wasting. In phosphoenolpyruvate carboxykinase promoter-driven Vpr-transgenic mice, in situ hybridization showed that Vpr mRNA was expressed in all nephron segments, including the distal convoluted tubule. Vpr-transgenic mice, compared with wild-type littermates, markedly increased urinary sodium excretion, despite similar plasma renin activity and aldosterone levels. Kidneys from Vpr-transgenic mice also markedly reduced protein abundance of the Na+-Cl- cotransporter (NCC), while mineralocorticoid receptor (MR) protein expression level was unchanged. In African green monkey kidney cells, Vpr abrogated the aldosterone-mediated stimulation of MR transcriptional activity. Gene expression of Slc12a3 (NCC) in Vpr-transgenic mice was significantly lower compared with wild-type mice, assessed by both qRT-PCR and RNAScope in situ hybridization analysis. Chromatin immunoprecipitation assays identified multiple MR response elements (MRE), located from 5 kb upstream of the transcription start site and extending to the third exon of the SLC12A3 gene. Mutation of MRE and SP1 sites in the SLC12A3 promoter region abrogated the transcriptional responses to aldosterone and Vpr, indicating that functional MRE and SP1 are required for the SLC12A3 gene suppression in response to Vpr. Thus, Vpr attenuates MR transcriptional activity and inhibits Slc12a3 transcription in the distal convoluted tubule and contributes to salt wasting in Vpr-transgenic mice.

Fig 1. Vpr gene expression in nephrons of Vpr-Tg mice analyzed by BaseScope.

(A) negative control image from the WT mouse kidney section which was hybridized using a Vpr probe. (B) The same Vpr probe was hybridized as (A) Vpr RNA (pink), indicated by arrows in images from Tg mouse kidney tissue sections. GL, glomerulus; PT, proximal tubule; LP, loop of Henle; DT, distal convoluted tubule; CD, collecting duct. Scale bar, 20 μm.

Fig 2. Kidney function and sodium homeostasis-related parameters in Vpr Tg mice.

(A) Urinary sodium excretion (UNaV, ratio of urinary sodium in mmol/Lto urine creatinine in mmol/L) was significantly elevated in Tg mice compared with WT littermates, before and after feeding a low salt diet. (B) Fractional excretion of sodium (FENa) was higher in Tg compared with WT mice before and after a low salt diet. (C) Serum creatinine concentrations and (D) creatinine clearances (Ccr) were not different among the groups. (E) Blood urea nitrogen (BUN) concentration was higher in Tg that WT mice before and after allow salt diet. (F) Plasma renin activity and (G) plasma aldosterone concentrations were increased on low salt diet but were similar in WT and Tg mice. n = 5–6. n.s., not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA, Bonferroni correction.

Fig 3. Expression of NCC, NaKa, and MR in kidney homogenates from wild-type (WT) and Vpr transgenic (Tg) mice.

(A) Protein expression of total NCC was markedly suppressed in kidneys from Tg mice compared with WT littermates. n = 6. **P < 0.01, Student’s t test. (B) Protein expressions of NaKa and MR were similar in WT and Tg mice. NaKa, Na⁺/K⁺-ATPase. n = 3. n.s., not significant, Student’s t test. (C) Immunohistochemical analysis of NCC distribution in kidney cortex sections demonstrated that NCC is located in the apical cellular regions of the renal distal convoluted tubule, with similar distribution in both WT and Tg mice. NCC immunostained tubules were scanned and NCC protein expression was found to be lower in Tg than WT mice both before and after salt depletion. n = 3–4. n.s., not significant; **P < 0.01, ***P < 0.001, ANOVA, Bonferroni correction.

Fig 4. Suppression of MR transcriptional activity by Vpr in African green monkey kidney (CV-1) cells.

(A) CV-1 cells, lacking MR, were transfected with Vpr- and MR-expressing plasmids. MR-mediated transcription activity, monitored by the MR-responsive MMTV-luciferase reporter, exhibited a sigmoidal dose-response to aldosterone and across the aldosterone concentration range, which was inhibited by Vpr. (B) Vpr markedly suppressed MR transcriptional activity in CV-1 cells transfected with MR and cultured with aldosterone (100 nM). (C) Both sVpr and MR antagonist spironolactone (1 μM) suppressed aldosterone-induced (100 nM) MR transcription activity; the spironolactone-mediated suppression was not further reduced by sVpr. n = 3–6. n.s., not significant; *P < 0.05, ***P < 0.001, ANOVA, Bonferroni correction.

Fig 5. Gene expression of Slc12a3 in the kidney of wild-type (WT) and Vpr transgenic (Tg) mice.

(A) Renal Slc12a3 gene expression, quantified by qRT-PCR, was significantly lower in Tg mice compared with WT mice. (B) Slc12a3 gene expression was markedly lower (fewer cells express Slc12a3) in Tg mice compared with WT mice, as determined in situ hybridization, using RNAScope. n = 12–13. ***P < 0.001, Student’s t test.

Fig 6. Characterization of putative transcription factor motifs within human SLC12A3 gene and the effect of sVpr on the association of MR to its binding regions.

(A) Human DCT cells were exposed to aldosterone or vehicle, with or without sVpr, and were processed for the ChIP assay, using anti-FLAG antibody, targeting FLAG-MR. Fifteen primer pairs (S2 Table), located around the transcription start site of the SLC12A3 gene, encoding NCC, were used for qRT-PCR. The fold-enrichment of the specific FLAG-MR-binding DNA sequences to that of the normal rabbit anti serum control was compared among four treatment groups: Control, aldosterone, sVpr, and aldosterone plus sVpr. As shown in the column graphs, the regions of highest Vpr-specific binding (determined by the ratio of aldosterone response to Vpr response) were amplicons P-9 and P-10. *P < 0.05 aldosterone vs control; **P < 0.05 aldosterone plus Vpr vs. aldosterone. ANOVA, Bonferroni correction. (B) CV-1 cells were transfected with WT or mutated SLC12A3 constructs. The construct pTSC-Luc contains one putative MR response element (MRE) motif (5’-CAATCAAATGGTGTTCTGC-3’, amplicon P-9) and one putative SP1 motif (5’- CCCTCCCTGGACACC-3’, amplicon P-10). Amplicons P-9 and P-10 exerted the largest suppressive effects by Vpr on transcriptional activity. Putative MRE and SP1 motifs flanking the SLC12A3 transcription start site were mutated with random sequences (S2 Table) to eliminate the putative regulatory motif sequences. Each reporter construct contained a fragment of the SLC12A3 gene. CV-1 cells were transfected with reporter constructs as shown and exposed to aldosterone and/or sVpr. sVpr attenuated aldosterone-stimulated reporter activity with the intact P-9 and P-10 constructs but had no effect with single or dual mutants of P-9 and P-10 amplicons. ***P < 0.001 vs control, ANOVA, followed by Bonferroni correction.

Fig 7. A schematic model of Vpr suppression of SLC12A3 transcription activity in the distal convoluted tubule.

(A) In the absence of Vpr, aldosterone binds MR in the cytoplasm. The activated MR translocates to the nucleus, where it binds the mineralocorticoid response element (MRE) in genomic DNA. This complex, together with SP1 and other transcription factor(s) and other co-regulator(s), activates the transcription initiation complex and thereby promotes SLC12A3 gene transcription and NCC protein translation. Although both MR (aldosterone) and GR (cortisol, corticosterone etc.) hormones potentially bind to MRE, the presence of 11βHSD2 in the DCT, especially DCT2, converts cortisol (humans)/corticosterone (rodents) to cortisone, which facilitates MR binding to MRE. (B) Vpr, binding either MR or SP1 and other transcription factors, abrogates the interaction of MR and MRE and thus prevents SLC12A3 transcription and translation. This reduces the abundance of NCC in the apical membranes of distal convoluted tubules. Considering that plasma aldosterone levels in Tg mice were similar to those of WT mice, and aldosterone concentrations were increased by salt depletion in both groups, Vpr did not impair aldosterone synthesis and regulation system in Tg mice. Of note, MREs can be located within intron or other regions of the gene, although it is illustrated here in the promoter region of SLC12A3.