Molecular Mechanism for Hypertensive Renal Disease: Differential Regulation of Chromogranin A Expression at 3'-Untranslated Region Polymorphism C+87T by MicroRNA-107

Abstract

Chromogranin A (CHGA) is coreleased with catecholamines from secretory vesicles in adrenal medulla and sympathetic axons. Genetic variation in the CHGA 3'-region has been associated with autonomic control of circulation, hypertension, and hypertensive nephropathy, and the CHGA 3'-untranslated region (3'-UTR) variant C+87T (rs7610) displayed peak associations with these traits in humans. Here, we explored the molecular mechanisms underlying these associations. C+87T occurred in a microRNA-107 (miR-107) motif (match: T>C), and CHGA mRNA expression varied inversely with miR-107 abundance. In cells transfected with chimeric luciferase/CHGA 3'-UTR reporters encoding either the T allele or the C allele, changes in miR-107 expression levels had much greater effects on expression of the T allele. Cotransfection experiments with hsa-miR-107 oligonucleotides and eukaryotic CHGA plasmids produced similar results. Notably, an in vitro CHGA transcription/translation experiment revealed that changes in hsa-miR-107 expression altered expression of the T allele variant only. Mice with targeted ablation of Chga exhibited greater eGFR. Using BAC transgenesis, we created a mouse model with a humanized CHGA locus (T/T genotype at C+87T), in which treatment with a hsa-miR-107 inhibitor yielded prolonged falls in SBP/DBP compared with wild-type mice. We conclude that the CHGA 3'-UTR C+87T disrupts an miR-107 motif, with differential effects on CHGA expression, and that a cis:trans (mRNA:miR) interaction regulates the association of CHGA with BP and hypertensive nephropathy. These results indicate new strategies for probing autonomic circulatory control and ultimately, susceptibility to hypertensive renal sequelae.

Keywords: CKD; hypertension; molecular genetics; progression of renal failure; systolic BP; transgenic mouse.

Figure 1.

Human CHGA 3′-UTR variant C+87T (rs7610): T allele has a superior match to micro-RNA hsa-miR-107. Alignment of human CHGA 3′-UTR C+87T variant alleles T versus C with miR hsa-miR-107. The T allele exhibits a superior match to hsa-miR-107 by both minimum free energy and alignment score. The scores are explained at the bottom of the panel.

Figure 2.

CHGA mRNA abundance: Inverse dependence on hsa-miR-107 across cell lines and tissues. Results were obtained by qRT-PCR in replicate samples and plotted±SEMs for each group. mRNA results were normalized to GAPDH mRNA in the same sample, whereas the miR-107 results are normalized to the small RNA SNORD61. CHGA mRNA abundance is inversely proportional to miR-107 abundance (R=0.96, P=0.005). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3.

Hsa-miR-107 regulates human CHGA 3′-UTR expression in human HEK293T cells. Luciferase reporter activity of CHGA 3′-UTR C+87T in human HEK293T cells: effects of hsa-miR-107. Results are analyzed by two-way ANOVA factoring for miR (present versus mock) and allele (T versus C). The values are expressed as ±SEMs. (A) Effect of hsa-miR-107 inhibitor. (B) Effect of hsa-miR-107 mimic.

Figure 4.

Hsa-miR-107 effect on CHGA protein expression in human HEK293T cells. CHGA 3′-UTR C+87T in the full-length cDNA: effects of hsa-miR-107. HEK293T cells were transfected with pCMV-hCHGA, in which the strong pCMV promoter drives expression of the full-length human CHGA cDNA. The electrophoresed/membrane-transferred proteins are detected by antibodies (anti-CHGA or anti-GAPDH) with intensity scored by ImageJ (http://rsbweb.nih.gov/ij/). Numbers show the ratio of CHGA to GAPDH. In a parallel experiment, when HEK293T cells were transfected with empty expression vector (pCMV plasmid) rather than the expression plasmid containing the human CHGA cDNA insert (pCMV→hCHGA), there was no expression of CHGA detected on immunoblots. The data are expressed as ±SEMs. (A) Effect of hsa-miR-107 inhibitor in human HEK293T cells. (B) Effect of hsa-miR-107 mimic in human HEK293T cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5.

Loss of CHGA results in higher eGFR and hyperfiltration. eGFR in the Chga(−/−) mouse. Plasma creatinine was determined by HPLC (to avoid the influence of noncreatinine chromogens in plasma), and eGFR was estimated from creatinine data. Nine Chga(+/+) (control) and 10 Chga(−/−) mice were evaluated. Because data were not normally distributed, results were analyzed by nonparametric (Mann–Whitney) statistical tests. The eGFR values are expressed as ±SEMs.

Figure 6.

Inhibition of hsa-miR-107 in vivo results in drop of BP. (A) Alignment of hsa-miR-107 with human and mouse versions of the CHGA 3′-UTR in the homologous region of C+87T. At C+87T in both human and mouse versions of CHGA in these models, the diploid genotype is T/T. (B) Differential effects of an miR-107 inhibitor/antagomir on mouse BP and HR in humanized (CHGA+/+;Chga−/−) versus wild-type (CHGA−/−;Chga+/+) mice. Male humanized (CHGA+/+;Chga−/−; n=7) and wild-type (CHGA−/−;Chga+/+; n=8) mice were studied at 16–18 weeks of age. The single-stranded oligonucleotide was delivered by intraperitoneal injection (at 3.3 µg/g body wt) at time 0 hours (10:00 hours) into mice with telemetric continuous recording of BP and HR. Values are expressed as ±SEMs. (C) Statistics were done by repeated measures ANOVA using linear mixed model, and data are presented.

Figure 7.

Hypothetical schema. Hypertension, hypertensive renal disease, and the CHGA pathway from 3′-UTR variation to disease. A sequence of proposed pathophysiologic events on the basis of our results and previous experimental results is shown.