Altered microRNA regulation of short chain fatty acid receptors in the hypertensive kidney is normalized with hydrogen sulfide supplementation

Abstract

Hypertension affects nearly one third of the adult US population and is a significant risk factor for chronic kidney disease (CKD). An expanding body of recent studies indicates that gut microbiome has crucial roles in regulating physiological processes through, among other mechanisms, one mode of short chain fatty acids (SCFA) and their target receptors. In addition, these SCFA receptors are potential targets of regulation by host miRNAs, however, the mechanisms through which this occurs is not clearly defined. Hydrogen sulfide (H₂S) is an important gasotransmitter involved in multiple physiological processes and is known to alleviate adverse effects of hypertension such as reducing inflammation in the kidney. To determine the role of host microRNAs in regulating short chain fatty acid receptors in the kidney as well as the gut, C57BL/6J wild-type mice were treated with or without Ang-II and H₂S donor GYY4137 (GYY) for 4 weeks to assess whether GYY would normalize adverse effects observed in hypertensive mice and whether this was in part due to altered gut microbiome composition. We observed several changes of SCFA receptors, including Olfr78, Gpr41/43 and predicted microRNA regulators in the kidney among the different treatments. Increased expression of inflammatory markers Il6 and Rorc2, along with Tgfβ, were found in the hypertensive kidney. The glomerular filtration rate (GFR) was improved in mice treated with Ang-II + GYY compared with Ang-II only, indicating improved kidney function. The Erysipelotrichia class of bacteria, linked with high fat diets, was enriched in hypertensive animals but reduced with GYY supplementation. These data point towards a role for miRNA regulation of SCFA receptors in hypertensive kidney and are normalized by H₂S supplementation.

Keywords: Gut microbiome; Hydrogen sulfide; MicroRNA; Short chain fatty acid.

Fig. 1

16S rRNA profiling of gut microbiome in mice treated with Ang-II and GYY. (A) Relative abundance of total bacteria at the genus level in each treatment group. (B) Relative abundance of total bacteria at the class level for each treatment group. Abundance is expressed as a percentage. C – Control, A – Ang-II, G – GYY, AG – Ang-II+GYY.

Fig. 2

Short chain fatty acid receptors are altered in the hypertensive kidney. (A) qPCR analysis of Olfr78, Gpr41, and Gpr43 expression in kidney of Ang-II and GYY treated mice. (B) Western blot analysis and (C) bar graph of protein levels of SCFA receptors. Band intensities were quantified using ImageJ. Bar graphs are the mean intensities ± SD. n=5/group. *p < 0.05 Control vs. Ang-II; (D–E) Immunostaining for Gpr41 and Gpr 43 showed increased expression in glomeruli and proximal tubules (white arrows). (F) Immunostaining for Olfr78 showed increased expression and localization in the glomerulus. Magnification, ×60. Scale bars, 40 μm.

Fig. 2

Short chain fatty acid receptors are altered in the hypertensive kidney. (A) qPCR analysis of Olfr78, Gpr41, and Gpr43 expression in kidney of Ang-II and GYY treated mice. (B) Western blot analysis and (C) bar graph of protein levels of SCFA receptors. Band intensities were quantified using ImageJ. Bar graphs are the mean intensities ± SD. n=5/group. *p < 0.05 Control vs. Ang-II; (D–E) Immunostaining for Gpr41 and Gpr 43 showed increased expression in glomeruli and proximal tubules (white arrows). (F) Immunostaining for Olfr78 showed increased expression and localization in the glomerulus. Magnification, ×60. Scale bars, 40 μm.

Fig. 2

Short chain fatty acid receptors are altered in the hypertensive kidney. (A) qPCR analysis of Olfr78, Gpr41, and Gpr43 expression in kidney of Ang-II and GYY treated mice. (B) Western blot analysis and (C) bar graph of protein levels of SCFA receptors. Band intensities were quantified using ImageJ. Bar graphs are the mean intensities ± SD. n=5/group. *p < 0.05 Control vs. Ang-II; (D–E) Immunostaining for Gpr41 and Gpr 43 showed increased expression in glomeruli and proximal tubules (white arrows). (F) Immunostaining for Olfr78 showed increased expression and localization in the glomerulus. Magnification, ×60. Scale bars, 40 μm.

Fig. 3

Ang-II induced inflammatory response in kidney. (A) Inflammatory markers were assessed by qPCR, including Tgfβ, Il6, Rorc1γ, Rorc2γ, and reported anti-inflammatory fatty acid receptor Gpr120. (B) Western blot analysis and (C) bar graph of protein levels inflammatory markers and long chain fatty acid receptor Gpr120. Bar graphs are the mean intensities ± SD. n=5/group. *p < 0.05 Control vs. Ang-II; (D) Immunostaining for Tgfβ and Il6 showed increased expression and localization in glomeruli (white arrows). (E) Immunostaining for Rorcγ showed increased expression and localization in the tubules and vessels around the glomerulus while (F) Gpr120 was less in hypertensive kidney (white arrows). GYY increased expression of GPR 120. Magnification, ×60. Scale bars, 40 μm.

Fig. 3

Ang-II induced inflammatory response in kidney. (A) Inflammatory markers were assessed by qPCR, including Tgfβ, Il6, Rorc1γ, Rorc2γ, and reported anti-inflammatory fatty acid receptor Gpr120. (B) Western blot analysis and (C) bar graph of protein levels inflammatory markers and long chain fatty acid receptor Gpr120. Bar graphs are the mean intensities ± SD. n=5/group. *p < 0.05 Control vs. Ang-II; (D) Immunostaining for Tgfβ and Il6 showed increased expression and localization in glomeruli (white arrows). (E) Immunostaining for Rorcγ showed increased expression and localization in the tubules and vessels around the glomerulus while (F) Gpr120 was less in hypertensive kidney (white arrows). GYY increased expression of GPR 120. Magnification, ×60. Scale bars, 40 μm.

Fig. 3

Ang-II induced inflammatory response in kidney. (A) Inflammatory markers were assessed by qPCR, including Tgfβ, Il6, Rorc1γ, Rorc2γ, and reported anti-inflammatory fatty acid receptor Gpr120. (B) Western blot analysis and (C) bar graph of protein levels inflammatory markers and long chain fatty acid receptor Gpr120. Bar graphs are the mean intensities ± SD. n=5/group. *p < 0.05 Control vs. Ang-II; (D) Immunostaining for Tgfβ and Il6 showed increased expression and localization in glomeruli (white arrows). (E) Immunostaining for Rorcγ showed increased expression and localization in the tubules and vessels around the glomerulus while (F) Gpr120 was less in hypertensive kidney (white arrows). GYY increased expression of GPR 120. Magnification, ×60. Scale bars, 40 μm.

Fig. 3

Ang-II induced inflammatory response in kidney. (A) Inflammatory markers were assessed by qPCR, including Tgfβ, Il6, Rorc1γ, Rorc2γ, and reported anti-inflammatory fatty acid receptor Gpr120. (B) Western blot analysis and (C) bar graph of protein levels inflammatory markers and long chain fatty acid receptor Gpr120. Bar graphs are the mean intensities ± SD. n=5/group. *p < 0.05 Control vs. Ang-II; (D) Immunostaining for Tgfβ and Il6 showed increased expression and localization in glomeruli (white arrows). (E) Immunostaining for Rorcγ showed increased expression and localization in the tubules and vessels around the glomerulus while (F) Gpr120 was less in hypertensive kidney (white arrows). GYY increased expression of GPR 120. Magnification, ×60. Scale bars, 40 μm.

Fig. 4

miRNAs target SCFA receptors in vitro. Mouse Glomerular Endothelial Cells were transfected with mimics, inhibitors, and controls, against miR-132 and miR-329. In silico analysis using target software predicted miR-132 would target Gpr43 and miR-329 binding to Gpr41. (A) One hundred μg of protein was resolved on SDS-PAGE gels and probed with appropriate antibodies overnight. Band intensities were quantified using ImageJ. (B) Bar graphs are the mean intensities ± SD. n=4/group. *p < 0.05.

Fig. 5

Glomeruluar filtration rate is improved in mice supplemented with GYY. Live, conscious mice were anesthetized with isoflurane and injected with FITC-sinistrin (15 mg/100 g) into the femoral artery. Detector was fixed to area removed of hair and mice were put back into cages while the detector took measurements for 2 hours. GFR was calculated after obtaining half-life of excreted FITC-sinistrin. Bar graph, n=5/group. *p < 0.05

Fig. 6

Intestinal barrier integrity is compromised in hypertensive mice. (A) Plasma samples were diluted 1/10,000 and added to ELISA sandwich with antibody to haptoglobin. The plate was washed and then exposed to substrate to reveal expression. Absorbance was detected at 480 nm wavelength. Bar graph, n=5/group. *p < 0.05 (B) gDNA extracted from kidney was purified and set up for a PCR reaction, using 100ng sample and primers specific to bacteria. Samples at the end of the PCR run were loaded onto a 1% agarose gel and imaged.

Fig. 7

Schematic or proposed model of hypertension-induced gut dysbiosis, leakage of contents into bloodstream and miRNA regulation of SCFA receptors in the kidney.