Renal ACE2 (Angiotensin-Converting Enzyme 2) Expression Is Modulated by Dietary Fiber Intake, Gut Microbiota, and Their Metabolites

HomeHypertensionVol. 77, No. 6Renal ACE2 (Angiotensin-Converting Enzyme 2) Expression Is Modulated by Dietary Fiber Intake, Gut Microbiota, and Their Metabolites Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBRenal ACE2 (Angiotensin-Converting Enzyme 2) Expression Is Modulated by Dietary Fiber Intake, Gut Microbiota, and Their Metabolites Matthew Snelson, Rikeish R. Muralitharan, Evany Dinakis, Michael Nakai, Hamdi A. Jama, Waled A. Shihata, Chad Johnson, David M. Kaye, Charles R. Mackay, Louise M. Burrell, Melinda T. Coughlan, Francine Z. Marques Matthew SnelsonMatthew Snelson https://orcid.org/0000-0003-4829-9550 Department of Diabetes, Central Clinical School, Faculty of Medicine Nursing and Health Sciences (M.S., M.T.C.), Monash University, Melbourne, Australia. Search for more papers by this author , Rikeish R. MuralitharanRikeish R. Muralitharan https://orcid.org/0000-0002-7577-2123 Hypertension Research Laboratory, School of Biological Sciences, Faculty of Science (R.R.M., E.D., M.N., H.A.J., F.Z.M.), Monash University, Melbourne, Australia. Institute for Medical Research, Ministry of Health Malaysia, Kuala Lumpur (R.R.M.). Search for more papers by this author , Evany DinakisEvany Dinakis Hypertension Research Laboratory, School of Biological Sciences, Faculty of Science (R.R.M., E.D., M.N., H.A.J., F.Z.M.), Monash University, Melbourne, Australia. Search for more papers by this author , Michael NakaiMichael Nakai https://orcid.org/0000-0002-6400-8007 Hypertension Research Laboratory, School of Biological Sciences, Faculty of Science (R.R.M., E.D., M.N., H.A.J., F.Z.M.), Monash University, Melbourne, Australia. Search for more papers by this author , Hamdi A. JamaHamdi A. Jama Hypertension Research Laboratory, School of Biological Sciences, Faculty of Science (R.R.M., E.D., M.N., H.A.J., F.Z.M.), Monash University, Melbourne, Australia. Search for more papers by this author , Waled A. ShihataWaled A. Shihata Heart Failure Research Laboratory, Baker Heart and Diabetes Institute, Melbourne, Australia (W.A.S., F.Z.M., D.M.K.). Search for more papers by this author , Chad JohnsonChad Johnson Monash Micro Imaging (C.J., D.M.K., F.Z.M.), Monash University, Melbourne, Australia. Search for more papers by this author , David M. KayeDavid M. Kaye https://orcid.org/0000-0003-4058-0372 Monash Micro Imaging (C.J., D.M.K., F.Z.M.), Monash University, Melbourne, Australia. Central Clinical School, Faculty of Medicine Nursing and Health Sciences (D.M.K.), Monash University, Melbourne, Australia. Heart Failure Research Laboratory, Baker Heart and Diabetes Institute, Melbourne, Australia (W.A.S., F.Z.M., D.M.K.). Department of Cardiology, Alfred Hospital, Melbourne, Australia (D.M.K.). Search for more papers by this author , Charles R. MackayCharles R. Mackay Infection and Immunity Program, Monash Biomedicine Discovery Institute (C.R.M.), Monash University, Melbourne, Australia. Department of Biochemistry and Molecular Biology (C.R.M.), Monash University, Melbourne, Australia. Search for more papers by this author , Louise M. BurrellLouise M. Burrell https://orcid.org/0000-0003-1863-7539 Department of Medicine, Austin Health, University of Melbourne, Australia (L.M.B.). Search for more papers by this author , Melinda T. CoughlanMelinda T. Coughlan Department of Diabetes, Central Clinical School, Faculty of Medicine Nursing and Health Sciences (M.S., M.T.C.), Monash University, Melbourne, Australia. Search for more papers by this author , Francine Z. MarquesFrancine Z. Marques Correspondence to: Francine Z. Marques, Hypertension Research Laboratory, School of Biological Sciences, Faculty of Science, Monash University, Melbourne, Australia. Email E-mail Address: [email protected] https://orcid.org/0000-0003-4920-9991 Hypertension Research Laboratory, School of Biological Sciences, Faculty of Science (R.R.M., E.D., M.N., H.A.J., F.Z.M.), Monash University, Melbourne, Australia. Monash Micro Imaging (C.J., D.M.K., F.Z.M.), Monash University, Melbourne, Australia. Heart Failure Research Laboratory, Baker Heart and Diabetes Institute, Melbourne, Australia (W.A.S., F.Z.M., D.M.K.). Search for more papers by this author Originally published19 Apr 2021https://doi.org/10.1161/HYPERTENSIONAHA.121.17039Hypertension. 2021;77:e53–e55Genetic deletion of the Ace2 (angiotensin-converting enzyme 2) gene is associated with renal injury, dysfunction, and fibrosis1 and more severe nephropathy in response to high blood pressure (BP).2 Conversely, treatment with exogenous recombinant ACE2 or transgenic overexpression of Ace2 has been demonstrated to limit kidney injury,1 providing evidence of the renoprotective effects of ACE2. A significant component of BP control is dietary modifications, such as the Dietary Approaches to Stop Hypertension and Mediterranean diets.3 Besides lower sodium and fat, these diets are high in fiber—an important nutrient for the gut microbiota, the microorganisms that inhabit the intestine.3 Recent studies have demonstrated that dietary fiber that is prebiotic (ie, feeds the microbiota) lowers BP by modulating the gut microbiota4,5 and that the microbiota that originates from a low-fiber diet can indeed increase BP.5 This is due to the release of short-chain fatty acids (SCFAs)—metabolites produced by the gut microbiota as a byproduct of fiber fermentation.4,5 Interventions with the most prevalent SCFAs, acetate, propionate, and butyrate can lower BP and improve cardiac function in animal models.4,5 SCFAs are sensed by G-protein–coupled receptors, particularly GPR41, GPR43, and GPR109A.3 Indeed, animals lacking these receptors in single or double (GPR43/GPR109A) knockout models were found to have different degrees of cardiac dysfunction.5 Here, we aimed to elucidate the impact of dietary fiber, the gut microbiota, SCFAs, and their receptors on renal ACE2 expression.A diet that is high in fiber is associated with an increase in renal Ace2 mRNA expression (Figure [A])—an effect that was not observed in either the heart or cecum (data not shown). ACE2 protein expression was quantified by immunohistochemistry in the kidneys and was significantly upregulated in the renal cortex with high-fiber diet (Figure [B]), but no significant effect was observed in the medulla (P=0.3, data not shown). To determine whether this was dependent on SCFA production as a result of fiber fermentation by the gut microbiota, we quantified renal Ace2 mRNA in mice that received a low-fiber diet supplemented with SCFAs in the drinking water. Acetate supplementation, but not butyrate or propionate, significantly increased renal Ace2 mRNA expression (Figure [A]). This is consistent with our recent findings that in an angiotensin II mouse model, acetate had the most potent BP-lowering effect.5 Previously, we determined that acetate was able to reduce renal hypertrophy and fibrosis in the DOCA/salt model.4 Future studies will need to determine whether differences in ACE2 due to fiber and acetate intake are also found in women and their impact on renal function.Download figureDownload PowerPointFigure. High fiber intake, via production of gut-derived metabolites, increases renal ACE2 (angiotensin-converting enzyme 2). All animal experimental protocols were approved by the Alfred Medical Research and Education Precinct and Monash Animal Experimentation Ethics Committees (ethics number: E1626_2016, E1670_2016, and 17465) and performed under the guidelines of the National Medical and Health Research Council of Australia. Six-week-old male C57/BL6J mice were fed a diet either low in fiber (SF09-028; Specialty Feeds) or high in fiber (SF11-025; Specialty Feeds), matched for all nutrients besides fiber, for a period of 7 wk.5 Additional groups of mice provided diets low in fiber had supplemental short-chain fatty acids (SCFAs) in the drinking water of 100 mmol/L magnesium acetate, sodium propionate, or sodium butyrate for 7 wk as described previously.5 SCFA-supplemented drinking water was refreshed twice per week. RNA was extracted using Trizol reagent (Thermo Fisher Scientific), treated with DNase (Qiagen), and cDNA was synthesized using the high-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Ace2 mRNA (custom assay: forward primer sequence, GGGCAAACTCTATGCTGACTGA; reverse primer, TGTGACCTTTGTACACATCTTGATTC; sequence and the probe 6-carboxyfluorescein, TTGTCTGCCACCCCACA) was determined using TaqMan reagents (Life Technologies) on a Quantstudio 5 (Applied Biosystems) in duplicates. Gene expression was normalized to Gapdh (assay ID 4352339E) using the ΔΔCT method and reported as fold change, relative to wild-type or low-fiber group. A, Renal Ace2 expression was increased with high fiber (n=6) or acetate (n=4) supplementation but not propionate (n=3) or butyrate (n=4) supplementation compared with a low-fiber diet (n=9). B, Kidney cortex was stained for ACE2 protein expression using anti-mouse ACE2 antibody (1:1000 dilution; catalog No. NBP1-76614; Novus Biologicals) in conjunction with the rabbit-specific horseradish peroxidase (HRP)/3, 3'-diaminobenzidine (DAB) Avidin Biotin Complex Detection IHC Kit (Abcam, Cambridge), in which the secondary antibody was included. The percentage of ACE2 protein levels was quantified in the renal cortex using the FIJI software (ImageJ 1.53c), specifically through a macro developed for the percentage level of DAB staining detected relative to unstained background over 6 to 8 images per animal. Negative control: no primary antibody. Scale bar, 50 μm. There was an increase in kidney cortex ACE2 staining with high fiber feeding (n=15) compared with low fiber feeding (n=4). Male mice deficient in G-protein receptor (GPR)41/GPR43/GPR109a (n=6 for real-time quantitative PCR and n=5 immunohistochemistry) fed a standard rodent chow (Barostoc 102119; Ridley Agriproducts) had reduced (C) renal Ace2 expression and a nonsignificant reduction in (D) kidney cortex ACE2 protein compared with wild-type mice (n=9 for qPCR and n=5 for immunohistochemistry). Negative control: no primary antibody. Scale bar, 50 μm. E, The gut microbiome was determined as published previously (n=15 samples; 612 639 reads in total).5 Network analysis was conducted using Calypso (version 8.84) to identify correlations between diets high or low in fiber and bacterial genera, using Spearman ρ correlation coefficients computed by 1000-fold permutations, with a false discovery rate (FDR) cutoff of 0.05 for significance. Genera associated with a high-fiber diet are colored in green, while those associated with a low-fiber diet are colored red. The relative brightness of each node indicates the significance level of the association. Edges between nodes indicate correlation, with blue edges indicating negative correlations, while positive correlations are shown as light red edges. The size of each node represents the relative abundance of each genus. F, Univariate correlation analysis with Ace2 expression was conducted using the Spearman correlation coefficient, with adjustment for multiple testing using an FDR cutoff of 0.05. Renal Ace2 expression was positively correlated with Clostridium symbiosum and Clostridium aldenese. No outliers in the data were identified using the robust regression and outlier removal (ROUT) method. Analyzed with 1-way ANOVA with Tukey post hoc test for multiple comparisons (A) and with unpaired t test (B–D). All data shown as mean±SD. *P<0.05, **P<0.01, and ****P<0.0001.An important mechanism of action of SCFAs is via ligation with metabolite-sensing G-protein–coupled receptors.5 Thus, we sought to determine whether these receptors were involved in Ace2 regulation. Given the significant redundancy between these receptors,5 we chose a newly developed CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) triple knockout model that lacks GPR41, GPR43, and GPR109A to explore this. We observed that deletion of these 3 receptors was associated with a significant decrease in renal Ace2 expression (Figure [C]). This was accompanied by less ACE2 staining of the renal cortex in the triple knockout animals compared with wild-type mice; however, this did not reach statistical significance (P=0.15, Figure [D]). The deletion of these 3 receptors did not alter ACE2 staining in the kidney medulla (data not shown). The effect of the deletion of these 3 receptors on BP and other cardiovascular parameters remains to be determined, as well as the impact of dietary fiber manipulations to ACE2 levels in their kidneys.Network analysis was performed at the genus level to identify associations between genera (determined by microbiome 16S rRNA sequencing) and intake of fiber (Figure [E]). Fiber intake was strongly associated with increased abundance of Clostridium, Bacteroides, Parabacteroides, Akkermansia, Prevotella, Proteus, and Ruminococcus genera. In contrast, animals fed a diet that was low in fiber had a comparative increased abundance of Bilophila, Oscillospira, Alistipes, Mucispirillum, and Subdoligranulum (all q<0.05). To determine whether the gut microbiota was associated with the changes observed in renal Ace2 levels, we performed Spearman correlations between Ace2 mRNA and the microbiome. Positive correlations were observed between renal Ace2 and Clostridium symbiosum (r=0.79, q=0.024; Figure [F]) and Clostridium aldenese (r=0.64, q=0.034; Figure [F])—a genus known as an SCFA producer.6 Inverse correlations were also observed between renal Ace2 expression and Alistipes massiliensis (r=−0.72, q=0.024), Mucispirillum schaedieri (r=−0.71, q=0.024), Clostridium methylpentosum (r=−0.70, q=0.024), and Subdoligranulum variabile (r=−0.68, q=0.024; data not shown). Some of these correlations were validated in animals supplemented with acetate in addition to a low-fiber diet, including Clostridium symbiosum (r=0.72, q=0.005), Mucispirillum schaedieri (r=−0.72, q=0.005), and Subdoligranulum variabile (r=−0.71, q=0.007; data not shown). Acetate supplementation also increased the levels of Clostridium symbiosum (q=0.042; fold change, 1.9). No significant correlations were observed between any genera and heart or cecum Ace2 expression.In conclusion, our data suggest that high fiber intake increases renal ACE2 via production of the SCFA acetate by the gut microbiota and sensing via G-protein–coupled receptors GPR41, GPR43, and GPR019A. Given the renoprotective effects observed with renal ACE2, these findings provide additional insights into how dietary fiber may act to protect the kidneys.Sources of FundingThis work was supported by the National Health and Medical Research Council of Australia and the National Heart Foundation.Disclosures None.FootnotesFor Sources of Funding and Disclosures, see page e55.Correspondence to: Francine Z. Marques, Hypertension Research Laboratory, School of Biological Sciences, Faculty of Science, Monash University, Melbourne, Australia. Email francine.[email protected]eduReferences1. Ortiz-Melo DI, Gurley SB. Angiotensin converting enzyme 2 and the kidney.Curr Opin Nephrol Hypertens. 2016; 25:59–66. doi: 10.1097/MNH.0000000000000182CrossrefMedlineGoogle Scholar2. Liu Z, Huang XR, Chen HY, Fung E, Liu J, Lan HY. Deletion of angiotensin-converting enzyme-2 promotes hypertensive nephropathy by targeting Smad7 for Ubiquitin degradation.Hypertension. 2017; 70:822–830. doi: 10.1161/HYPERTENSIONAHA.117.09600LinkGoogle Scholar3. Muralitharan RR, Jama HA, Xie L, Peh A, Snelson M, Marques FZ. Microbial peer pressure: the role of the Gut microbiota in hypertension and its complications.Hypertension. 2020; 76:1674–1687. doi: 10.1161/HYPERTENSIONAHA.120.14473LinkGoogle Scholar4. Marques FZ, Nelson E, Chu PY, Horlock D, Fiedler A, Ziemann M, Tan JK, Kuruppu S, Rajapakse NW, El-Osta A, et al.. High-fiber diet and acetate supplementation change the Gut microbiota and prevent the development of hypertension and heart failure in hypertensive mice.Circulation. 2017; 135:964–977. doi: 10.1161/CIRCULATIONAHA.116.024545LinkGoogle Scholar5. Kaye DM, Shihata WA, Jama HA, Tsyganov K, Ziemann M, Kiriazis H, Horlock D, Vijay A, Giam B, Vinh A, et al.. Deficiency of prebiotic fiber and insufficient signaling through Gut metabolite-sensing receptors leads to cardiovascular disease.Circulation. 2020; 141:1393–1403. doi: 10.1161/CIRCULATIONAHA.119.043081LinkGoogle Scholar6. Guo P, Zhang K, Ma X, He P. Clostridium species as probiotics: potentials and challenges.J Anim Sci Biotechnol. 2020; 11:24. doi: 10.1186/s40104-019-0402-1CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails June 2021Vol 77, Issue 6Article InformationMetrics Download: 200 © 2021 American Heart Association, Inc.https://doi.org/10.1161/HYPERTENSIONAHA.121.17039PMID: 33866801 Originally publishedApril 19, 2021 Keywordsfibrosismicrobiotaacetatedietary fibermetabolitePDF download SubjectsACE/Angiotensin Receptors/Renin Angiotensin System