Toll-like receptor 4 mutation mitigates gut microbiota-mediated hypertensive kidney injury

Abstract

Hypertension-associated dysbiosis is linked to several clinical complications, including inflammation and possible kidney dysfunction. Inflammation and TLR4 activation during hypertension result from gut dysbiosis-related impairment of intestinal integrity. However, the contribution of TLR4 in kidney dysfunction during hypertension-induced gut dysbiosis is unclear. We designed this study to address this knowledge gap by utilizing TLR4 normal (TLR4N) and TLR4 mutant (TLR4M) mice. These mice were infused with high doses of Angiotensin-II for four weeks to induce hypertension. Results suggest that Ang-II significantly increased renal arterial resistive index (RI), decreased renal vascularity, and renal function (GFR) in TLR4N mice compared to TLR4M. 16 S rRNA sequencing analysis of gut microbiome revealed that Ang-II-induced hypertension resulted in alteration of Firmicutes: Bacteroidetes ratio in the gut of both TLR4N and TLR4M mice; however, it was not comparably rather differentially. Additionally, Ang-II-hypertension decreased the expression of tight junction proteins and increased gut permeability, which were more prominent in TLR4N mice than in TLR4M mice. Concomitant with gut hyperpermeability, an increased bacterial component translocation to the kidney was observed in TLR4N mice treated with Ang-II compared to TLR4N plus saline. Interestingly, microbiota translocation was mitigated in Ang-II-hypertensive TLR4M mice. Furthermore, Ang-II altered the expression of inflammatory (IL-1β, IL-6) and anti-inflammatory IL-10) markers, and extracellular matrix proteins, including MMP-2, -9, -14, and TIMP-2 in the kidney of TLR4N mice, which were blunted in TLR4M mice. Our data demonstrate that ablation of TLR4 attenuates hypertension-induced gut dysbiosis resulting in preventing gut hyperpermeability, bacterial translocation, mitigation of renal inflammation and alleviation of kidney dysfunction.

Keywords: Gut dysbiosis; Hypertension; Kidney function; MMP; TIMP; Tight junction.

Fig. 1.. Echography determination of mean renal arterial blood velocity in TLR4N, TLR4M mice treated with or without Ang-II.

(A): representative waveforms of TLR4N and TLR4M mice treated with or without Ang-II. Velocity was detected from pulsed Doppler waveforms that were acquired from the renal artery in isoflurane-anesthetized mice. (B) resistive index calculated as (PSV - EDV)/PSV, using Vevo 2100 Workstation Software. Bars are mean ± SD (n = 6); *p <0.05 vs. TLR4N+Saline, †p <0.05 vs. TLR4N+Ang-II.

Fig. 2.. TLR4 mutation improves Ang-II-induced attenuation of vascular density.

Renal vascular architecture was measured by using the Carestream Molecular Imaging In-vivo Multispectral system after infusion of 0.6 ml of barium sulfate (0.1 mg/ml) in the infrarenal aorta through a PE10 tube. (A) i) Barium Angiography ii) VesSeg analysis. Vascular density was quantified utilizing Vessel Segmentation software. Loss of interlobular arteries is shown by red arrows and recovery by yellow arrows (B) Bar diagram represents the mean percentage change ± SD (n = 6). Values were obtained after background subtraction and plotted using TLR4N+Saline as control; *p<0.05 vs. TLR4N+Saline, †p<0.05 vs. TLR4N+Ang-II.

Fig. 3.. TLR4 deficiency improved Ang-II induced attenuation of glomerular filtration rate (GFR).

Live, conscious mice were anesthetized with isoflurane and injected with FITC-sinistrin (15 mg/100 g) into the femoral artery. The detector was fixed to the area removed of hair and mice were put back into cages while the detector recorded the data for 2 hours. GFR was calculated after obtaining the half-life of excreted FITC-sinistrin. Bar graphs are mean ± SD, n=6/group; *p<0.05 vs. TLR4N+Saline, †p<0.05 vs. TLR4N+Ang-II.

Fig. 4.. 16 S rRNA profiling of gut microbiome in TLR4N and TLR4M mice treated with or without Ang-II.

(A) Relative abundance of total bacteria at the phylum level for each group. (B) Relative abundance of total bacteria at the class level for each group. Abundance is expressed as a percentage; n=6/group.

Fig. 5.. Ang-II treatment led to gut barrier dysfunction and promoted bacterial translocation in the kidney which is prevented by TLR4 deficiency.

Immunostaining for tight junction proteins (A) ZO-1, (B) Claudin-1, and Occludin showed reduced expression and localization in the colon (white arrows) in TLR4N mice treated with Ang-II, while TLR4M preserves the expression of these proteins. Magnification 60X. Scale bars, 40 μm. (C) FITC-dextran level in the plasma. Mice were fed FITC-dextran by oral gavage at a concentration of 44 mg/100 g. Mice were starved of water 12 hours before the procedure to minimize variability. After 4 hours, mice were anesthetized, and blood was drawn from the inferior vena cava. Plasma was subsequently separated from the blood, diluted 1:2 with PBS, and the concentration of FITC was determined. Bar graphs represent plasma FITC-dextran level in all mice groups showing that Ang-II treatment promoted increased gut leakiness in TLR4N mice compared to TLR4M mice. Data mean ± SD, n=6/group; *p<0.05 vs. TLR4N+Saline, †p<0.05 vs. TLR4N+Ang-II. D) Bacterial translocation from gut to kidney. The presence of bacteria was determined by PCR using 16 S rRNA primers specific to bacterial rRNA. Genomic DNA (gDNA) was extracted from mice kidneys, purified, and set up for PCR reaction using a 100 ng sample. At the end of the PCR run, samples were loaded onto 1.5 % agarose gel and imaged. 18 s rRNA primers were used as a control. (E) Bar diagram data mean ± SD, n=6/group; *p<0.05 vs. TLR4N+Saline, †p<0.05 vs. TLR4N+Ang-II.

Fig. 6.. TLR4M attenuated Ang-II-induced renal inflammation.

(A) Immunostaining of localized M1 macrophage marker CD40 and M2 macrophage marker CD206 in the kidney. Magnification 60X. Scale bars, 40 μm. (B) Inflammatory markers IL-1β, IL-6, and IL-10 gene expression were assessed by qPCR. Data mean ± SD, n=6/group; *p<0.05 vs. respective TLR4N+Saline, †p<0.05 vs. respective TLR4N+Ang-II.

Fig. 7.. TLR4M mitigated Ang-II-induced metalloproteinases expression in the kidney.

(A) MMP-2, –9, –14 and (B) TIMP-1, –2 gene expression was assessed by qPCR. Bar graphs represent mean ± SD, n=6/group; *p<0.05 vs. respective TLR4N+Saline, †p<0.05 vs. respective TLR4N+Ang-II. (C) MMP-2, –9, –14, and TIMP-1, –2 protein expression were assessed by Western blot. (D) and (E) Data were normalized to GAPDH and bar diagrams are presented as mean ± SD, n=6/group; *p<0.05 vs. respective TLR4N+Saline, †p<0.05 vs. respective TLR4N+Ang-II.

Fig. 8.. Schematic of findings:

Ang-II hypertension induced gut dysbiosis resulting in leakage of bacterial components into the kidney. The bacterial translocation induced kidney inflammation and remodeling leading to renal dysfunction. TLR4 mutation preserves the gut integrity and thus protects the kidney from dysbiosis-related kidney dysfunction in hypertension.