Gut Bacteria Products Prevent AKI Induced by Ischemia-Reperfusion

Abstract

Short-chain fatty acids (SCFAs) are fermentation end products produced by the intestinal microbiota and have anti-inflammatory and histone deacetylase-inhibiting properties. Recently, a dual relationship between the intestine and kidneys has been unraveled. Therefore, we evaluated the role of SCFA in an AKI model in which the inflammatory process has a detrimental role. We observed that therapy with the three main SCFAs (acetate, propionate, and butyrate) improved renal dysfunction caused by injury. This protection was associated with low levels of local and systemic inflammation, oxidative cellular stress, cell infiltration/activation, and apoptosis. However, it was also associated with an increase in autophagy. Moreover, SCFAs inhibited histone deacetylase activity and modulated the expression levels of enzymes involved in chromatin modification. In vitro analyses showed that SCFAs modulated the inflammatory process, decreasing the maturation of dendritic cells and inhibiting the capacity of these cells to induce CD4(+) and CD8(+) T cell proliferation. Furthermore, SCFAs ameliorated the effects of hypoxia in kidney epithelial cells by improving mitochondrial biogenesis. Notably, mice treated with acetate-producing bacteria also had better outcomes after AKI. Thus, we demonstrate that SCFAs improve organ function and viability after an injury through modulation of the inflammatory process, most likely via epigenetic modification.

Keywords: SCFA; epigenetic modifications; ischemia-reperfusion; kidney disease; microbiota.

Figure 1.

SCFA ameliorates renal function. Mice (n=5) were subjected to kidney IRI and treated with acetate (Ac), propionate (Prop), and butyrate (But) at 0.5 hour before ischemia and at the moment of reperfusion (200 mg/kg each). Serum creatinine (A) and urea (B) were measured after 24 hours. (C) Quantification of necrosis in tubular epithelial cells. Ctl, control group.

Figure 2.

Acetate treatment decreases cellular stress, cytokine and chemokine production, and cellular infiltrates. Mice (n=5) were subjected to kidney IRI and treated with acetate. (A) Glutathione reduced (GSS) and glutathione oxidized (GSSH) ratio. mRNA levels measured by real-time PCR (B) and protein levels measured by BioPlex (C) of proinflammatory cytokines and chemokines in kidney tissue and serum protein levels of the proinflammatory cytokines and chemokines (D). (E) mRNA levels of TLR4 and biglycan measured by real-time PCR in kidney tissue. (F) Right, Western blot analysis in kidney tissue for IκBα and β-actin; each band quantified (optical densitometry) for each protein in the group and normalized by quantification of β-actin for the respective group. (G) Myeloperoxidase (MPO) levels in kidney tissue. (H) Percentage of activated DCs (CD11c⁺/CD40⁺) and macrophages (CD11b⁺/F4/80⁺) in kidney tissue analyzed by flow cytometry. Ac, acetate; Ctl, control; KC, Cxcl1; MCP-1, monocyte chemotactic protein-1.

Figure 3.

Acetate treatment decreases apoptosis levels and increases tubular proliferating cells and activation of the autophagy pathway. (A) Immunofluorescence of apoptosis levels measured by TUNEL in kidney tissue. The percentage of apoptosis was measured relative to the photographic area (original magnification, ×20). (B) Immunohistochemistry for proliferating cell nuclear antigen in kidney tissue. The percentage of positive staining for the molecule was measured relative to the photographic area (original magnification, ×20). (C) Real-time PCR in kidney tissue for BCL-2. (D) Western blot analysis in kidney tissue for ATG-7 and β-actin. The ratio of ATG-7/β-actin was calculated through quantification of each band (optical densitometry) in the group and normalized to the quantification of β-actin. (E) Ratio of mitochondrial DNA (mtDNA) and genomic DNA (gDNA) in kidney tissue. n=5 per group. Ac, acetate; Ctl, control; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling.

Figure 4.

Acetate treatment modulates epigenetic modification and upregulates GPR43 expression. (A) Real-time PCR of GPR41 and GPR43 in kidney tissue. (B) PCR-based array for the expression of genes encoding chromatin modification enzymes in kidney tissue. Comparison of gene expression levels for these genes between IR versus control (left), IR plus acetate versus IR (middle) and IR plus acetate versus IR (right). Up- and downregulated gene expression is shown in relation to the y axis group. (C) Measurement of histone deacetylase activity in kidney tissue. (D) Global DNA methylation in kidney tissue. n=5 per group Ac, acetate; Ctl, control.

Figure 5.

SCFA treatment decreases activation of BM-DCs and inhibits APC function. (A) BM-DCs were generated with GM-CSCFs (20 ng/ml) and on the seventh day were stimulated with LPS (20 ng/ml in the presence of acetate (Ac), propionate (Pr), and butyrate (But) for 24 hours. Histogram of the percentage of positive cells (A) and the mean fluorescence intensity (B) for the costimulatory molecules CD80, CD86, and CD40 (C) Spleen cells from RAGKO mice (APCs) were pretreated with LPS (20 ng/ml) in the presence of acetate, propionate, and butyrate for 24 hours. After, APCs were washed and cocultivated with carboxyfluorescein succinimidyl ester (CSFE)–labeled spleen cells from BALB/c mice for 4 days. (D) Percentage of proliferating CFSE-labeled CD4 and CD8 cells. NP, nonproliferating cells; PROL, proliferating cells. **P<0.01; ***P<0.001.

Figure 6.

SCFA treatment inhibits NFκb activation and nitric oxide production in epithelial kidney cell line. MM55.k kidney epithelial cells were stimulated with inflammatory cocktail (LPS, 10 µg/ml; zymosan, 10 µg/ml; IL-6, 50 ng/ml; IL-1β, 50 ng/ml, and TNF-α, 100 ng/ml) in the presence of acetate (Ac; 25 mM), propionate (Pr; 12 mM), and butyrate (But; 3.2 mM) for 24 hours and were evaluated by flow cytometry for (A) left: overlay histogram of NFκb activation measured through phosphorylation of the p65 subunit (percentage of p65⁺ cells) and right: percentage of the p65⁺ cells and (B) left: overlay histogram nitric oxide production (DAF-FM Diacetate) [4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate] and right: mean fluorescence intensity of the DAF-positive cells. *P<0.05.

Figure 7.

SCFA treatment inhibits ROS production in an epithelial kidney cell line after hypoxia. HK-2 human kidney epithelial cells were seeded on a cover slip and subjected to hypoxia for 24 hours in the presence of acetate (Ac; 25 mM), propionate (Pr; 12 mM), and butyrate (But, 3.2 mM). Thirty minutes before completion of the 24 hours, cells were incubated with Hypoxyprobe and MitoSOX markers and analyzed with confocal microscopy. Scale bar, 10 µm.

Figure 8.

SFCA treatment inhibits HIF-1α translocation to the nucleus, lactate production, and VEGF expression under hypoxia. HK-2 human kidney epithelial cells were seeded on a cover slip and subjected to hypoxia for 24 hours in the presence of acetate (Ac; 25 mM), propionate (Pr; 12 mM), and butyrate (But, 3.2 mM). (A) Cells were labeled with anti-human HIF-1α followed by FITC-labeled secondary antibodies and analyzed with confocal microscopy. Scale bar, 10µm. (B) Left lactate levels after hypoxia were measured in culture supernatant. Right relative expression of VEGF in normoxia and hypoxia. *P<0.05; ***P<0.001.

Figure 9.

Acetate-producing bacteria ameliorate AKI induced by IR. Mice (n=5 per group) were administered B. longum or B. adolescentis (10⁸ bacteria) by gavage for 10days before euthanasia and were subjected to kidney IRI. (A) Weight change and (B) food intake during 10 days before surgery induction. (C) Acetate levels measured in the intestinal (colon) content. (D) Acetate levels measured from plasma samples harvested pre and post probiotic treatment. (E) Serum levels of creatinine and urea in mice subjected to kidney IRI. (F) Serum levels of proinflammatory cytokines and chemokines in mice subjected to kidney IRI, treated or untreated with B. longum (BL) or B. adolescentis (BA). Ctl, control. **P<0.01.