Male Wild-type (WT) C57BL/6 mice were obtained from the Animal Resource Centre (Perth, Western Australia). Gpr43 ^−/− and Gpr109A ^−/− mice on a C57BL/6 background were bred and maintained in our facility. Animals were housed in a specific pathogen-free facility at the University of Sydney. Mice aged 7–9 weeks old were used. All experiments were performed in accordance with approval from the University of Sydney Animal Ethics Committee. Animals from in vivo experiments are from a sample examined previously, however with additional unpublished histological assessment (Li et al., 2020).

Mice were fasted for 4 h prior to intraperitoneal injection of Streptozotocin (STZ), 55 mg/kg daily, for 5 consecutive days. Non-diabetic controls received volume and pH matched citrate buffer. Mice with a blood glucose level (BGL) > 20 mmol/L were used to assess diabetic kidney injury. Blood glucose was measured with an Accu-Check glucometer (Roche Diagnostics) using tail vein blood. All mice were euthanized 12 weeks after injection.

Three weeks after STZ injection, mice were randomized to receive sodium acetate (SA) 100mM, sodium butyrate (SB) 50 mM or sodium propionate (SP) 100 mM (Sigma-Aldrich) ad libitum in drinking water. Control mice received pH and sodium matched water. Drinking solutions were refreshed three times per week.

Periodic acid-Schiff’s (PAS) and Picro-Sirius Red (PSR) staining were performed on 3 μm and 5 μm formalin-fixed kidney sections respectively. Glomerular tuft area (A_G) was measured in 20 glomerular profiles per mouse using DP2-BSW v2.2 (OLYMPUS). Glomerular volume (V_G) was calculated using the formula: VG = (β/κ) × (AG) ^3/2, where β = 1.38 (shape coefficient for spheres) and κ = 1.1 (size distribution coefficient) (Weibel and Gomez, 1962). Glomerular extracellular matrix (ECM) was defined as the PAS-positive area quantified by image analysis software (ImagePro Premier 9). Interstitial collagen was assessed by point counting using an ocular grid at ×400 magnification in 20 consecutive fields of renal cortex. Fibrosis score was expressed as percentage of positive crosses (McWhinnie et al., 1986).

Mouse kidney TECs were isolated and cultured from WT C56BL/6, Gpr43−/− and Gpr109A−/− mice as described previously (Li et al., 2020). In brief, kidneys were perfused with saline then removed. Kidney cortices were dissected into 1 mm³ pieces and digested in HBSS containing 3 mg/mL of collagenase at 37°C for 25 min, followed by washing in DMEM/F12 medium (Invitrogen). The kidney digest was washed through a series of sieves (mesh diameters: 250, 150, 75 and 40 µm) then spun down at 300 g for 5 min. The cell pellet was re-suspended in defined K1 medium: DMEM/F12 supplemented with 25 ng/mL epidermal growth factor, 1 ng/mL PGE1, 5 x 10-11M triiodothyronine, 5 x 10-8M hydrocortisone (Sigma-Aldrich), ITSS media supplement, 1% penicillin/streptomycin, 25 mM HEPES, and 5% FCS (Invitrogen). Cell suspension was then seeded on cell culture Petri dishes and incubated at 37°C for 2–3 h to facilitate adherence of contaminating glomeruli. The non-adherent tubules were collected and cultured on collagen-coated Petri dishes (BD Biosciences) in K1 medium. Expression of the epithelial cell marker cytokeratin was verified by immunofluorescent staining with an anti-cytokeratin antibody (Sigma-Aldrich). Cells were >95% cytokeratin positive.

Kidneys were perfused with 10⁷ Dynabeads and the cortex was cut into small pieces (1–2 mm³) and digested in 2 mg/mL collagenase at 37°C for 30 min. The collagenase-digested tissue was passed through a 100 µm sieve and centrifuged at 200g. The pellet was resuspended and glomeruli-containing Dynabeads were gathered in a magnetic field. The glomeruli were pipetted onto a 40 µm nylon sieve to remove free Dynabeads and collected by washing through an inverted nylon sieve. Isolated glomeruli were seeded onto collagen-coated culture dishes (BD Biosciences) in DMEM/F-12 medium containing 5% fetal bovine serum supplemented with 0.5% insulin-transferrin-sodium selenite (ITSS), 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen) and incubated at 37°C. The cultured cells were examined for the podocyte markers podocin and nephrin by immunofluorescent staining. Cells were >95% positive for these markers.

Cultured podocytes or TECs at 80% confluence were rinsed and incubated with serum-free DMEM/F12 medium with 0.5% ITSS supplement for podocytes, or serum free K1 medium for TECs for 48 h. Cells were then exposed to 30 mM D-glucose (Invitrogen) or mannitol (5.5 mM glucose + 24.5 mM mannitol) in the presence and/or absence of acetate (25 mM), propionate (12 mM) or butyrate (3.2 mM) for 12 h (Andrade-Oliveira et al., 2015). After stimulation, cells were harvested for RNA extraction and assessment of mRNA expression by RT-PCR.

Total RNA was extracted from kidney tissue or cells using TRIzol reagent (Invitrogen, CA) according to the manufacturer’s instructions. cDNA was synthesized using oligo (dT)₁₆ primers (Applied Biosystems) and the SuperScript III reverse transcriptase kit (Invitrogen) according to the manufacturer’s instructions. cDNA was amplified in universal Master Mix (Applied Biosystems) with gene-specific primers and probes, using the Roche Lightcycler 480 (Roche Applied Science). Specific TaqMan primers and probes for IL6, TNF-α, CCL2, CXCL10, TGF-β, fibronectin and GAPDH were used. Results were normalised to GAPDH expression.

All results are expressed means ± SD or mean ± SEM. Data was analysed using student’s two-tailed t-tests, or one- or two-way analysis of variance (ANOVA) with posthoc Bonferroni’s correction (Graph Pad Software, San Diego, CA, United States of America) where appropriate. A p-value of < 0.05 was considered statistically significant.

WT, Gpr43−/− and Gpr109A−/− mice were equally susceptible to STZ -induced diabetes, displaying similar profiles in progression of hyperglycaemia (Figure 1A) and weight change (Figure 1B) over the 12-week experimental period. SCFA treatment did not alter the glucose profile relative to diabetic controls.

In a previous study, we demonstrated that SCFAs protected against development of albuminuria and histological damage in STZ induced DN (4). SCFAs are known to exert some of their effects through metabolite sensing GPRs to elicit cellular responses. To determine whether these signaling pathways are involved in SCFA mediated renoprotection, we treated diabetic WT, Gpr43−/− and Gpr109A−/− mice with their primary ligands acetate and butyrate, respectively, supplemented in drinking water.

WT B6 mice treated with acetate were protected from development of DN with a lower kidney to body weight ratio, less glomerular hypertrophy and mesangial cellularity compared to WT diabetic controls (Figures 2A–C). However, acetate treatment in diabetic Gpr43−/− mice was ineffective, with similar histological injury severity to control WT diabetic mice, as evidenced by increased glomerular volume and mesangial cellularity (Figure 2E). Gpr109A−/− diabetic mice treated with butyrate exhibited similar kidney to bodyweight ratios to diabetic WT controls but developed less glomerular hypertrophy and mesangial cellularity compared to WT diabetic controls (Figures 3A–C, E). However there remained a significant difference between WT and Gpr109A−/− butyrate treated diabetic mice, indicating that butyrate provided partial protection against DN in the absence of Gpr109A−/−.

Quantification of interstitial fibrosis using Picro-Sirius Red staining revealed substantial interstitial collagen deposition in diabetic WT kidneys compared to non-diabetic WT kidneys. Diabetic Gpr43−/− kidneys revealed significant tubulointerstitial fibrosis with collagen deposition of similar severity compared with WT diabetic controls despite acetate treatment (Figures 2D, F). In contrast, diabetic Gpr109A−/− kidneys from butyrate treated mice revealed modest degrees of fibrosis which was reduced in severity compared with control WT diabetic kidneys (Figures 3D, F).

To confirm whether SCFAs directly mediate protective responses through these signaling pathways in intrinsic kidney cells under hyperglycaemic conditions, primary tubular epithelial cells (TEC) and podocyte cultures from WT, Gpr43−/− and Gpr109A−/− kidneys were treated with acetate, butyrate, or propionate under normal or high glucose conditions. Exposure to high glucose increased mRNA expression of pro-inflammatory genes (IL6, TNFa) and pro-fibrotic genes (TGFβ, Fibronectin) 2-7-fold compared to osmotic controls (Figures 4, 5).

We have previously shown that SCFAs attenuated pro-inflammatory and pro-fibrotic responses in primary TECs exposed to high glucose. Acetate, butyrate, and propionate attenuated glucose induced upregulation of IL6, Fibronectin and TGFβ in WT TECs (Li et al., 2020). However, this effect was not seen in acetate treated Gpr43−/− TECs, with no reduction in IL6, Fibronectin or TGFβ expression compared to osmotic controls, suggesting a GPR43 dependent signaling mechanism (Figure 4A). Acetate treatment of Gpr109A−/− TECs resulted in diminished expression of IL6, CCL2, CXCL10, TGFβ and Fibronectin, consistent with the effect seen in WT TECs (Figure 4B).

Butyrate’s effects on pro-fibrotic responses appear GPR109A dependent, with butyrate unable to downregulate Fibronectin or TGFβ expression in Gpr109A−/− TECs (Figure 4D). In contrast, butyrate treatment in the absence of GPR109A or GPR43 had no effect pro-inflammatory gene expression. Butyrate attenuated IL6 expression in WT, Gpr109A−/− and Gpr43−/− derived TECs (Figures 4C, D), indicating butyrate facilitates its beneficial effects via both GPCRs and additional pathways.

RT-PCR demonstrated glucose induced upregulation of pro-inflammatory and pro-fibrotic mRNA gene expression in primary podocytes (Figure 5). Whilst butyrate treatment attenuated expression of IL6, TNFα and CXCL10 expression in WT podocytes, it was ineffective Gpr109A−/− podocytes (Figure 5D). Absence of GPR109A did not alter expression of pro-inflammatory genes following treatment with acetate under high glucose conditions, as compared to WT controls (Figure 5B).

In contrast, whilst acetate and butyrate both suppressed expression of pro-fibrotic genes (Fibronectin, TGFβ) compared to osmotic controls in WT podocytes, this effect appeared independent of GPR109A, and was instead mediated through GPR43. Both SCFAs were able to suppress Fibronectin and TGFβ mRNA expression in Gpr109A−/− podocytes (Figures 5B, D) but were unable to alter TGFβ and Fibronectin expression in Gpr43−/− podocytes (Figures 5A, C).