Exploring the role of the metabolite-sensing receptor GPR109a in diabetic nephropathy

Alterations in gut homeostasis may contribute to the progression of diabetic nephropathy. There has been recent attention on the renoprotective effects of metabolite-sensing receptors in chronic renal injury, including the G-protein-coupled-receptor (GPR)109a, which ligates the short chain fatty acid butyrate. However, the role of GPR109a in the development of diabetic nephropathy, a milieu of diminished microbiome-derived metabolites, has not yet been determined. This study aimed to assess the effects of insufficient GPR109a signalling via genetic deletion of GPR109a on the development of renal injury in diabetic nephropathy. Gpr109a−/− mice or their wildtype littermates (Gpr109a+/+) were rendered diabetic with streptozotocin (STZ). Mice received a control diet or an isocaloric high fiber diet (12.5% resistant starch) for 24 weeks and gastrointestinal permeability and renal injury were determined. Diabetes was associated with increased albuminuria, glomerulosclerosis and inflammation. In comparison, Gpr109a−/− mice with diabetes did not show an altered renal phenotype. Resistant starch supplementation did not afford protection from renal injury in diabetic nephropathy. Whilst diabetes was associated with alterations in intestinal morphology, intestinal permeability assessed in vivo using the FITC-dextran test was unaltered. GPR109a deletion did not worsen gastrointestinal permeability. Further, 12.5% resistant starch supplementation, at physiological concentrations, had no effect on intestinal permeability or morphology. These studies indicate that GPR109a does not play a critical role in intestinal homeostasis in a model of type 1 diabetes or in the development of diabetic nephropathy.


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Diabetic nephropathy is a major microvascular complication of diabetes, occurring in up 50 to 30% of patients with type 1 diabetes [1]. Concomitant with the rise in diabetes and 51 obesity, the prevalence of diabetic nephropathy has been increasing rapidly, with 52 diabetic nephropathy now the leading cause of end stage renal disease worldwide [1]. 53 Despite optimal conventional management with pharmacological inhibition of the renin 54 angiotensin system (RAS), glycemic and blood pressure control, a significant proportion 55 of patients with DKD still progress over time to end stage renal failure. Thus, there is an 56 urgent need for the identification of new therapeutic options to help limit the progression 57 of this disease. 58 Recently there has been an increasing interest in the diet-gut-kidney axis, 59 whereby elements derived from the diet alter the composition of the gut microbiota and 60 production of microbial metabolites which induce effects at extra-intestinal sites, 61 including the kidneys [2,3]. It has been noted that patients with diabetes [4] and end 62 stage renal disease [5,6] have a contraction in the bacterial taxa that produce beneficial 63 short chain fatty acids (SCFAs). Furthermore, during end stage renal disease, there is 64 an increase in intestinal permeability and subsequent inflammation [7]. SCFAs act via 65 local metabolite-sensing receptors in order to reduce intestinal permeability and 66 inflammation [8,9]. The use of dietary therapies that directly target the gut microbiota to 67 increase SCFA production, including probiotics and prebiotics, have been recently 68 considered as potential adjunct interventions to limit injury in diabetic nephropathy [10]. 69 Butyrate acts as a ligand for the G-protein-coupled-receptor (GPR)109a, 70 decreasing intestinal inflammation and promoting gut epithelial barrier integrity, thus 71 GPR109a activation is considered to be protective [11]. A recent study showed that 72

Induction of Diabetes 97
Diabetes was induced at six weeks of age by five daily intraperitoneal injections of 98 streptozotocin (55 mg/kg Sigma Aldrich) in sodium citrate buffer. Diabetes was 99 confirmed by a glycated haemoglobin (GHb) greater than 8%. Two mice failed to meet 100 this cutoff and were excluded from any further analysis. Of those diabetic mice that 101 were included in the analysis, the mean GHb was 11.7% (median 11.9%).

Diet Intervention 104
Since previous studies exploring the role of resistant starch (RS) on the development of 105 renal injury have used supraphysiological doses over a short period of time, we sought 106 to supplement a dose of resistant starch that could be more reasonably expected to be 107 consumed by people (25% HAMS Hi maize 1043, equivalent to 12.5% RS) over a 108 longer time frame (24 weeks). From six weeks of age, mice received either a custom-109 made control diet (CON) or a high fiber diet supplemented with resistant starch 110 prepared by Speciality Feeds (Perth, Western Australia, Australia). Both of these semi-111 pure diets were formulated based on a modified AIN93G growth diet for rodents. These 112 diets were isocaloric, had equivalent protein, provided as 20% g/g casein, and fat, 113 provided as 7% g/g canola oil. Each diet contained 5% g/g sucrose, 13.2% g/g 114 dextrinised starch and 7.4% g/g cellulose. A resistant starch supplemented diet (SF15-115 015) was formulated with 25% g/g Hi-maize 1043, whilst the CON diet (SF15-021) 116 contained an additional 20% g/g regular starch and 5% g/g cellulose in order to maintain 117 caloric equivalency between diets. Hi-maize 1043, an RS2 starch prepared from high 118 amylose maize starch (HAMS) which contains 50% resistant starch [12], was provided 119 as a raw ingredient by Ingredion (Westchester, IL, USA). Mice received these 120 experimental diets ad libitum for 24 weeks. 121 122

Tissue collection 123
At the end of the study period, mice were anaesthetised by an intraperitoneal injection 124 of 100 mg/kg body weight sodium pentobarbitone (Euthatal; Sigma-Aldrich, Castle Hill, 125 Australia) followed by cardiac exsanguination. Following cardiac exsanguination, blood 126 was immediately centrifuged at 6000 rpm for 6 minutes and plasma was snap frozen on 127 dry ice and stored at -80°C. Kidney sections were fixed in neutral buffered formalin 128 Glycated haemoglobin 137 Glycated haemoglobin (GHb) was measured in blood collected at cull using a Cobas b 138 101 POC system (Roche Diagnostics, Forrenstrasse, Switzerland) according to the 139 manufacturer's instructions. The Cobas b 101 POC system has a detection range of 140 between 4-14%, with any sample with a GHb less than 4% designated as low and 141 samples with a GHb of greater than 14% designated high. 142 143

In Vivo Intestinal Permeability Assay 144
Intestinal permeability was assessed in vivo using the previously described dextran 145 FITC technique [13], during the week prior to cull. In brief, mice were fasted for a 146 minimum of four hours and received an oral gavage of a 125 mg/mL solution of dextran 147 FITC equivalent to 500 mg/kg body weight. After one hour, approximately 120 μL was 148 collected from the tail vein using heparinised capillary tubes. Blood was centrifuged at 149 6000 rpm for 6 minutes, plasma collected and the fluorescence in plasma samples was 150 determined in relation to a standard dilutions set, using a fluorescence 151 spectrophotometer (BMG Labtech, Ortenberg, Germany) set to excitation 490nm, 152 emission 520nm. The intra-and interassay coefficients of variation were 3.2 and 8.9%, 153 respectively. 154 155

Body Composition 156
Fat mass and lean body mass were determined using a 4-in-1 EchoMRI body 157 composition analyser (Columbus Instruments, Columbus, OH, USA), which measures 158 fat mass, lean mass and total water content using nuclear magnetic resonance 159 relaxometry [14]. The weight of mice prior to being placed in the body composition 160 analyser was used for calculation of percentage fat and lean mass. 161 162

Metabolic Caging, Urine and Plasma Analyses 163
After 23 weeks of experimental diet, mice were housed individually in metabolic cages 164 (Iffa Credo, L'Arbresle, France) for 24 hours for urine collection and measurement of 9 urine output and food and water intake. The animals received ad libitum access to food 166 and water during this period. Urine was stored at -80°C until required for analyses. 167 Urinary albumin was determined using a mouse specific ELISA (Bethyl Laboratories,168 Montgomery, TX, USA) according to the kit protocol. The intra-and interassay 169 coefficients of variation were 7.3 and 8.9%, respectively. Urinary monocyte 170 chemoattractant protein-1 (MCP-1) was measured using a commercially available 171 ELISA kit (R&D Systems, Minneapolis MN, USA) as per the kit protocol. The intra-and 172 interassay coefficients of variation were 2.8 and 4.0%, respectively. Blood urea nitrogen 173 was analysed using a commercially available colorimetric urea assay (Arbor Assays, 174 Ann Arbor, MI, USA) as per the kit protocol. The intra-and interassay coefficients of 175 variation were 4.5 and 3.9%, respectively. Plasma cystatin C was determined using a 176 commercially available ELISA from R&D Systems. The intra-and interassay coefficients 177 of variation were 3.8 and 8.3%, respectively 178 179

Kidney and Ileum Histology 180
Kidneys were fixed in 10% (v/v) neutral buffered formalin prior to embedding in paraffin. 181 Kidney sections (3 μm) were stained with periodic acid-Schiff (PAS) and assessed in a 182 semiquantitative manner, whereby a blinded researcher assessed the level of 183 glomerulosclerosis for each glomerulus and assigned an integer score of between 1 and 184 4, indicative of the level of severity of glomerulosclerosis. Twenty-five glomeruli were 185 scored per animal, and these scores were averaged to provide a glomerulosclerosis 186 score index (GSI) for each animal, as previously described [15]. Ileal sections were 187 fixed in 4% paraformaldehylde for 24 hours, followed by a transfer to 4% sucrose, and 188 subsequent embedding in paraffin. Ileal sections (5 μm) were stained with haemotoxylin 189 and eosin (H&E) and images were captured using a brightfield microscope (Nikon 190 Eclipse-Ci; Nikon, Tokyo, Japan) coupled with a digital camera Nikon,191 Tokyo, Japan). Morphological measurements of villus height and crypt depth were 192 conducted using ImageJ (Version 1.52a). Villus height was measured from the topmost 193 point of the villus to the crypt transition, whilst the crypt depth was measured as the 194 invagination between two villi to the basement membrane.

Metabolic and Phenotypic parameters 211
Consistent with the diabetic phenotype, mice with STZ-induced diabetes had increased 212 glycated haemoglobin and decreased body weight (Table 1). Diabetes was associated 213 with a decrease in relative fat mass and an increased lean mass and increased 24-hour 214 urine output and water intake (Table 1). Neither deletion of the GPR109a receptor nor 215 consumption of the high fiber (resistant starch) diet was associated with changes in 216 glycated haemoglobin, body weight or body composition. Diabetes was associated with 217 an increased liver weight and a decreased spleen weight (Table 1). Mice with diabetes 218 had an increase in small intestine length (p<0.001, Fig 1A) caecum length (p<0.0001, 219 Fig 1B), small intestine weight (p<0.0001, Fig 1D), caecum weight (p<0.0001, Fig 1E) 220 and colon weight (p<0.0001, Fig 1F). Consumption of the fiber supplement led to an 221 increase in caecal weight and length in diabetic mice, but not in non-diabetic mice (Fig  222   1B, 1E). Resistant starch supplementation was also associated with an increase in 223 colon weight (Fig 1F). 224 225

Renal Injury and Inflammation 226
Diabetic mice exhibited the hallmarks of diabetic nephropathy, including albuminuria 227 (p<0.0001, Fig 2A), increased blood urea nitrogen (p<0.0001, Fig 2B), renal 228 hypertrophy (p<0.0001, Fig 2C) and hyperfiltration (p<0.0001, Fig 2D). Genetic ablation 229 of Gpr109a resulted in no change in the renal phenotype in diabetic mice (Fig 2A-D). 230 Likewise, the high fiber diet in the context of diabetes did not alter hallmarks of diabetic 231 nephropathy (Fig 2A-D). Diabetes was associated with an increase in the inflammatory 232 marker MCP-1, whilst there was no effect of either resistant starch supplementation or 233 deletion of Gpr109a (p<0.0001, Fig 2E). Assessment of renal histology revealed an 234 overall increase in glomerulosclerosis in the diabetic setting, however, genetic ablation 235 of GPR109a or supplementation with the high fiber diet did not impact on renal 236 structural injury (p<0.0001, Figure 3A  with a trend towards increased villi height (p=0.06, Fig 4A). The high fiber diet led to a 244 trend towards an increase in villi height in non-diabetic wildtype and in Gpr109a-/-245 diabetic mice (Fig 4A). Despite these morphological changes in the ileum with diabetes, 246 diabetes did not induce any alteration in intestinal permeability, as measured by an in 247 vivo intestinal permeability procedure (dextran-FITC, Fig 4D) or as determined by gene 248 expression of the tight junction proteins occludin (Ocln, Fig 4E) or zonulin (Tjp-1) (Fig  249   4F). 250

Discussion 251
The current study explored the effects genetic deletion of the butyrate receptor, 252 GPR109a, had on the development of chronic renal injury in diabetic nephropathy. No 253 effect on the diabetic renal phenotype was observed with deletion of Gpr109a. 254 Surprisingly, there was no change in intestinal permeability or morphometry as a result 255 of Gpr109a deletion. Furthermore, a high fiber 12.5% resistant starch diet was not 256 effective in reducing renal injury in the setting of diabetes. 257 This is the first study to assess the effects of deletion of GPR109a on diabetic 258 nephropathy and these findings show that deletion of this receptor was not associated 259 with any change in renal injury in long term studies (24 weeks). Given the hypothesis 260 that renal injury would occur downstream of alterations in intestinal permeability and 261 there was no effect of Gpr109a deletion on in vivo assessment of intestinal permeability, 262 this should perhaps not come as a surprise finding. In vitro, Gpr109a knockdown inhibits 263 butyrate-induced increases in the tight junction protein Claudin-3, suggesting that 264 GPR109a may have a role in the integrity of the intestinal epithelial barrier [9]. However 265 in vivo, whilst deletion of GPR109a was associated with a trend towards an increase in 266 intestinal permeability in an induced food allergy model [16] there was no effect in 267 otherwise healthy mice [8], indicating that deletion of the GPR109a receptor alone is 268 insufficient to alter intestinal permeability. There is redundancy in the metabolite-269 sensing GPCR family, with butyrate being recognised by GPR109a, GPR41 and GPR43 270 receptors. Indeed, it has been suggested that given this redundancy, single knockout 271 models are insufficient to fully elucidate the effects of these receptors, and that double 272 or triple knockout models are required [17].