Novel Differences in Renal Gene Expression in a Diet Induced Obesity Model of Diabetic Nephropathy
Authors: Kuhns Victoria L Halperin , Pluznick Jennifer L 
Published: November 2023

Diabetic nephropathy (DN) is a complication of diabetes mellitus. To identify novel genes differentially regulated in the early stages of DN using an unbiased approach, male C57bl/6 mice were fed high fat diet (HFD, 60% kcal fat) or matched control diet (CD, 10% kcal fat) for 9 weeks starting at 6 weeks of age to induce metabolic syndrome and DN. HFD mice had impaired glucose tolerance in a glucose tolerance test (Area Under the Curve, p=0.01, n=5 mice per group), demonstrating a pre‐diabetic like phenotype. HFD mice trended towards enlarged glomeruli (p=0.09, n= >50 glomeruli from 4 mice per group) and an increase in glomerular collagen infiltration. No differences were seen in mesangial matrix deposition between HFD and CD mice. HFD mice also had an increased glomerular filtration rate/body weight (p=0.01, n= 4 mice per group). These findings are consistent with early DN. Next, RNASeq was performed on RNA from the renal cortex (n=4 CD, n=4 HFD). We identified 1134 genes differentially expressed in the renal cortex on HFD; of the 22 genes with the greatest fold changes, only 12 have been previously been studied in a diabetic context. We then utilized Taqman real‐time (RT) PCR arrays to confirm our initial findings. This secondary screen included the 9 most upregulated and 11 most downregulated genes from the RNA Seq data, with an additional 11 genes altered by RNA Seq and of interest to our group (primarily sensory receptors and G proteins). These arrays (31 genes + 18S control) were used to screen additional tissue samples (renal medulla, liver, heart, n=3 CD, n=3 HFD), and an expanded cohort of renal cortex samples (n=5 CD, n=5 HFD). All 9 of the genes significantly upregulated with HFD by RNA Seq were also upregulated by RT‐PCR (Atp12a, Ccl28, Ctxn3, Cyp2b10, Lhx2, Popdc3, Ptpn5, Sorcs1, Synpr; p<0.05). However, of the remaining 22 genes (downregulated + other genes of interest) only 2 were confirmed by RT‐PCR (Gsta2, Slc7a12; p<0.05). Other tissues (renal medulla, heart, and liver) were then screened for these 11 genes to determine if these changes are cortex‐specific. There were 3 genes for which changes in the renal medulla mirrored changes seen in the renal cortex. However, only 1 gene changed (p<0.05) in the same direction as in the renal cortex in both the liver (Gsta2) and the heart (Cyp2b10). These data demonstrate that our RNA Seq data regarding upregulation were more reproducible than those regarding downregulation, and that changes are largely kidney specific. Furthermore, these data imply that renal changes are downstream of metabolic changes, and not non‐specific global changes due to an alteration in diet. Changes were then examined in samples from CD and HFD female mice, which were either diet matched (DM; fed the diet described above) or weight matched (WM; fed diet for 12 weeks from weaning). Only 1 gene (Cyp2b10, p<0.05) was altered as expected in the DM females, while a different gene (Synpr, p<0.05) was altered in the WM females, indicating that the changes seen in our initial cohort are likely male‐specific, in agreement with the fact that female mice fail to develop metabolic syndrome on this diet. Thus, we have identified genes in the renal cortex which are differentially regulated in DN, the majority of which are sex‐specific, and many of which have never before been studied in a diabetic context.