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Charting Novel Cellular Subpopulations in the Renal Cortex of Diabetic Nephropathy in a Mouse Model Through Single-Cell RNA Sequencing

ABSTRACT: Type 1 Diabetes Mellitus (T1DM) is a prevalent autoimmune disorder character ized by the destruction of insulin-producing cells, often leading to diabetic kidney disease (DKD). Despite extensive research, the cellular mechanisms underpinning DKD remain poorly understood. This study aims to elucidate the cellular het erogeneity and specific cell populations involved in the renal cortex of diabetic mice using single-cell RNA sequencing. Kidney cortex cells from streptozotocin (STZ)-induced diabetic mice and healthy controls were isolated and subjected to single-cell RNA sequencing. Statistical analyses were performed to identify distinct cellular subsets and evaluate changes in cell population dynamics. Immunohistochemistry and renal function evalua tions via Masson and Hematoxylin and Eosin staining were employed to validate the sequencing data. We first identified 11 distinct cellular subsets within the renal cortex. Diabetic mice exhibited a significant increase in distal convoluted tubule cells compared to controls. Additionally, a marked decrease in immune-related cells, including T and Blymphocytes, neutrophils, and macrophages, was observed. Cell communication analysis highlighted the role of the VISFATIN signaling pathway, particularly the Nampt-Insr gene interaction, in the diabetic renal cortex. Our findings reveal a unique cellular and molecular landscape in the renal cortex of diabetic mice, emphasizing altered cell populations and signaling path ways critical in DKD progression. This study enhances our understanding of T1DM-induced renal alterations and suggests potential targets for therapeutic intervention to mitigate kidney complications in diabetes.

ORGANISM(S): Mus musculus

PROVIDER: GSE301966 | GEO | 2026/07/06

REPOSITORIES: GEO

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Project description:Dyslipidemia is a significant risk factor for progression of diabetic kidney disease (DKD). To identify individual lipids and lipid networks that may be involved in DKD progression, we performed untargeted lipidomic analysis of kidney cortex tissue from diabetic db/db and db/db eNOS-/- mice along with nondiabetic littermate controls. A subset of mice were treated with the renin-angiotensin system (RAS) inhibitors, lisinopril and losartan, which improves the DKD phenotype in the db/db eNOS-/- mouse model. Of the three independent variables in this study, diabetes had the largest impact on overall lipid levels in the kidney cortex, while eNOS expression and RAS inhibition had smaller impacts on kidney lipid levels. Kidney lipid network architecture, particularly of networks involving glycerolipids such as triacylglycerols, was substantially disrupted by worsening kidney disease in the db/db eNOS-/- mice compared to the db/db mice, a feature that was reversed with RAS inhibition. This was associated with decreased expression of the stearoyl-CoA desaturases, Scd1 and Scd2, with RAS inhibition. In addition to the known salutary effect of RAS inhibition on DKD progression, our results suggest a previously unrecognized role for RAS inhibition on the kidney triacylglycerol lipid metabolic network. Keywords: Dyslipidemia is a significant risk factor for progression of diabetic kidney disease (DKD). To identify individual lipids and lipid networks that may be involved in DKD progression, we performed untargeted lipidomic analysis of kidney cortex tissue from diabetic db/db and db/db eNOS-/- mice along with non-diabetic littermate controls. A subset of mice were treated with the renin-angiotensin system (RAS) inhibitors, lisinopril and losartan, which improves the DKD phenotype in the db/db eNOS-/- mouse model. Of the three independent variables in this study, diabetes had the largest impact on overall lipid levels in the kidney cortex, while eNOS expression and RAS inhibition had smaller impacts on kidney lipid levels. Kidney lipid network architecture, particularly of networks involving glycerolipids such as triacylglycerols, was substantially disrupted by worsening kidney disease in the db/db eNOS-/- mice compared to the db/db mice, a feature that was reversed with RAS inhibition. This was associated with decreased expression of the stearoyl-CoA desaturases, Scd1 and Scd2, with RAS inhibition. In addition to the known salutary effect of RAS inhibition on DKD progression, our results suggest a previously unrecognized role for RAS inhibition on the kidney triacylglycerol lipid metabolic network.

Project description:Background: Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus and a leading cause of chronic kidney disease and end stage renal disease. One potential mechanism underlying cellular dysfunction contributing to kidney disease is aberrant post-translational modifications, such as lysine acetylation. Lysine acetylation is associated with cellular metabolic flux and is thought to be altered in patients with diabetes and dysfunctional renal metabolism. Methods: A novel technique was adapted to quantify sites of N-acetylation from formalin-fixed paraffin-embedded kidney explant tissue from patients with diabetic kidney disease and non-diseased donors (n=5 and n=7, respectively). Multiple methodologies were integrated to extract viable proteins from formalin-fixed paraffin-embedded kidney explant tissue followed by enrichment and isolation. Proteomic and acetylomic profiles were then quantified via LC-MS/MS analysis. Results: Quantitative proteomic analysis of FFPE tissues identified 840 total proteins with 260 of those significantly changing and our acetylomic analysis quantified 290 acetylated peptides with 98 of those significantly changing in human diabetic kidneys. Lysine acetylation is known to regulate protein function, providing a mechanism by which proteins respond to cellular metabolic status. Protein pathways found to be impacted in DKD patients revealed an association with numerous metabolic pathways, specifically mitochondrial function, EIF2 signaling, oxidative phosphorylation, lipid metabolism, sirtuin signaling, and LXR/RXR activation, each of which are intimated to play a significant role in the pathogenesis of DKD. Differential protein acetylation in DKD patients reflected biochemical processes associated with sirtuin signaling, valine, leucine, and isoleucine degradation, lactate metabolism, oxidative phosphorylation, and ketogenesis. Conclusions: Here, we establish a quantitative acetylomics platform for protein biomarker discovery in formalin-fixed and paraffin-embedded biopsies of kidney transplant patients suffering from diabetic kidney disease. Our analysis provides a novel platform for quantifying enriched post-translational modifications in preserved explant tissues and includes optimizations for addressing protein crosslinking, paraffin removal, and formalin-derived formaldehyde protein adducts combined with immunoprecipitation of acetyl-peptides and total protein quantitation.

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Charting Novel Cellular Subpopulations in the Renal Cortex of Diabetic Nephropathy in a Mouse Model Through Single-Cell RNA Sequencing

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