Department of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea.
Department of Pediatrics, Ajou University School of Medicine, Suwon, Korea.
Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea.
Division of Nephrology, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.
Division of Endocrinology and Metabolism, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.
Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
Department of Pediatrics, Seoul National University College of Medicine, Seoul, Korea.
Abstract
BACKGRUOUND Due to the limited availability of therapeutic agents for type 2 diabetic kidney disease (T2DKD), there is a need for further knowledge derived from experimental models and innovative techniques. In addressing this issue, single-cell RNA sequencing (scRNA-seq) has been exclusively applied to a genetically modified diabetic kidney disease model, but not to an induced model representing T2DKD. Herein, we analyzed scRNA-seq and other experiments from an induced T2DKD model and validated the results in human-derived biospecimens.
METHODS The model was induced by combining a high-fat diet with streptozotocin to simulate induced T2DKD. scRNA-seq, histological, and flow cytometric analyses were conducted, and the results were compared with control mice. The findings were then applied to human T2DKD kidneys.
RESULTS Biochemical and histological analyses unveiled early-stage T2DKD features, such as hyperfiltration, increased proteinuria, glomerulomegaly, and interstitial fibrosis. scRNA-seq identified that proximal tubules secreted a variety of chemokines, potentially in response to crosstalk with glomeruli. Notably, C-X-C motif chemokine 12 (CXCL12) emerged as a key player in potentially promoting T-cell recruitment. Flow cytometry substantiated T-cell infiltration into the kidney of the T2DKD model. This finding was further corroborated in human biopsied kidney tissues, showing a correlation between elevated CXCL12 levels and T2DKD progression.
CONCLUSION The induced T2DKD model highlights the pivotal role of CXCL12-mediated T-cell infiltration, stemming from the crosstalk between proximal tubules and glomeruli. This data serves as a foundation for future studies, promising a therapeutic target for T2DKD.
Fig. 1.Biochemical and histological parameters in type 2 diabetic kidney disease (T2DKD) and control kidneys. (A) Serum cholesterol and glucose levels over 6 months. (B) Total body and kidney weights. (C) Homeostatic model assessment of insulin resistance (HOMA-IR) scores. (D) Trend in serum creatinine over 6 months and creatinine clearance at 6 months of the model. (E) Urinary protein-to-creatinine ratio (uPCR) over 6 months. (F) Representative images of periodic acid–Schiff-stained glomeruli, and measurements of glomerular volume and mesangial expansion. Scale bar=50 μm. (G) Representative images of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL)-stained kidneys, and their expression levels. Scale bar=100 μm. (H) Representative Sirius red-stained images, and their expression levels. Scale bar=100 μm. aP<0.05, bP<0.001.Fig. 2.Exploring crosstalk between glomeruli and tubules using single-cell RNA sequencing. (A) Uniform Manifold Approximation and Projection (UMAP) visualization of 115,412 cells, categorized by cell type, including proximal tubule segments 1, 2, and 3 (PT-S1, PT-S2, PT-S3), thin limb of Henle’s loop (tL), thick ascending limb (TAL), combined TAL and distal convoluted tubule (DCT), connecting tubule (CNT), collecting duct (CD), glomerular endothelial cell (gEC), peritubular endothelial cell (ptEC), arteriolar endothelial cell (Arteriole), mesangial cell and pericyte (MES and PERI), podocyte (PODO), myeloid cell, T-cell and natural killer (NK) cell, B cell, and plasma cell (PC). (B) Dot plot highlighting canonical marker genes for each identified cell type, with the color intensity reflecting the mean scaled expression of each gene and the dot size representing the proportion of cells expressing the gene. (C) Expression profiles of canonical marker genes for each cell types, with color intensity denoting mean scaled gene expression. (D) Number of upregulated differentially expressed genes in type 2 diabetic kidney disease (T2DKD) and control kidneys. (E) Circle plots showing predicted ligand-receptor interactions between PODO or gECs and PT segments in T2DKD and control kidneys. (F) Visualization of hallmark pathways significantly activated in PT segments of T2DKD kidneys. TNFα, tumor necrosis factor-α; NFκB, nuclear factor-κB; IL, interleukin; STAT, signal transducer and activator of transcription; TGFβ, transforming growth factor-β; UV, ultraviolet.Fig. 3.Immune cell-infiltrated milieu in type 2 diabetic kidney disease (T2DKD) kidneys. (A) Representative images of CD45- and CD3-stained kidneys, and their respective expression levels. Scale bar=100 μm. (B) Proportions of whole immune cells, and T and natural killer (NK) cell cluster, calculated from single-cell RNA sequencing data. (C) Flow cytometric analysis of cell number of lymphocytes. (D) Flow cytometric analysis of cell number of myeloid cells. (E) Flow cytometric analysis of proportions of CD4+ (left) and CD8+ (right) T-cell subsets. (F) Flow cytometric analysis of cytokine production in CD4+ and CD8+ T cells. Mϕ, macrophage; Nϕ, neutrophil; MHCII, major histocompatibility complex class II; DC, dendritic cell; IFNγ, interferon γ; IL, interleukin; TNFα, tumor necrosis factor-α. aP<0.05, bP<0.01, cP<0.001.Fig. 4.Recruitment of T and natural killer (NK) cells in type 2 diabetic kidney disease (T2DKD) kidneys. (A) Uniform Manifold Approximation and Projection (UMAP) plot illustrating cell distributions of T and NK subsets. (B) Dot plot highlighting canonical marker genes for each cell type, with the color intensity reflecting the mean scaled expression of each gene and the dot size representing the proportion of cells expressing the gene. (C) Selected chemokine ligands and receptors on segments of proximal tubule (PT) and T and NK cell subsets in T2DKD kidneys compared to control kidneys. (D) Mapping of C-X-C motif chemokine 12 (Cxcl12) and C-X-C motif chemokine receptor 4 gene (Cxcr4) genes in PT segments and T and NK cell subsets, respectively. (E) Representative images of CXCL12-stained kidneys, and their expression levels. Scale bar=100 μm. (F) Representative image of kidney sections immunostained for CXCL12 and CD3 from patients with T2DKD. Scale bars=100 μm (left) and 50 μm (right). (G) Flow cytometric analysis of T-cell numbers following administration with anti-CXCL12 antibody (αCXCL12 Ab) or control Ab. (H) Flow cytometric analysis of CD4+ (left) and CD8+ (right) T-cell subset proportions following administration with αCXCL12 Ab or control Ab. PT-S, proximal tubule segment; EM, effector memory; DAPI, 4´,6-diamidino-2-phenylindole. aP<0.05, bP<0.01, cP<0.001.Fig. 5.Human translation of C-X-C motif chemokine 12 (CXCL12) expression. (A) Representative image of CXCL12-stained kidneys, and their expression levels in tubules. Scale bar=100 μm. (B) Representative image of kidney sections immunostained for CXCL12 and CD3. Scale bar=100 μm. (C) Correlation between tubular CXCL12 expression and estimated glomerular filtration rate (eGFR) or urinary protein-to-creatinine ratio (uPCR). Line and gray area indicate the trend line and 95% confidence interval, respectively. (D) Relationship between tubular CXCL12 expression and cumulative rate curves of kidney progression. T2DKD, type 2 diabetic kidney disease; DAPI, 4´,6-diamidino-2-phenylindole. aP<0.001.
그림 출처: PubMed Central 공개 논문 (PMC12436036)
Conflict of interest statement
CONFLICTS OF INTEREST . Soo Heon Kwak has been an associate editor of the Diabetes & Metabolism Journal since 2022. He was not involved in the review process of this article. Otherwise, there was no conflict of interest.
