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. 2021 Oct;70(10):2377-2390.
doi: 10.2337/db21-0110. Epub 2021 Jul 7.

Profile of Podocyte Translatome During Development of Type 2 and Type 1 Diabetic Nephropathy Using Podocyte-Specific TRAP mRNA RNA-seq

Yinqiu Wang  1   2 , Aolei Niu  1   2 , Yu Pan  1   2 , Shirong Cao  1   2 , Andrew S Terker  1   2 , Suwan Wang  1   2 , Xiaofeng Fan  1   2 , Cynthia L Toth  3 , Marisol A Ramirez Solano  3 , Danielle L Michell  3 , Danielle Contreras  3 , Ryan M Allen  3 , Wanying Zhu  3 , Quanhu Sheng  3 , Agnes B Fogo  1   4 , Kasey C Vickers  3 , Ming-Zhi Zhang  5   2 , Raymond C Harris  5   2   6
Affiliations

Profile of Podocyte Translatome During Development of Type 2 and Type 1 Diabetic Nephropathy Using Podocyte-Specific TRAP mRNA RNA-seq

Yinqiu Wang et al. Diabetes. 2021 Oct.

Abstract

Podocyte injury is important in development of diabetic nephropathy (DN). Although several studies have reported single-cell-based RNA sequencing (RNA-seq) of podocytes in type 1 DN (T1DN), the podocyte translating mRNA profile in type 2 DN (T2DN) has not previously been compared with that of T1DN. We analyzed the podocyte translatome in T2DN in podocin-Cre; Rosa26fsTRAP; eNOS-/-; db/db mice and compared it with that of streptozotocin-induced T1DN in podocin-Cre; Rosa26fsTRAP; eNOS-/- mice using translating ribosome affinity purification (TRAP) and RNA-seq. More than 125 genes were highly enriched in the podocyte ribosome. More podocyte TRAP genes were differentially expressed in T2DN than in T1DN. TGF-β signaling pathway genes were upregulated, while MAPK pathway genes were downregulated only in T2DN, while ATP binding and cAMP-mediated signaling genes were downregulated only in T1DN. Genes regulating actin filament organization and apoptosis increased, while genes regulating VEGFR signaling and glomerular basement membrane components decreased in both type 1 and type 2 diabetic podocytes. A number of diabetes-induced genes not previously linked to podocyte injury were confirmed in both mouse and human DN. On the basis of differences and similarities in the podocyte translatome in T2DN and T1DN, investigators can identify factors underlying the pathophysiology of DN and novel therapeutic targets to treat diabetes-induced podocyte injury.

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Figures

Figure 1
Figure 1
Generation of podocin-Cre TRAP mice with T2DN and T1DN. A: Schematic for when mice were sacrificed for TRAP analysis. B: ACR was increased in 3-month-old podocin-Cre; Rosa26fsTRAP; eNOS−/−; db/db mice. *P < 0.05 vs. corresponding eNOS−/− mice; †P < 0.05 vs. 3-month-old group, n = 6. C: ACR was increased as early as 3 weeks after injection of STZ in podocin-Cre; Rosa26fsTRAP; eNOS−/− mice. *P < 0.05 vs. corresponding eNOS−/− mice, n = 6. D: GFP-L10a was exclusively expressed in podocytes in podocin-Cre; Rosa26fsTRAP; eNOS−/− mice. Original magnification ×600.
Figure 2
Figure 2
Podocyte enriched translating mRNAs identified by TRAP. A: Distribution of RNA detected from TRAP samples. B: The number of protein-coding genes enriched in podocytes (fold >3 and FPKM >1.5) and the highly enriched and expressed genes in podocytes (fold >15 and FPKM >15) in nondiabetic mice. C: The fold enrichment of translating mRNA levels of selected podocyte marker genes compared with whole kidney mRNA samples. D: Scatterplot shows nonenriched, enriched, and highly enriched genes in podocytes. E: Venn diagram indicates shared podocyte genes identified by different approaches. F: Top 20 GO_BP, GO_MF, and KEGG pathways enriched in the 126 highly enriched and expressed genes in podocytes were related to the podocyte development and differentiation pathway, among others.
Figure 3
Figure 3
DEGs in podocytes in type 2 diabetic podocin-Cre; Rosa26fsTRAP; eNOS−/−; db/db mice. A: Heat map of the expression of enriched genes in podocytes in early (3 months) and advanced (6 months) T2DN. Genes were ranked by the fold enrichment, with the most enriched genes at the top. B: The number of DEGs in podocytes in early and advanced T2DN. C: Scatterplot shows enriched and highly enriched genes in podocytes in early and advanced T2DN. D: Heat map of the top 20 upregulated genes and top 20 downregulated genes in early and advanced T2ND, with the highest P value at the top. E: Venn diagram shows the numbers of unique and shared upregulated and downregulated genes in podocytes in early and advanced T2DN.
Figure 4
Figure 4
DEGs in podocytes in podocin-Cre; Rosa26fsTRAP; eNOS−/− mice with STZ-induced type 1 diabetes. A: Heat map of the expression of enriched genes in podocytes in early (3 weeks) and later (24 weeks) T1DN. Genes were ranked by the fold enrichment, with the most enriched genes at the top. B: The number of DEGs in podocytes in early and advanced T1DN. C: Scatterplot shows enriched and highly enriched genes in podocytes in early and later T1DN. D: Heat map of top 20 most upregulated genes and top 20 most downregulated genes in early and later T1DN, with the highest P value at the top. E: Venn diagram shows the numbers of unique and shared upregulated and downregulated genes in podocytes in early and advanced T1DN.
Figure 5
Figure 5
Comparison of enriched genes in podocytes between T2DN and T1DN. Type 2 diabetic podocin-Cre; Rosa26fsTRAP; eNOS−/−; db/db mice and podocin-Cre; Rosa26fsTRAP; eNOS−/− mice with STZ-induced type 1 diabetes were used. A: Heat map of the expression of the 126 highly enriched and expressed genes in podocytes in early and later stages of type 1 and type 2 diabetes in mice. B. Venn diagram shows the unique and shared upregulated and downregulated genes in podocytes in T2DN and T1DN.
Figure 6
Figure 6
Validation of the translation of representative DEGs in podocytes in DN. A: Podocyte-translating Ngb levels were decreased in early and later stages of T2DN and T1DN. *P < 0.05; n = 3–5. B: Neuroglobin expression in podocytes was decreased in eNOS−/−; db/db mice compared with eNOS−/− mice. C: In normal wild-type mice, neuroglobin was primarily colocalized with WT1, a marker of podocyte nucleus (arrows). Magnification ×600 for all.
Figure 7
Figure 7
Validation of the representative DEGs in glomeruli from T2DN mice and in human T2DN. A: Glomerulus Wt1, Ngb, Mgat5b, Cldn5, Nr4a3, Nr4a1, and Magi2 expression was lower in eNOS−/−; db/db mice than eNOS−/− mice at 3 months old. *P ≤ 0.05; n = 4 and 5. B: The percentage of NGB-expressing podocytes (NGB and WT1 double-positive cells [arrows]) was decreased in human T2DN. *P ≤ 0.05; n = 7 and 4. C: The percentage of TSPAN2-expressing podocytes (arrows) was markedly increased in human T2DN. *P ≤ 0.05; n = 4.
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