Gene expression profiles of glomerular endothelial cells support their role in the glomerulopathy of diabetic mice

Abstract

Endothelial dysfunction promotes the pathogenesis of diabetic nephropathy (DN), which is considered to be an early event in disease progression. However, the molecular changes associated with glomerular endothelial cell (GEC) injury in early DN are not well defined. Most gene expression studies have relied on the indirect assessment of GEC injury from isolated glomeruli or renal cortices. Here, we present transcriptomic analysis of isolated GECs, using streptozotocin-induced diabetic wildtype (STZ-WT) and diabetic eNOS-null (STZ-eNOS^-/-) mice as models of mild and advanced DN, respectively. GECs of both models in comparison to their respective nondiabetic controls showed significant alterations in the regulation of apoptosis, oxidative stress, and proliferation. The extent of these changes was greater in STZ-eNOS^-/- than in STZ-WT GECs. Additionally, genes in STZ-eNOS^-/- GECs indicated further dysregulation in angiogenesis and epigenetic regulation. Moreover, a biphasic change in the number of GECs, characterized by an initial increase and subsequent decrease over time, was observed only in STZ-eNOS^-/- mice. This is consistent with an early compensatory angiogenic process followed by increased apoptosis, leading to an overall decrease in GEC survival in DN progression. From the genes altered in angiogenesis in STZ-eNOS^-/- GECs, we identified potential candidate genes, Lrg1 and Gpr56, whose function may augment diabetes-induced angiogenesis. Thus, our results support a role for GEC in DN by providing direct evidence for alterations of GEC gene expression and molecular pathways. Candidate genes of specific pathways, such as Lrg1 and Gpr56, can be further explored for potential therapeutic targeting to mitigate the initiation and progression of DN.

Keywords: diabetic nephropathy; endothelial nitric oxide synthase; glomerular endothelial cells; transcriptional profiling.

Figure 1. Isolation of glomerular endothelial cells from Flk1-H2B-EYFP mice

(A) Kidney sections of Flk-1-H2B-EYFP mice were immunostained with antibodies against CD31 or WT-1 and counterstained with DAPI. Scale bar: 20μm. Rectangular area on the right panel is further magnified to visualize the colocalization of EYFP with CD31, but not with WT-1. (B) Single-cell suspension of isolated glomeruli from Flk1-H2B-EYFP mice was subjected to fluorescence-activated cell sorting (FACS) for EYFP+ cells. Typical FACS profile is shown. Approximately 15.7% EYFP+ GECs are obtained from dissociated glomerular cells. (C) Real-time PCR analysis of endothelial-specific or podocyte-specific genes show a robust enrichment of endothelial cell markers (Cdh5, Pecam1, and Icam1) in EYFP+ fraction, but were depleted of podocyte (Nphs1, Nphs2, and synaptopodin) and tubular cell (Pax8) markers as compared with EYFP- fraction.

Figure 2. PCA and heatmap of RNA-seq data from diabetic and control GECs

(A, B) Principal-component analysis (PCA) of RNA-seq results comparing STZ-WT vs. Vehicle-WT (A) and STZ-eNOS^−/− vs. Vehicle-eNOS^−/− (B) GECs. (C, D) Heat-map of top 50 differentially expressed genes between STZ-WT vs. Vehicle-WT (C) and STZ-eNOS^−/− vs. Vehicle-eNOS^−/− (D) GECs.

Figure 3. Gene set enrichment analysis comparing GECs from diabetic vs. control mice

DEGs were examined for their known biological functions and grouped in the respective functional category using DAVID and Enrichr analyses. Similar results were obtained between the two analyses, and results from Enrichr analysis is shown. (A, B) GO terms of upregulated genes in diabetic mouse GECs: GO terms of upregulated genes in STZ-WT vs. Vehicle-WT (A), and GO terms of upregulated genes in STZ-eNOS^−/− vs. Vehicle-eNOS^−/− (B). (C, D) GO terms of downregulated genes diabetic mouse GECs: GO terms of dowregulated genes in STZ-WT vs. Vehicle-WT (C), and GO terms of downregulated genes in STZ-eNOS^−/− vs. Vehicle-eNOS^−/− (D). Significance is expressed as a p-value calculated using the Fisher’s exact test (p<0.05) and shown as -log₁₀ (p-value).

Figure 4. Comparison of STZ-eNOS^−/− vs. STZ-WT GECs

(A, B) GO terms of upregulated genes (A) or downregulated genes (B) in direct comparison of STZ-eNOS^−/− vs. STZ-WT GECs. Significance is expressed as a p-value calculated using Fisher’s exact test (p<0.05) and shown as -log₁₀ (p-value).

Figure 5. Comparison of DEGs between STZ-eNOS^−/− vs. STZ-WT GECs

(A) Number of upregulated (left) and downregulated (right) DEGs in two diabetic mouse models in comparison to their respective vehicle controls are shown in a Venn diagram. Total number of DEGs is shown on the bottom, and the number of overlapping genes is shown in the overlapping regions. (B) Gene set enrichment analysis of overlapping genes between DEGs[STZ-eNOS^−/− vs. Vehicle-eNOS^−/−] versus DEGs[STZ-WT vs. Vehicle-WT]. (C) Gene set enrichment analysis of DEGs[STZ-eNOS^−/− vs. Vehicle-eNOS^−/−] that do not overlap with DEGs[STZ-WT vs. Vehicle-WT]. Significance is expressed as a p-value calculated using Fisher’s exact test (p<0.05) and shown as -log₁₀ (p-value).

Figure 6. Augmented oxidative stress and apoptosis in GECs of diabetic eNOS^−/− mice

(A, B) Representative images of 8-oxoG and CD31 immunofluorescence in glomeruli of STZ-WT and STZ-eNOS^−/− mice (A) and semi-quantification of 8-oxoG intensity per CD31+ area (B). (C, D) Representative images of cleaved Caspase-3 and CD31 immunofluorescence in glomeruli of STZ-WT and STZ-eNOS^−/− mice (C) and semi-quantification of cleaved Caspase-3 intensity per CD31+ area (D). Scale bar: 50μm. Results are mean±SEM of at least 60 glomeruli evaluated per mouse (n=3 mice for STZ-WT and n=5 for STZ-eNOS^−/− mice).

Figure 7. Decreased GEC and podocyte numbers in glomeruli of diabetic eNOS^−/− mice

Representative images of WT-1 immunostaining and EYFP signal in glomeruli of diabetic mice. Scale bar: 25μm. Quantification of the number of EYFP-positive cell nuclei representing endothelial cells, WT1-positive cell nuclei representing podocytes, and the number of total glomerular cells (DAPI+) in Vehicle-WT and STZ-WT mice (A) and in Vehicle-eNOS^−/− and STZ-eNOS^−/− mice (B) at 10 weeks post-injection of either citrate buffer or STZ. Results are mean±SEM of at least 60 glomeruli evaluated per mouse (n=3 per group). n.s., not significant.

Figure 8. Quantification of GEC and podocyte numbers over time during the early diabetic injury

(A) Experimental design for time course study. Serial biopsies were performed at 4 and 6 week post STZ-injection and compared with 10 weeks post STZ-injection in both WT and eNOS^−/− mice. (B, C) Quantification of EYFP+ cells (left) vs. WT-1+ cells (right) per glomerular cross section in STZ-WT vs. vehicle-WT mice (B) and in STZ- eNOS^−/− vs. vehicle-eNOS^−/− mice (C). (D, E) Ratio of EYFP+ cells (left) and WT-1+ cells (right) per total DAPI+ cells per glomerular cross section in STZ-WT vs. vehicle-WT mice (D) and in STZ- eNOS^−/− vs. vehicle-eNOS^−/− mice (E). Results are mean ±SEM of at least 40 glomeruli evaluated per group (n=7 mice per group). *P<0.05 and **P<0.01 when compared to the respective vehicle control at each time point.

Figure 9. Validation of genes involved in angiogenesis and epigenetic regulation diabetic eNOS^−/− mice

(A, B) Real-time PCR analysis of select genes in the angiogenesis GO term (Lrg1, Gpr56, Angptl4, Itgb3, and Serpine 1) in GECs of STZ-eNOS^−/− mice (left panel) and in GECs of STZ-WT mice (right panel) relative to its respective vehicle control (n=3 mice per group, *P<0.05, **P<0.01, and ***P<0.001, when compared with vehicle-control). (B) Real-time PCR analysis of select genes in the epigenetic regulation GO term (Ddb2, Prmt5, Kdm6a, Zfp57, and Prdm2) in GECs of STZ-eNOS^−/− mice (left panel) and in GECs of STZ-WT mice (right panel) relative to its respective vehicle control (n=3 mice per group, *P<0.05, when compared with vehicle-control). (C–E) Representative images showing immunostaining of LRG1 (C), GPR56 (D), or ZFP57 (E) in the glomeruli of STZ-eNOS^−/− (left panel) or in STZ-WT (right panel) mice.

Figure 10. High glucose induces alteration in Lrg1, Gpr56 and Zfp57 expression in mGECs in vitro

(A) Real-time PCR analysis of Lrg1 and Gpr56 in mGECs under high mannitol (HM) or high glucose (HG) conditions relative to normal glucose (NG) control (n=3). (B) Real-time PCR analysis of Zfp57 in mGECs under high mannitol (HM) or high glucose (HG) conditions relative to normal glucose (NG) control (n=3). (C, D) Representative Western blot image (C) and densitometric analysis (D) of LRG1 expression and under high glucose conditions in mGECs. Increased LRG1 expression is detected in the supernatant (secreted) and in cells under high glucose conditions. (E, F) Representative western blot image (E) and densitometric analysis (F) of GRP56 expression under high glucose conditions in mGECs. Increased full-length GPR56 (GPR56^FL) and mature cleaved N-terminal fragment (GPR56^NTF) are both detected in response to high glucose. (G, H) Representative Western blot image (G) and densitometric analysis of ZFP57 expression in mGECs under high glucose conditions. All experiments were repeated at least three times.

Figure 11. Restoration of LRG1, GPR56 and ZPF57 expression limits high glucose-induced angiogenesis in vitro

(A, B) Representative western blot image (A) and densitometric analysis (B) shows knockdown efficiencies of lentivector expressing shRNA against Lrg1 (shLrg1#1 and shLrg1#2) in comparison to scrambled shRNA (shScr) or uninfected cells (control). (C, D) Representative western blot image (C) and densitometric analysis (D) shows the knockdown efficiencies of lentivector expressing shRNA against Gpr56 (shGpr56#1, shGpr56#2 and shGpr56#3) in comparison to scrambled shRNA (shScr) or uninfected cells (control). (E, F) Representative western blot image (E) and densitometric analysis shows the expression of ZFP57 in mGECs transduced with lentivirus expressing ZFP57 (ZFP57^OE) or unrelated mCherry protein (mCherry^OE) in comparison to uninfected cells (control). All experiments were repeated at least three times. (G–H) In vitro angiogenesis assay. mGECs exposed to high glucose or were seeded into the 8-well multiwall chamber slides coated with matrigel. Images were taken 6 hours post-plating. Scale bar: 50μm. The effects of Lrg1 or Gpr56 knockdown on angiogenesis in vitro are shown in (G), and the effects of ZFP57^OE is shown in (H).