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BRIEF COMMUNICATION www.jasn.org Comparison of Glomerular and Podocyte mRNA Profiles in Streptozotocin-Induced Diabetes † † † ‡ † Jia Fu,* Chengguo Wei, Kyung Lee, Weijia Zhang, Wu He, Peter Chuang, Zhihong Liu,* † and John Cijiang He § *National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China; †Division of Nephrology, Department of Medicine, ‡Flow Cytometry Shared Resource Facility, Icahn School of Medicine at Mount Sinai, New York; §Renal Program, James J. Peters VA Medical Center at Bronx, New York. ABSTRACT Evaluating the mRNA profile of podocytes in the diabetic kidney may indicate STZ induction alone.10 In order to spe- genes involved in the pathogenesis of diabetic nephropathy. To determine if the cifically label and isolate podoctyes, we 2 2 podocyte-specific gene information contained in mRNA profiles of the whole crossed the eNOS / mice with the IRG glomerulus of the diabetic kidney accurately reflects gene expression in the isolated mice11 that ubiquitously express a red 2/2 podocytes, we crossed Nos3 IRG mice with podocin-rtTA and TetON-Cre mice fluorescent protein prior to Cre-mediated for enhanced green fluorescent protein labeling of podocytes before diabetic injury. recombination and an enhanced green Diabetes was induced by streptozotocin, and mRNA profiles of isolated glomeruli fluorescent protein (EGFP) following re- and sorted podocytes from diabetic and control mice were examined 10 weeks combination. These mice were further later. Expression of podocyte-specific markers in glomeruli was downregulated in bred with podocin-rtTA and TetON-Cre diabetic mice compared with controls. However, expression of these markers was (LC1) transgenic mice for inducible not altered in sorted podocytes from diabetic mice. When mRNA levels of glomeruli podocyte-specific EGFP expression. Mice were corrected for podocyte number per glomerulus, the differences in podocyte were fed with doxycycline to induce EGFP marker expression disappeared. Analysis of the differentially expressed genes in expression permanently in podocytes diabetic mice also revealed distinct upregulated pathways in the glomeruli (mito- prior to diabetes induction by at 8 weeks 2 2 chondrial function, oxidative stress) and in podocytes (actin organization). In con- of age (STZ-eNOS / ). Body weight, clusion, our data suggest reduced expression of podocyte markers in glomeruli is blood glucose, and urine excretion of al- a secondary effect of reduced podocyte number, thus podocyte-specific gene ex- bumin were monitored every 2 weeks pression detected in the whole glomerulus may not represent that in podocytes in (Supplemental Figure 1, A–D). Urine the diabetic kidney. albumin-to-creatinine ratio steadily in- J Am Soc Nephrol 27: 1006–1014, 2016. doi: 10.1681/ASN.2015040421 creased in the diabetic mice starting from 2 weeks post-STZ injection, and by 8 weeks a 10-fold increase was observed in comparison to the control mice A large body of evidence suggests that performed to ascertain differential regula- podocyte injury is a key event in diabetic tion of genes involved in DN pathogene- nephropathy (DN). The reduction in sis.6,7 However, due to the heterogeneity Received April 18, 2015. Accepted July 12, 2015. podocyte density is the strongest predictor in cell types, these data provide limited J.F. and C.W. contributed equally to the work. of progressive DN1 and its extent corre- information specifically on podocyte in- fi Published online ahead of print. Publication date lates directly with the magnitude of pro- jury. Recently, mRNA pro les obtained available at www.jasn.org. teinuria.2 Apoptosis, detachment of directly from podocytes have been report- podocytes from the glomerular basement ed,8,9 but such information from the di- Correspondence: Dr. John Cijiang He, Division of Nephrology, Box 1243, Mount Sinai School of membrane, and epithelial-mesenchymal abetic kidney has not been determined. Medicine, One Gustave L. Levy Place, New York NY transition (EMT) are potential mecha- In this study we compared the mRNA 10029, or Dr. Zhihong Liu, National Clinical Re- nisms for podocyte injury and loss in profiles of glomeruli and podocytes be- search Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, 3–5 DN ; however, its exact mechanism of tween diabetic and control mice. We China, 210002. Email: [email protected] or loss remains unclear. Gene expression employed streptozotocin (STZ)-induced [email protected] fi 2/2 pro ling of glomeruli or cortex of animal diabetes in eNOS mice, resulting in a Copyright © 2016 by the American Society of or human diabetic kidneys have been more pronounced DN phenotype than Nephrology 1006 ISSN : 1046-6673/2704-1006 JAmSocNephrol27: 1006–1014, 2016 www.jasn.org BRIEF COMMUNICATION (Supplemental Figure 1D). By 10 weeks recognize only the extracellular domain However, whether these processes occur 2 2 STZ-eNOS / mice developed typical of nephrin) and/or to the loss of podo- mainly in podocytes, mesangial cells, or histologic findings of DN, including me- cytes. Immunostaining of diabetic kid- glomerular endothelial cells cannot be sangial expansion and foot process efface- neys for nephrin and podocin in fact concluded. ment (Supplemental Figure 2, A and B). showed a reduction in staining area, Analysis of the DEGs in sorted podo- We obtained whole glomeruli or sor- rather than of staining intensity (Figure cytes showed that many upregulated ted podocytes from diabetic and control 2B). We also did not detect any changes genes were involved in the actin organi- mice at 10 weeks post-STZ injection for in EMT marker expression in podocytes zation (Supplemental Figure 9), suggest- mRNA sequencing (RNA-seq). Sorted by qPCR analysis (Figure 3A) and by im- ing that significant alteration of the actin podocytes were viable and had enriched munostaining (Supplemental Figure 5). cytoskeleton occurs in the early stage of expression of podocyte differentiation RNA-seq data further revealed that the diabetes-induced podocyte injury, markers (Supplemental Figure 3, A and expression of cell death-related genes which is consistent with the foot process B). Each glomerular RNA sample was Bid, Dapk1,andCd40 were significantly effacement (Supplemental Figure 2B). In from an individual mouse (n=4), while increased in diabetic podocytes, which persistent or aggravated injury, the early RNA samples from sorted podocytes was confirmed by qPCR analysis (Figure cytoskeletal changes may eventually lead were pooled from five mice per sample 3B). In addition, a modest increase in to podocyte detachment or death, ensu- (n=3 diabetic, n=2 controls; one sample cleaved Caspase-3 expression was ob- ing in their loss in DN. It also suggests from a control mouse was discarded served by immunostaining in EGFP- that intervention to prevent podocyte de- from analysis due to a technical issue). positive cells, suggesting an increased tachment or death in the early stages of The top 50 differentially expressed genes podocyte apoptosis in the diabetic kid- injury may be an effective therapy against (DEGs) in diabetic glomeruli or podo- ney (Supplemental Figure 6). diabetes-induced podocyte loss. Most of cytes are listed in Supplemental Table 1. The above findings, from an unbiased the downregulated genes in diabetic podo- The principle component analysis and the approach, suggest that the decreased cytes were involved in the RNA processing heatmapofDEGsareshowninFigure1, podocyte marker expression in diabetic and endoplasmic reticulum function A–C. Consistent with the previous stud- glomeruli is likely a secondary effect of (Supplemental Figure 10), suggesting a ies, the expression of podocyte-specific podocyte loss, rather than a direct result possible alteration in the mTOR pathway markers WT-1, nephrin, and synaptopo- of podocyte dedifferentiation or EMT. and autophagy/ER stress response in po- din was significantly downregulated in the This is an important step in better un- docytes, which are known to be involved diabetic glomeruli (Table 1). To our sur- derstanding DN pathogenesis, and it also in podocyte injury in DN.14,15 prise, this downregulation was not ob- indirectly supports the notion that termi- The above pathway analyses of DEGs served in sorted podocytes of diabetic nally differentiated podocytes do not fur- in diabetic mice indicate significant dif- mice (Table 1). The discrepancy in podo- ther dedifferentiate, similar to neurons. ferences in the altered pathways between cyte marker expression level between iso- Nevertheless, we cannot rule out the isolated glomeruli versus podocytes. In- lated glomeruli and sorted podocytes was possibility that podocytes may undergo terestingly, the regulation of actin cyto- further validated by real-time quantitative dedifferentiation or EMT in other diabetic skeleton-related genes differs between PCR (qPCR) (Figure 2A). We confirmed animal models with more advanced DN. glomeruli and podocytes (i.e., some gene by immunostaining that EGFP labeling Analysis of the DEGs in isolated expression changes in opposite direc- co-localizes with WT-1 (Supplemental glomeruli revealed that the upregulated tions), suggesting that changes of actin Figure 4A), and consistent with the pre- genes in diabetic glomeruli were mostly cytoskeleton-related genes in other glo- vious findings3 podocyte number per glo- involved in the regulation of mitochon- merular cells may mask their changes in merulus was significantly lower in the drial function