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Proteome Analysis of Isolated Podocytes Reveals Stress Responses in Glomerular Sclerosis

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Proteome Analysis of Isolated Podocytes Reveals Stress Responses in Glomerular Sclerosis BASIC RESEARCH www.jasn.org Proteome Analysis of Isolated Podocytes Reveals Stress Responses in Glomerular Sclerosis Sybille Koehler,1,2 Alexander Kuczkowski,1 Lucas Kuehne,1 Christian Jüngst ,3 Martin Hoehne ,1,3 Florian Grahammer,4 Sean Eddy ,5 Matthias Kretzler ,5,6 Bodo B. Beck,7 Jörg Höhfeld,8 Bernhard Schermer,1,3 Thomas Benzing,1,3 Paul T. Brinkkoetter,1 and Markus M. Rinschen1,3,9 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Understanding podocyte-specific responses to injury at a systems level is difficult because injury leads to podocyte loss or an increase of extracellular matrix, altering glomerular cellular composi- tion. Finding a window into early podocyte injury might help identify molecular pathways involved in the podocyte stress response. Methods We developed an approach to apply proteome analysis to very small samples of purified podo- cyte fractions. To examine podocytes in early disease states in FSGS mouse models, we used podocyte fractions isolated from individual mice after chemical induction of glomerular disease (with Doxorubicin or LPS). We also applied single-glomerular proteome analysis to tissue from patients with FSGS. Results Transcriptome and proteome analysis of glomeruli from patients with FSGS revealed an under- representation of podocyte-specific genes and proteins in late-stage disease. Proteome analysis of puri- fied podocyte fractions from FSGS mouse models showed an early stress response that includes perturbations of metabolic, mechanical, and proteostasis proteins. Additional analysis revealed a high correlation between the amount of proteinuria and expression levels of the mechanosensor protein Filamin-B. Increased expression of Filamin-B in podocytes in biopsy samples from patients with FSGS, in single glomeruli from proteinuric rats, and in podocytes undergoing mechanical stress suggests that this protein has a role in detrimental stress responses. In Drosophila, nephrocytes with reduced filamin homo- log Cher displayed altered filtration capacity, but exhibited no change in slit diaphragm structure. Conclusions We identified conserved mechanisms of the podocyte stress response through ultrasensitive proteome analysis of human glomerular FSGS tissue and purified native mouse podocytes during early dis- ease stages. This approach enables systematic comparisons of large-scale proteomics data and phenotype- to-protein correlation. JASN 31: 544–559, 2020. doi: https://doi.org/10.1681/ASN.2019030312 Received March 28, 2019. Accepted December 4, 2019. Podocytes are specialized epithelial cells at the kid- P.T.B. and M.M.R. shared senior authorship. ney filtration barrier that enwrap the glomerular capillaries.1 Upon injury, podocytes dedifferenti- Published online ahead of print. Publication date available at www.jasn.org. ate, lose their unique three-dimensional morphol- ogy, and detach into the urine. This response to Correspondence: Dr. Paul Brinkkoetter, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of injury of any kind is morphologically followed Cologne, Faculty of Medicine and University Hospital Cologne, and accompanied by glomerular scarring and Kerpener Str.62, Köln, Germany 50931, or Dr. Markus Rinschen, Center for Metabolomics and Mass Spectrometry, The Scripps Re- FSGS. Various molecular, chemical, and genetic search Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037. Email: stressors can induce such a response. Diverse animal [email protected] or [email protected] models are used to study the disease. Commonly Copyright © 2020 by the American Society of Nephrology 544 ISSN : 1046-6673/3103-544 JASN 31: 544–559, 2020 www.jasn.org BASIC RESEARCH used models for identifying cellular pathways during podo- Significance Statement cyte injury include genetic models, and chemically induced podocyte damage such as the Doxorubicin nephrosis and LPS Analyses of entire glomeruli using a proteomic, transcriptomic, or models. Although these models are widely used, it is currently other “omic” approach may obscure the molecular footprints of not clear which parts of human podocyte disease are reflected early and decisive processes in podocytes responding to injury. To pinpoint mechanisms underlying glomerulosclerosis, the authors in the animal models. Even with an increased understanding performed ultrasensitive proteomics of purified podocyte fractions of the genomic landscape of FSGS,2,3 the immediate molec- at early injury stages in mouse models of glomerular disease in- ular response of podocytes in response to injury is still in- duced by doxorubicin or LPS. These analyses revealed an early completely understood at a systems level. stress response that involves upregulation of metabolic, proteo- fi Although proteomics technology is increasingly used to static, and mechanoresponsive mechanisms. They also identi ed conserved upregulated proteins involved in the podocyte stress 4–7 study glomerular disease, the three-cell architecture of the response, including the mechanosensor Filamin-B, and found a high glomerulus limits data interpretation of glomerular omics correlation between proteinuria and Filamin-B levels. The work data: podocyte injury leads to podocyte loss, and thereby alters demonstrates that proteome integration at the single glomerulus the cellular composition of the glomerulus.8 In addition, the and the individual organism levels can link “omics” datasets to technical nature of proteomics requires analysis of pooled ma- physiological function at high resolution. terial from several animals with variable phenotypes, limiting feasibility of these studies. To improve this, we adapted an ultra- HBSS) and 500 ml Dynabeads in digestion buffer (containing sensitive proteome analysis9,10 of pure podocyte fractions from collagenase 300 U/ml [Collagenase Type II; Worthington], individual mice comprising as few as 10,000 cells to identify 1 mg/ml pronase E [P6911; Sigma, Germany], and DNase I proteins that are regulated in the podocytes’ damage response. 50 U/ml [A3778; Applichem, Germany]). Kidneys were The use of mouse models allows studying of early disease minced into 1-mm3 pieces and incubated in digestion buffer stages, when transcriptomic and proteomic changes are al- at 37°C for 15 minutes. The suspension was mildly pressed ready measurable, but podocyte cell number is not yet dimin- through a 100-mm straining sieve for 15 minutes with enough ished. The technique described herein can therefore be used to HBSS buffer (approximately 20 ml). The suspension was then compare different disease stages in mouse models in order to pelleted by mild centrifugation (3000 rpm, 5 minutes), and the identify genes and proteins involved in the early and late dis- solution was resuspended. For primary podocyte isolation, ease responses of podocytes. glomeruli were resuspended in digestion buffer. For RNA iso- lation, glomeruli were transferred into Trizol until further processing. For proteomic analysis, glomeruli were digested METHODS until a single-cell suspension was obtained which was further used for FACs sorting. For this purpose, glomeruli were in- Transgenic Mouse Models cubated at 37°C for 40 minutes, and the suspension was mixed The Doxorubicin study was performed with R26mTmG mice, by pipetting up and down every 10 minutes. Magnetic parti- which were mated with hNphs2.PodCre mice to achieve GFP cles were discarded. Purity of cells was checked by fluorescence expression exclusively in podocytes.11,12 For the LPS study analysis. Cell suspension (2 ml) was sieved through a 40-mm Podocin.2A.iCre.2A.mTomato mice were used, which express mesh and washed with 10 ml HBSS. Cells were collected by tomato only in podocytes.13 In the Doxorubicin study we used centrifugation at 1500 rpm for 5 minutes at 4°C, resuspended only male mice, whereas in the LPS study we included mice in 0.5 ml HBSS, and supplemented with 0.1% BSA plus DAPI 1 from both sexes. Animals used in the Doxorubicin study were (1 mg/ml). To separate GFP-expressing (GFP )andGFP- 2 on a pure CD-1 background, whereas mice used in the LPS negative (GFP ) cells, glomerular cells were sorted by FACS study were backcrossed for nine generations from C57BL/6 to for the respective dyes. The minimum number of sorted po- CD-1 (95% CD-1). The mouse holding was done in the Uni- docytes was approximately 12,500 podocytes/animal. versity of Cologne animal facility according to standardized specific pathogen–free conditions. The experimental protocol RNA Isolation and Quantitative PCR was examined and approved by the LANUV NRW (Landesamt Glomeruli used for quantitative PCR were isolated from für Natur, Umwelt und Verbraucherschutz Nordrhein- 12-week-old pure Balb/C mice either treated with Doxorubi- Westfalen, State Agency for Nature, Environment and Consumer cin (12 mg/kg body wt) or without treatment. RNA was iso- Protection North Rhine-Westphalia, AZ 84–02.04.2013.A375). lated using the Direct-Zoll RNA MiniPrep Kit according to manufacturer’s instructions (Zymo, Irvine). A primer pair Isolation of Primary Podocytes specific for murine Filamin-B was used to assess Filamin-B Isolation of primary podocytes was performed after euthaniz- mRNA levels in glomeruli: sense primer: 59-CAAAGCTGG ing mice and glomerular preparation was as previously de- GTCCAACATGC-39, anti-sense primer: 59-CGAGTCAAG scribed.14 A detailed protocol was described before. Mice TCTAGGGCACC-39. For normalization, a primer pair spe- were killed by
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