Artigo Acesso aberto Revisado por pares

JAK-STAT signaling is activated in the kidney and peripheral blood cells of patients with focal segmental glomerulosclerosis

2018; Elsevier BV; Volume: 94; Issue: 4 Linguagem: Inglês

10.1016/j.kint.2018.05.022

ISSN

1523-1755

Autores

Jianling Tao, Laura H. Mariani, Sean Eddy, Holden T. Maecker, Neeraja Kambham, Kshama Mehta, John R. Hartman, Weiqi Wang, Matthias Kretzler, Richard A. Lafayette,

Tópico(s)

Vasculitis and related conditions

Resumo

Focal segmental glomerular sclerosis (FSGS) is a devastating disease with limited treatment options and poor prognosis. Activated JAK-STAT signaling has been implicated in other kidney diseases. Since new technologies allow us to better evaluate changes in systemic and renal JAK-STAT activity as it relates to kidney function, we examined this in 106 patients with biopsy-proven FSGS compared to 47 healthy control individuals. Peripheral immune function was assessed in peripheral blood mononuclear cells by phosphoflow studies before and after cytokine stimulation. Kidney JAK-STAT activity was measured by immunofluorescence and by transcriptomics. A STAT1 activity score was calculated by evaluating message status of downstream targets of pSTAT 1. Peripheral blood mononuclear cells were found to be upregulated in terms of pSTAT production at baseline in FSGS and to have limited reserve to respond to various cytokines. Increased staining for components of the JAK-STAT system in FSGS by microscopy was found. Furthermore, we found transcriptomic evidence for activation of JAK-STAT that increased pSTAT 1 and pSTAT 3 in glomerular and tubulointerstitial sections of the kidney. Some of these changes were associated with the likelihood of remission of proteinuria and progression of disease. JAK-STAT signaling is altered in patients with FSGS as compared to healthy controls with activated peripheral immune cells, increased message in the kidney and increased activated proteins in the kidney. Thus, our findings support immune activation in this disease and point to the JAK-STAT pathway as a potential target for treatment of FSGS. Focal segmental glomerular sclerosis (FSGS) is a devastating disease with limited treatment options and poor prognosis. Activated JAK-STAT signaling has been implicated in other kidney diseases. Since new technologies allow us to better evaluate changes in systemic and renal JAK-STAT activity as it relates to kidney function, we examined this in 106 patients with biopsy-proven FSGS compared to 47 healthy control individuals. Peripheral immune function was assessed in peripheral blood mononuclear cells by phosphoflow studies before and after cytokine stimulation. Kidney JAK-STAT activity was measured by immunofluorescence and by transcriptomics. A STAT1 activity score was calculated by evaluating message status of downstream targets of pSTAT 1. Peripheral blood mononuclear cells were found to be upregulated in terms of pSTAT production at baseline in FSGS and to have limited reserve to respond to various cytokines. Increased staining for components of the JAK-STAT system in FSGS by microscopy was found. Furthermore, we found transcriptomic evidence for activation of JAK-STAT that increased pSTAT 1 and pSTAT 3 in glomerular and tubulointerstitial sections of the kidney. Some of these changes were associated with the likelihood of remission of proteinuria and progression of disease. JAK-STAT signaling is altered in patients with FSGS as compared to healthy controls with activated peripheral immune cells, increased message in the kidney and increased activated proteins in the kidney. Thus, our findings support immune activation in this disease and point to the JAK-STAT pathway as a potential target for treatment of FSGS. Focal segmental glomerulosclerosis (FSGS) is a group of diseases sharing a common glomerular lesion of segmental glomerular sclerosis and hyalinosis.1Malaga-Dieguez L. Bouhassira D. Gipson D. Trachtman H. Novel therapies for FSGS: preclinical and clinical studies.Adv Chronic Kidney Dis. 2015; 22: e1-e6Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar It accounts for approximately 20% of cases of nephrotic syndrome in children and nearly 40% in adults.2Kitiyakara C. Kopp J.B. Eggers P. Trends in the epidemiology of focal segmental glomerulosclerosis.Sem Nephrol. 2003; 23: 172-182Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Over the last 30 years, the prevalence of FSGS in the United States (US) has increased from 14% to 20%3McGrogan A. Franssen C.F. de Vries C.S. The incidence of primary glomerulonephritis worldwide: a systematic review of the literature.Nephrol Dial Transplant. 2011; 26: 414-430Crossref PubMed Scopus (316) Google Scholar among primary glomerulonephritis, and it is the leading glomerular disorder causing end-stage renal disease (ESRD)4Maisonneuve P. Agodoa L. Gellert R. et al.Distribution of primary renal diseases leading to end-stage renal failure in the United States, Europe, and Australia/New Zealand: results from an international comparative study.Am J Kidney Dis. 2000; 35: 157-165Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar in the US. Traditionally, idiopathic FSGS, a podocytopathy,5Fogo A.B. Causes and pathogenesis of focal segmental glomerulosclerosis.Nat Rev Nephrol. 2015; 11: 76-87Crossref PubMed Scopus (201) Google Scholar is thought to be caused, at least in part, by a disorder of circulating lymphocytes or T cell dysfunction, potentially through the release of an unidentified "permeability factor,"6Bao L. Haas M. Pippin J. et al.Focal and segmental glomerulosclerosis induced in mice lacking decay-accelerating factor in T cells.J Clin Invest. 2009; 119: 1264-1274Crossref PubMed Scopus (32) Google Scholar which is toxic to the glomerular ultrafiltration barrier.7Shalhoub R.J. Pathogenesis of lipoid nephrosis: a disorder of T-cell function.Lancet. 1974; 2: 556-560Abstract PubMed Scopus (742) Google Scholar Many attempts have been made to elucidate the nature of this factor, although it remains elusive.8Wada T. Nangaku M. A circulating permeability factor in focal segmental glomerulosclerosis: the hunt continues.Clin Kidney J. 2015; 8: 708-715Crossref PubMed Scopus (51) Google Scholar Knowledge about disorders of circulating lymphocytes in FSGS is even more limited due to limitations in research technology. Human peripheral blood mononuclear cells exist in a heterogeneous pool comprising 70% T cells, 10% B cells, and another 10% monocytes. In 2000, utilizing 3 channels as the most advanced technology for flow cytometry at that time, Stachowski et al.9Stachowski J. Barth C. Michalkiewicz J. et al.Th1/Th2 balance and CD45-positive T cell subsets in primary nephrotic syndrome.Pediatr Nephrol. 2000; 14: 779-785Crossref PubMed Scopus (51) Google Scholar found the activity of T-helper-1 (Th1) and T-helper-2 (Th2) cells and specific lymphocyte subsets, namely CD45RA+CD4+ ("naive" helper T cells, suppressor-inducer), CD45RA+CD8+ ("naive" cytotoxic T cells, cytotoxic-effector), and CD45RO+CD4+ ("memory" helper T cells) were predictive of steroid sensitivity in FSGS. The work suggested a strategy to further study the function of peripheral blood mononuclear cells (PBMCs) as a disease pathway and biomarker of FSGS. Phospho-flow is a more recently developed research strategy based on flow cytometry. It can simultaneously distinguish cell subsets in PBMCs and quantify the phosphorylation levels of intracellular signaling proteins such as signal transducer and activator of transcription (STAT).10Krutzik P.O. Crane J.M. Clutter M.R. Nolan G.P. High-content single-cell drug screening with phosphospecific flow cytometry.Nat Chem Biol. 2008; 4: 132-142Crossref PubMed Scopus (166) Google Scholar Present phospho-flow platforms allow us to precisely separate PBMCs into subsets as CD4+, CD4+CD45RA+, CD4+CD45RA–, CD8+, CD8+CD45RA+, CD8+CD45RA–, natural killer cells, B lymphocytes, and monocytes. Janus kinase (JAK)-STAT is a major pathway that responds to and transduces inflammatory signals from cells through extracellular ligands such as cytokines and chemokines.11O'Shea J.J. Plenge R. JAK and STAT signaling molecules in immunoregulation and immune-mediated disease.Immunity. 2012; 36: 542-550Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar Recently, compelling preliminary data in kidney cell–specific over-expression and knockout transgenic animal models, as well as in genetic profile analysis in human samples, have revealed that JAK-STAT is extensively activated in diabetic nephropathy, autosomal dominant polycystic kidney disease, HIV-associated nephropathy, acute kidney injury, and obstructive uropathy,12Brosius III, F.C. He J.C. JAK inhibition and progressive kidney disease.Curr Opin Nephrol Hypertens. 2015; 24: 88-95Crossref PubMed Scopus (68) Google Scholar in which the JAK2-STAT3 pathway is the most involved. This has already resulted in active trials of medications that inhibit the JAK-STAT pathway in diabetic nephropathy and kidney transplantation.13Brosius F.C. Tuttle K.R. Kretzler M. JAK inhibition in the treatment of diabetic kidney disease.Diabetologia. 2016; 59: 1624-1627Crossref PubMed Scopus (91) Google Scholar, 14Vincenti F. Silva H.T. Busque S. et al.Evaluation of the effect of tofacitinib exposure on outcomes in kidney transplant patients.Am J Transplant. 2015; 15: 1644-1653Crossref PubMed Scopus (40) Google Scholar Thus far, in primary glomerular disease, only 1 study analyzing the genomic profile of PBMCs from 3 Ig A nephropathy patients highlighted the role of STAT1 in the exacerbation of gross hematuria.15Cox S.N. Sallustio F. Serino G. et al.Activated innate immunity and the involvement of CX3CR1-fractalkine in promoting hematuria in patients with IgA nephropathy.Kidney Int. 2012; 82: 548-560Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar It thus seemed prudent to explore JAK-STAT in mediating the immune response systemically or locally in the kidney as a major role in the pathogenesis of idiopathic FSGS.16Reggiani F. Ponticelli C. Focal segmental glomerular sclerosis: do not overlook the role of immune response.J Nephrol. 2016; 29: 525-534Crossref PubMed Scopus (8) Google Scholar In this study, we, for the first time, examined the phosphorylation of STAT1, STAT3, and STAT5 in each of these subsets with or without in vitro cytokine stimulation. We also explored JAK-STAT expression and content in the kidney and associated it with disease activity and outcomes (Figure 1). In the initial part of this study, 15 adult patients with biopsy-proven idiopathic FSGS were evaluated and provided their blood for analysis of PBMCs (Figure 1). All subjects provided informed consent. Healthy controls (n = 20) matched for age (42.9 ± 14.56 years) and gender (10 men and 10 women) were also studied. Patients were not on any active immunotherapy for at least the prior 6 months. Their general characteristics are listed in Table 1. Kidney tissue from 9 FSGS patients were studied by immunohistochemistry for JAK1, JAK2, STAT3, pSTAT3, and pSTAT1. Their baseline characteristics are also summarized in Table 2. Five patients had both PBMCs assayed and biopsy slides stained. A separate group of 72 patients (Table 3) with biopsy-proven FSGS from NEPTUNE were evaluated. NEPTUNE, part of the National Institutes of Health (NIH) Rare Disease Clinical Research Network (RDCRN), is a multicenter prospective cohort study enrolling patients with proteinuric glomerular disease and performing comprehensive clinical and molecular phenotyping. Patients with secondary glomerular disease (such as diabetic kidney disease, lupus nephritis, and amyloidosis) were excluded. Comparable healthy tissue was obtained from living transplant donors for comparison with NEPTUNE (n = 6) FSGS biopsy samples (transcriptional group, with added patients from European Renal cDNA Bank; Figure 1). Biopsy material from both cohorts was micro-dissected into glomerular and tubulointerstitial compartments prior to transcriptional profiling as previously described.17Gadegbeku C.A. Gipson D.S. Holzman L.B. et al.Design of the Nephrotic Syndrome Study Network (NEPTUNE) to evaluate primary glomerular nephropathy by a multidisciplinary approach.Kidney Int. 2013; 83: 749-756Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 18Sampson M.G. Robertson C.C. Martini S. et al.Integrative genomics identifies novel associations with APOL1 risk genotypes in black NEPTUNE subjects.J Am Soc Nephrol. 2016; 27: 814-823Crossref PubMed Scopus (84) Google Scholar Twelve of the NEPTUNE subjects had biopsy slides available from that same biopsy further assessed for JAK-STAT component and CD3 staining (immunochemistry group; Figure 1). The institutional review boards of Stanford University Medical Center and participating centers in NEPTUNE approved all of these studies.Table 1Baseline characteristics of 15 patients with FSGS with PBMCs assayed in phospho-flowAge at biopsy (yr)37 (24–61)Female9 (60%)African American2 (13%)Asian4 (26%)White4 (26%)Hispanic5 (33%)Sampling time after biopsy (ds)351 (63–2884)eGFR at PBMC sampling (ml/min per 1.73 m2)50 (26–133)Urine protein excretion at PBMC sampling (g/g)2.8 (0.5–6.1)eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; PBMC, peripheral blood mononuclear cell. Open table in a new tab Table 2Baseline characteristics of 9 patients with FSGS with kidneys stained for JAK1, JAK2, STAT3, pSTAT3, and pSTAT1Age at biopsy (yr)56 (28–69)Female4 (44%)White Hispanic4 (44%)White non-Hispanic5 (56%)eGFR (ml/min per 1.73 m2)76 (24–124)Urine protein excretion (g/g)3.5 (1.7–7.2)eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis. Open table in a new tab Table 3Baseline characteristics of 72 patients with FSGS with kidney gene analysisAge at biopsy (yr)29 (16–55)Female23 (32%)African American27 (38%)HispanicNAeGFR (ml/min per 1.73 m2)77 (51–103)Urine protein excretion (g/g)1.7 (0.9–4)eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; NA, not applicable. Open table in a new tab eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; PBMC, peripheral blood mononuclear cell. eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis. eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; NA, not applicable. PBMCs were isolated using density gradient separation Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK) and then cryopreserved. For evaluation, cells were thawed, stored at room temperature for 1 hour, and counted. Then, 0.5 x 106 viable cells/ml were plated in a 96-well plate. These cells were then stimulated with 104 U/ml of interferon-α, 50 ng/ml of interferon-γ, 50 ng/ml of interleukin (IL)-6, 50 ng/ml of IL-7, 50 ng/ml of IL-10, 50 ng/ml of IL-2, and 50 ng/ml of IL-21 prior to paraformaldehyde fixation and methanol permeabilization. Each well was then bar-coded using a combination of Pacific Orange and Alexa-750 dyes (Invitrogen, Carlsbad, CA), pooled in tubes, washed with FACS buffer (phosphate-buffered saline supplemented with 2% fetal bovine serum and 0.1% sodium azide), and stained with the following antibodies: BV421 to CD3, PerCP-Cy5.5 to CD4, PerCp-Cy5.5 to CD20, PE-Cy7 to CD33, BV510 to CD45RA, AlexaFluor488 to pSTAT-1, AlexaFluor 647 to pSTAT-3, and PE to pSTAT-5 (all from BD Biosciences, San Jose, CA). Following resuspension in FACS buffer, 100,000 cells per stimulation condition were collected using DIVA 6.0 software on an LSRII flow cytometer (BD Biosciences). Data acquisition was performed using FlowJo v 9.3, gating on live cells based on forward versus side scatter profiles, then on singlets using forward scatter area versus height, followed by cell subset-specific gating. Unstimulated cell populations were compared after log normalization. Following cytokine stimulation, the fold change was calculated as any stimulant status/nonstimulant status. Fold change in any subset under any stimulant between control and FSGS was compared. The subset percentage of PBMCs is shown in Supplementary Table S1. Phosphorylation of individual STAT1, 3, and 5 proteins in PBMCs from FSGS (n = 15) stimulated in vitro by interferon-α, interferon-γ, IL-6, IL-7, IL-10, IL-2, and IL-21 were compared with healthy controls (n = 20) (Figure 2). Starting with unstained paraffin fixed slides, antigen retrieval was performed with citrate solution at pH 6.0. Normal serum was used to block the antigens for 30 minutes at room temperature. Primary antibodies to JAK1 (Cell Signaling Technology [Danvers, MA] product no. 3344), JAK2 (Cell Signaling Technology product no. 3230), STAT3 (Cell Signaling Technology product no. 9139), pSTAT3 (Cell Signaling Technology product no. 9145), CD3 (Dako CD3 Clone F7.28, Agilent, Santa Clara, CA), and pSTAT1 (Cell Signaling Technology product no. 8826) were applied with a dilution of 1:200, 1:100, 1:100, 1:2000, 1:200, and 1:200, respectively, for 60 minutes at room temperature. Second antibodies to rabbit Ig or mouse Ig (Vector Labs, Burlingame, CA) were incubated for 30 minutes at room temperature. DAB was incubated for 3 minutes at room temperature to develop the staining. Hematoxylin was counter-stained for 1 minute at room temperature. Kidney tissue was obtained by biopsy, stored in RNAlater (Thermo Fisher Scientific, Waltham, MA), and manually micro-dissected into tubulointerstitial (TI) and glomerular compartments. In terms of glomerular injury, manual glomerular microdissection captures glomeruli with an open Bowman's space. Therefore, across samples, our glomerular expression studies are enriched for the transcriptomes of functioning glomeruli, rather than for globally sclerosed glomeruli. Microdissected renal biopsy specimens were processed and analyzed using Affymetrix Human Gene ST 2.1 Array platforms (Thermo Fisher Scientific). Probe sets were annotated to Entrez Gene IDs using custom CDF version 19 generated from the University of Michigan Brain Array group.19Sandberg R. Larsson O. Improved precision and accuracy for microarrays using updated probe set definitions.BMC Bioinformatics. 2007; 8: 48Crossref PubMed Scopus (125) Google Scholar Kidney tissue was processed as previously described.15Cox S.N. Sallustio F. Serino G. et al.Activated innate immunity and the involvement of CX3CR1-fractalkine in promoting hematuria in patients with IgA nephropathy.Kidney Int. 2012; 82: 548-560Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Expression data was quantile-normalized and batch-corrected using COMBAT.20Chen C. Grennan K. Badner J. et al.Removing batch effects in analysis of expression microarray data: an evaluation of six batch adjustment methods.PLoS One. 2011; 6: e17238Crossref PubMed Scopus (334) Google Scholar Differential gene expression across the transcriptome was compared between patients with FSGS and living donors using the SAM method;21Rapaport F. Khanin R. Liang Y. et al.Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data.Genome Biol. 2013; 14: R95Crossref PubMed Scopus (464) Google Scholar genes were defined as differentially expressed with q value ≤ 0.05. CEL files are accessible in GEO20Chen C. Grennan K. Badner J. et al.Removing batch effects in analysis of expression microarray data: an evaluation of six batch adjustment methods.PLoS One. 2011; 6: e17238Crossref PubMed Scopus (334) Google Scholar, 22Edgar R. Domrachev M. Lash A.E. Gene expression omnibus: NCBI gene expression and hybridization array data repository.Nucleic Acids Res. 2002; 30: 207-210Crossref PubMed Scopus (8467) Google Scholar for the ERCB data set (GSE10498, GSE104948) and for the NEPTUNE data set under reference series GSE104066. Differential expression analysis was performed using the SAM function in Multiple Experiment Viewer (MeV) v4.9. Functional enrichment and upstream regulator analyses were performed in Ingenuity Pathways Analysis (Qiagen, Hilden, Germany). A STAT1-dependent signature was derived using STAT1 target genes identified from 1441 potential target genes from ChiP-Seq and limiting the search space to the 20 genes with the highest fold change after treatment of HeLa cells with interferon gamma.23Shoguchi E. Tanaka M. Shinzato C. et al.A genome-wide survey of photoreceptor and circadian genes in the coral, Acropora digitifera.Gene. 2013; 515: 426-431Crossref PubMed Scopus (27) Google Scholar Of the 20 genes, only 17 were annotated on a test data set and carried forward. The 17 gene set was used to generate STAT1 signature scores across samples from the transcriptional data. The STAT1 scores were generated by creating Z-scores for each of the 17 genes in the network and then using the average Z-score of 17 genes as a transcriptional measure of STAT1 activation. Descriptive statistics, including mean and SD for normally distributed variables, median and interquartile range (IQR) for skewed variables, and proportions for categorical variables were used to characterize participant characteristics. Differences between subjects and controls in baseline and stimulated phospho-flow activity were compared with Wilcoxon rank-sum analysis. Spearman's rank correlation coefficient (rho) was used to evaluate the relationship between JAK-STAT1 activation score and baseline estimated glomerular filtration rate (eGFR) and urine protein-to-creatinine ratio. Kaplan-Meier survival curves were generated by tertile of JAK-STAT1 activation score for time to complete remission (defined as first urine protein-to-creatinine ratio <0.3 mg/mg) and the composite of 40% decline in eGFR from baseline or ESRD. Differences between the curves were tested using the log-rank test. Additionally, separate Cox proportional hazards models were fit to estimate the hazard ratio for complete remission and the composite of 40% decline in eGFR from baseline or ESRD using JAK-STAT1 activation score as a continuous variable. Unadjusted models and models adjusted for baseline eGFR and urine protein-to-creatinine ratio were fit. Analyses were performed using both STATA v. 12 and R. Fifteen subjects with FSGS were compared with 20 healthy, adult controls. Baseline subsets of PBMCs were similar, with a trend toward fewer CD4+ lymphocytes and more CD8+ lymphocytes in FSGS (Supplementary Table S1). Under nonstimulated conditions, pSTAT1 was significantly greater in FSGS subjects in CD4+T cells, CD8+T cells, and monocytes. A similar trend was seen for pSTAT3 but did not reach significance. pSTAT5 was significantly increased in B cells and in CD4+ lymphocytes, restricted to those that were CD45RA marker–positive (Figure 2 and Supplementary Table S2). However, with cytokine stimulation, there was a generalized, highly significant pattern of reduced ability to increase pSTATs 1, 3, and 5 in FSGS patients as compared with controls, suggesting a generalized defect or fatigue in the system. This was most notable among the CD4+ lymphocytes, and again mainly within the CD45RA+ subset (Figure 3). It was variable as to whether the decreased fold change left the cellular pSTAT higher or lower than control subjects after stimulation, but frequently the levels were lower or the same (Supplementary Table S3). There was correlation between higher creatinine in these subjects and reduced ability to increase STAT phosphorylation in the CD4 and CD45+ lymphocytes. Greater proteinuria was significantly and inversely correlated with the ability of cytokines to stimulate phosphorylation of STAT1 or STAT3 in a variety of PBMC subtypes, shown by heat maps (Supplementary Figure S1). The generalized pattern of increased basal STAT activation and decreased fold change could be seen in other glomerular disease patients, such as patients with lupus nephritis and membranous nephropathy, but cell lines involved and specific cytokine pathways behaved differently (Supplementary Figure S2).Figure 3(a) Changes of pSTAT1 and pSTAT3 of B lymphocytes, CD4+, CD4+CD45RA+, CD4+CD45RA–, CD8+, CD8+CD45RA+, CD8+CD45RA–, natural killer cells, and monocytes stimulated by interferon (IFN)α, IFNγ, interleukin (IL)-6, IL-7, IL-10, IL-2, and IL-21 were compared between focal segmental glomerular sclerosis (FSGS) and control. P values were determined by Wilcoxon rank sum test. P values lower than 0.05 were filtered and shown in a heat map format (left panel). Direction of increased or decreased signal transducer and activator of transcription phosphorylation in comparison with controls is represented by blue or red. Correlation analysis between 24-hour urine protein excretion or serum creatinine with any fold change described above were conducted, and (b,c,d,e) the 4 significant correlations that overlapped with the significant change of pSTAT1 are shown. Detailed raw data in change of pSTAT1 and pSTAT3 in any cell subset under any stimulant between control and FSGS are expressed as mean ±SEM in a supplementary file provided on request. P values from those comparisons are also available in a supplementary file provided on request.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Unstained slides of 9 subjects with FSGS were assessed for STAT3, JAK2, pSTAT1, and pSTAT3 and compared with staining in control subjects. As demonstrated in Figure 4, staining was somewhat decreased for JAK 2 but increased for STAT 3 as well as for pSTAT1 and pSTAT3. Semiquantitative analysis (Figure 5) demonstrated significantly greater staining for pSTAT1 and pSTAT3 in FSGS subjects in the glomerulus, tubules, and vessels of the kidney. While pSTAT3 appears restricted to glomerular endothelial cells, vasculature, and tubule cells, pSTAT1 staining was more generalized, including podocytes and mesangial cells.Figure 5Semiquantification of the JAK2, pSTAT1, STAT3, and pSTAT3 immunohistochemistry staining in focal segmental glomerular sclerosis (FSGS) patients (n = 9). Healthy non-neoplastic kidney dissected from tumor nephrectomy was used as a control. (a) For JAK2, the scoring was defined as 0 = none; +1 = <25% cells positive with weak intensity; +2 = 26% to 100% cells positive with weak intensity; +3 = 26% to 100% cells positive with strong intensity. (b) For pSTAT1, the scoring was defined as 0 = none; +1 = <50% cells positive with weak intensity; +2 = 51% to 100% cells positive with a stronger intensity; +3 = 51% to 100% cells positive with further stronger intensity. (c) For STAT3, the scoring was defined as 0 = none; +1 = 100% cells positive with weak intensity; +2 = 100% cells positive with a stronger intensity and similar intensity between nuclear staining and cytoplasmic staining; +3 = 51% to 100% cells positive with a strong intensity and stronger intensity in nuclear staining than cytoplasmic staining. (d) For pSTAT3, the scoring was defined as 0 = none; +1 = <10% cells positive with weak intensity; +2 = 11% to 74% cells positive with weak intensity; +3 = >75% cells positive with a strong intensity. glom, glomerular; IF, interstitial; PTC, peritubular capillaries.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Glomerular (n = 48) and tubulointerstitial (n = 67) profiles from 72 subjects with FSGS were compared with profiles from healthy kidney donor biopsies (n = 6 for glomeruli and n = 5 for tubulointerstitium). Analysis of the differentially expressed genes in FSGS demonstrated a significant enrichment of the JAK-STAT signaling pathway by Ingenuity Pathway Analysis (Qiagen) in the tubulointerstitium (P = 1.08E-04). In addition to JAK-STAT pathway enrichment, upstream analysis in Ingenuity Pathway Analysis identified predicted activation of JAK-STAT signaling pathway nodes, IFNG, and STAT1 in the tubulointerstitium (activation Z-score: 4.7 and 3.106; enrichment P value = 2.56E-07) (Figure 6). A similar finding was demonstrated in the glomerular tissue (Figure 7). To represent a predicted activation at the patient level, a transcriptional assessment of JAK-STAT activation was performed. We used this composite score comprising 17 genes as representative of STAT1 activation (Supplementary Table S4). In the NEPTUNE cohort, the STAT1 activation score was elevated in FSGS patients relative to healthy living donor biopsies in both glomerular and TI sections (Supplementary Figure S3). Evaluation of an independent cohort of FSGS patients, from the European Renal cDNA Bank (baseline characteristics seen in Supplementary Table S5), confirmed this elevation in STAT1 activation scores in tissue transcriptomic data from FSGS patients compared with living donors, seen in both glomerular and TI sections. Within the European Renal cDNA Bank, an analysis of diabetic subjects with similar chronic kidney disease and proteinuria (Supplementary Table S6) demonstrated increased STAT1 activations scores as well (Supplementary Figure S4).Figure 7Differentially expressed transcripts in focal segmental glomerular sclerosis (FSGS) were enriched in the Janus kinase–signal transducer and activator of transcription (JAK-STAT) pathway in the glomeruli. (a) Signaling network nodes highlighted in purple indicate those genes that were differentially expressed in FSGS relative to living donor controls, while shading of the nodes indicates up-regulation (red) or down-regulation (green) in FSGS relative to living donor controls. (b) Predicated activation of STAT-related signaling nodes in the glomeruli, with (c) evidence supporting activation of STAT1 indicated. Network supporting STAT1 activation in the glomeruli. Orange and blue connections indicate that STAT1 can induce or inhibit expression of downstream genes. These findings are consistent with STAT1 activation. Yellow connections indicate findings that are inconsistent with STAT1 activation, while gray lines are

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