Cytokine-Driven Janus Kinase Signal Transducer and Activator of Transcription Pathway Hyperactivity Predicts Disease Severity in Pediatric FSGS

Visual Abstract

Abstract

Key Points

• We identify broad hyperactivity of the Janus kinase signal transducer and activator of transcription pathway in pediatric patients with FSGS as compared with healthy controls.

• We discover a distinct pattern of STAT3 hyperactivity in T cell subsets, which correlates with worse clinical outcomes.

• We describe broad cytokine dysregulation corresponding with Janus kinase signal transducer and activator of transcription hyperactivity.

Background

FSGS accounts for approximately 20% of cases of pediatric nephrotic syndrome and is the leading cause of ESKD among glomerular disorders in the United States. Primary FSGS is thought to be caused by a circulating factor which causes podocyte, glomerular, and endothelial injury and immune dysregulation. There is growing evidence that the Janus kinase signal transducer and activator of transcription (JAK/STAT) immunoregulatory pathway is involved in various kidney diseases; however, its role in FSGS has not been thoroughly studied. We recently identified JAK1 gain-of-function mutation in a pediatric patient with membranous nephropathy and multisystem immune dysregulation, which was successfully treated with a JAK inhibitor. We hypothesized that JAK/STAT pathway hyperactivity may play a role in the pathogenesis of pediatric primary FSGS and may correlate with worse clinical outcomes.

Methods

We recruited eleven individuals age 2–18 years with primary FSGS and ten age- and sex-matched healthy controls. Mass cytometry with antibodies against STAT proteins was performed on heparinized whole blood samples to characterize JAK/STAT pathway activity in immune cell subsets. Multiplex immunoassay was performed on plasma samples to quantify circulating cytokine levels. Unsupervised clustering by machine learning algorithms and statistical analyses were performed to determine clinically relevant patterns.

Results

The JAK/STAT pathway is overactive in 60% patients despite immunosuppression, predominantly showing increased phosphorylated STAT3 activity in T cell subsets. The level of STAT3 phosphorylation in T cells predicts lower kidney function, lower serum albumin, and increased proteinuria 1 year later. Various cytokines upstream of STAT3 phosphorylation are increased in patient plasma.

Conclusions

JAK/STAT pathway overactivity is present in pediatric patients with primary FSGS and predicts the severity of disease. JAK/STAT hyperactivity is likely driven by cytokine signaling and may be targeted by JAK inhibition.

Introduction

FSGS is the leading cause of ESKD among glomerular disorders in the United States.¹ It accounts for approximately 20% of cases of nephrotic syndrome in children and nearly 40% in adults.² Rather than a discrete clinicopathologic entity, FSGS is a group of conditions characterized by segmental glomerular sclerosis and podocyte effacement on histology. On the basis of the etiology, FSGS can be classified into primary, secondary, or genetic forms. Primary FSGS is presumed to be caused by a circulating factor which targets podocytes, leading to deranged podocyte function, increased glomerular permeability, and endothelial cell injury, ultimately resulting in the characteristic progressive focal and segmental sclerotic lesions in the glomerulus.³ The release of such a permeability factor is thought to play a role in disordered T cell function in FSGS⁴; however, despite several molecules being proposed as the offending permeability factor over the years,⁵ the identity of this factor and the role of immune system dysregulation in the pathogenesis of primary FSGS remain undetermined.

The Janus kinase signal transducer and activator of transcription (JAK/STAT) signaling pathway regulates a wide variety of cellular processes, principally inflammation and immune responses, but also proliferation, metabolism, and others.⁶ It consists of four JAK members (JAK1-3 and TYK2 2) and seven signal transducer and activator of transcription (STAT) members (STAT1-STAT4, STAT5a, STAT5b, and STAT6). STAT proteins act downstream of cytokines and other stimulatory factors by translocating into the nucleus in their phosphorylated form and acting as transcription factors for downstream immune functions. There is a large body of evidence linking the JAK/STAT pathway to the differentiation and maturation of the immune system. Various STATs have been implicated in T cell differentiation and activity⁷ and in innate immunity.⁸ Animal model and human genetic studies have linked gene polymorphisms in JAK2, STAT3, and STAT4 to autoimmune diseases such as inflammatory bowel disease⁹ and type 1 diabetes.¹⁰ The discovery of the involvement of JAK/STAT signaling in important disease processes has led to the development of JAK inhibitors, which are used for various clinical indications such as treatment of rheumatoid arthritis and transplantation immunosuppression.¹¹

There is growing evidence that the JAK/STAT pathway is involved in various kidney diseases, such as diabetic nephropathy, autosomal dominant polycystic kidney disease (ADPKD), HIV-associated nephropathy, and AKI; however, the in vivo mechanisms are not well understood.¹² In a murine model of induced diabetic nephropathy, a transgenic strain with a reduced capacity for STAT3 activation developed less proteinuria, mesangial expansion, cell proliferation, macrophage infiltration, inflammation, and abnormal matrix synthesis during early diabetic kidney disease compared with controls.¹³ In human patients with diabetic nephropathy, transcriptomic studies demonstrated increased expression of JAK1, JAK2, and JAK3 and STAT1 and STAT3 in affected patients compared with normal individuals.¹⁴ A randomized phase 2 trial of baricitinib (a JAK1/2 inhibitor) in individuals with diabetic kidney disease demonstrated reduced kidney disease progression in those receiving treatment.¹⁵ The JAK/STAT pathway has also been studied in ADPKD. Studies provide evidence that the activation of the STAT3 and STAT6 pathways promoted proliferation of cyst-lining cells in human and murine models,^16,17 and knockout of STAT6 led to improved kidney function and decreased cyst size in murine models.¹⁸ In HIV-associated nephropathy, inhibition of STAT3 activity attenuated the progression of glomerular and tubulointerstitial injury.¹⁹ In a kidney microorganoid model of coronavirus disease 2019–associated nephropathy,²⁰ cytokine-induced STAT1, STAT2, and STAT3 activation was found to induce APOL1 expression, which is a known risk allele for progressive kidney disease.

Despite growing interest in the involvement of the JAK/STAT pathway in several kidney diseases, few studies have investigated the role of JAK/STAT in primary FSGS. A study of 15 adult patients with FSGS showed upregulation of phosphorylated STAT1, STAT3, and STAT5 in cytokine-stimulated CD4⁺ T lymphocytes, and immunohistochemistry of kidney biopsy samples and transcriptomic analysis of JAK/STAT and downstream targets suggest overactivity of JAK/STAT in FSGS compared with controls.²¹ A study using an in vitro assay of albumin permeability in rat glomeruli demonstrated upregulation of phosphorylation of STAT3 in rat glomeruli by cardiotrophin-like cytokine factor 1, a member of the IL 6 family.²² A transcriptomic study linking high-risk APOL1 genotype and increased JAK/STAT expression²³ has informed an ongoing clinical trial investigating the use of baricitinib in APOL1-associated FSGS (NIH Project number 1R01MD016401-01).

We recently investigated a case of multiorgan immune dysregulation in a pediatric patient with nephrotic syndrome caused by membranous nephropathy and identified a mosaic, gain-of-function mutation (S703I) in JAK1.²⁴ Mass cytometry (CyTOF) immunophenotyping was performed on whole blood from the patient to analyze the phosphorylation of all STATs downstream of JAK1 in all of the major immune cells. We observed heightened phosphorylation of STAT1, STAT3, STAT4, STAT5, and STAT6 in certain subsets of immune cells. After identification and classification of the mutation and after ex vivo treatment on the patient's blood demonstrated inhibition of STAT phosphorylation by tofacitinib, clinical treatment using tofacitinib was elected. After treatment, the patient's dermatitis and gastrointestinal symptoms completely resolved; kidney outcomes could not be observed because the patient had progressed to dialysis dependence preceding the treatment. In light of these findings, we concluded that the genetic and immunologic basis of dysregulation of the JAK/STAT pathway in other forms of nephrotic syndrome merited further investigation. In this work, we investigate the hypothesis that the JAK/STAT pathway is dysregulated in pediatric primary FSGS and may be associated with clinical outcomes.

Methods

Sample Collection

The study was approved by the Icahn School of Medicine at Mount Sinai Institutional Review Board, and written informed consent for all individuals in this study was provided. All samples from patients were drawn in the context of an outpatient nephrology visit; samples were deferred if a participant exhibited signs of infection (such as fever, runny nose, cough, and sore throat). Age- and sex-matched healthy controls (HCs) were identified from the nephrology clinic as individuals with normal kidney function and without proteinuria or glomerular disease, such as those with hypertension or a solitary kidney. Peripheral blood was drawn into a cell preparation tube with sodium heparin (BD Vacutainer). Plasma was isolated from Ficoll separation and stored at −80°C until use. Whole blood was either directly fixed using Proteomic Stabilizer PROT1 (SmartTube) and frozen at −80°C, or fresh whole-blood samples were stained by adding 270  µl blood directly to the Maxpar Direct Immune Profiling Assay (Standard Biotools) tube containing the lyophilized antibody panel, mixing, and incubating for 15 minutes at room temperature, followed by stabilization with PROT1 and freezing.

Whole Exome Sequencing

Genomic DNA was isolated from whole blood (QIAamp DNA Mini Kit) and sequenced on the Illumina platform at a depth of >30×. Variant interpretation was performed using Emedgene software.

CyTOF

The protocol for CyTOF was previously described.²⁵ Frozen stabilized blood samples were thawed according to the manufacturer's recommended protocol and then washed with barcode permeabilization buffer (Fluidigm). The samples were uniquely barcoded using the Cell-ID 20-Plex Pd Barcoding Kit (Fluidigm) and pooled together. For previously unstained samples, cells were incubated with an antibody cocktail for surface markers to identify major immune populations, followed by methanol permeabilization, heparin block, and staining with a cocktail of antibodies against intracellular targets, including markers of phosphorylation and signaling. All of the antibodies used were purchased from Fluidigm. After washing, cells were incubated in freshly diluted 2.4% formaldehyde containing 125 nM Ir Intercalator (Fluidigm), 0.02% saponin, and 30  nM OsO4 (ACROS Organics) for 30 minutes at room temperature. Samples were then washed and acquired immediately.

For acquisition, samples were washed with PBS+0.2% BSA, PBS, and then cell acquisition solution buffer (Fluidigm). The final solution in cell acquisition solution buffer consisted of 1 million cells per millilitre and a 1/20 dilution of EQ beads (Fluidigm). After routine instrument optimization, samples were acquired at a rate of <300 events per second on a Helios mass cytometer (Fluidigm) with a modified wide-bore injector (Fluidigm).

FCS files of acquired events were normalized and concatenated with Fluidigm acquisition software and deconvoluted using a MATLAB-based debarcoding application, and the resulting files were analyzed using Cytobank. Cell events were identified as Ir191/193-positive and Ce140-negative events. Doublets were excluded on the basis of Mahalanobis distance and barcode separation and with the Gaussian parameters calculated using the Helios CyTOF software. Downstream data analysis was performed on Cytobank by biaxial gating of immune populations according to a previously published gating scheme.²⁵ Patients with FSGS (n=9) and age- and sex-matched HC individuals (n=8) were included.

Multiplex Cytokine Analysis

Cytokine plasma collected by Ficoll isolation from heparinized whole blood was thawed and centrifuged. Magnetic Luminex assays with the MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel (MilliporeSigma, HCYTA-60K-PX38) were run according to the manufacturer's protocol. The samples were quantified on the MAGPIX xMAP Instrument (Luminex). For each sample, >50 beads were collected per analyte. The median fluorescence intensity of these beads was recorded and used for analysis with the Milliplex Analyst software using a 5P regression algorithm. In the absence of signal, the assay's lower limit of detection for each cytokine was used as the intensity value for analysis. Patients with FSGS (n=10) and age- and sex-matched HC individuals (n=6) were included.

Machine Learning and Statistical Analysis

To discern clinically meaningful patterns, patients were clustered according to phosphorylated STAT3 (pSTAT3) patterns across immune cell types by unsupervised learning algorithms. Unsupervised hierarchical clustering was performed with one-minus-Pearson correlation as the similarity measure, using the publicly available online software Morpheus (Broad Institute).²⁶ Principal component analysis (PCA) was performed using Prism (Graphpad). Unpaired two-tailed Mann–Whitney U tests were used to compare eGFR, urine protein/creatinine ratio, and serum albumin between groups. Linear regression was performed to determine the association between pSTAT3 and eGFR, urine protein/creatinine ratio, and serum albumin. Statistical analyses were performed using Excel and Stata.

Results

The ten patients with FSGS had a mean age of 13 years, 50% were female, and they had a variety of racial backgrounds (Table 1). All patients were steroid resistant, and all but one patient was receiving immunotherapy. None of the patients had the APOL1 high-risk genotype (G1/G2, G1/G2, or G2/G2). The proportion of major immune cell populations delineated by CyTOF did not differ significantly between patients and controls (Figure 1A). However, when CD4 T cells were further divided into subtypes, patients with FSGS had decreased proportions of naïve and memory regulatory CD4 T cells compared with controls (Figure 1B).

Table 1.

Characteristics of patients with primary FSGS (N=10) and age- and sex-matched controls (N=10)

Demographics	Patients N=10 (%)	Controls N=10 (%)
Mean age in years (SEM)	13.0 (6.1)	14.1 (1.6)
Female	5 (50%)	5 (50%)
Race
Asian	10 (10%)	2 (20%)
Black	4 (40%)	4 (40%)
White	3 (30%)	0 (0%)
Other	2 (20%)	2 (20%)
Hispanic	3 (30%)	3 (10%)
Steroid-resistant	10 (100%)	0
Immunosuppression
Tacrolimus	8 (72.7%)	0
Mycophenolate mofetil	2 (18.2%)	0
Mean eGFR (SEM; ml/min per 1.73 m2)	87 (11.5)	99 (8.2)

Figure 1.

Immune profiling with CyTOF indicates hyperactivity in the JAK/STAT pathway in most individuals with FSGS. Frequency of (A) all immune cell subtypes and (B) CD4⁺ T cell subtypes in patients with FSGS (n=9) and age- and sex-matched controls (n=10) expressed as the log₂-transformed percentage of live cells. Significance was assessed using Mann–Whitney U test; *P < 0.05. (C) Relative intensity of phosphorylated STAT proteins measured by CyTOF expressed as log₂ ratio of patients (n=9) compared with the median value of controls (n=10). Unsupervised clustering of patients using the complete method (one-minus-Pearson correlation metric). (D) PCA of raw phospho-STAT3 values from CyTOF. CyTOF, mass cytometry; JAK/STAT, Janus kinase/signal transducer and activator of transcription; PCA, principal component analysis; Phospho-STAT, phosphorylated STAT; STAT, signal transducer and activator of transcription.

Despite having an overall ordinary immune cell profile, patients with FSGS had increased JAK/STAT pathway activity when compared with controls. Basal phosphorylated STAT1 levels were upregulated in a subset of patients with FSGS compared with controls (three of ten), and basal pSTAT3 levels were increased in most of the patients with FSGS (six of ten) compared with controls, particularly in T cell subsets (Figure 1C). There were no significant differences in phosphorylated STAT5 and phosphorylated STAT6 levels between patients and controls. Patients were clustered according to pSTAT3 patterns across immune cell types (Figure 1C). PCA generated pSTAT3 high (pSTAT3 hi), intermediate (pSTAT3 int), and low groups (pSTAT3 lo; Figure 1D). The pSTAT3 hi group consisted of four patients with FSGS, the intermediate group had four patients with FSGS and five controls, and the low group had one patient with FSGS and three controls.

The clinical outcomes of the pSTAT3 hi group were compared with those of the combined pSTAT3 lo and int groups (Table 2). At enrollment, none of the clinical outcomes measured—eGFR, urine protein-to-creatinine ratio (UPC), or serum albumin—differed between the pSTAT3 hi group versus the combined pSTAT3 lo and int group. However, after 1 year of follow-up, patients in the pSTAT3 hi group had worse clinical outcomes. The pSTAT3 hi group had worse kidney function (eGFR 29 versus 100 ml/min per 1.73 m², P < 0.01), worse proteinuria (UPC 46 versus 1.8 mg/g Cr, P < 0.01), and worse serum albumin (0.9 versus 4.0 mg/dl, P < 0.01) versus the combined pSTAT3 lo and int group. Notably, although the clinical status of the pSTAT3 hi group worsened over 1 year, the pSTAT lo and int group stayed relatively stable as measured by eGFR, UPC, and serum albumin. The correlation of clinical parameters with pSTAT3 levels within more specific T cell subsets found pSTAT3 levels in CD4, CD8, and CD4⁻ CD8⁻ T cells positively associated with UPC and negatively associated with albumin and eGFR at 1 year of follow-up (Supplemental Figure 1), whereas other cell types did not reveal significant associations. These results suggest that pSTAT3 level measured across all immune cell types can be used to stratify patients into high- and low-risk groups and may predict future progression of kidney disease (as measured by eGFR, UPC, and albumin). This effect is predominantly represented by pSTAT3 in T cells, which specifically may be used as a marker of disease severity.

Table 2.

Phosphorylated STAT3 levels predict future worsening of FSGS severity

Outcome	pSTAT3 hi mean	pSTAT3 lo and int mean	Difference (pSTAT3 lo and int.−pSTAT3 hi; 95% CI)	P Value
eGFR at enrollment	69	89	20.5 (−39.2 to 80.2)	0.45
UPCR at enrollment	9.2	1.6	−7.6 (−16.0 to 0.9)	0.07
Albumin at enrollment	2.4	3.6	1.2 (−0.7 to 3.1)	0.18
eGFR at 1 yr	29	100	71.5 (−30.9 to 112.1)	<0.01
UPCR at 1 yr	46	1.8	−44.2 (−65.0 to −23.4)	<0.01
Albumin at 1 yr	0.9	4.0	3.15 (2.3 to 4.1)	<0.01

Mann–Whitney U test comparing mean clinical outcomes between pSTAT3 high cluster (n=4) versus other participants (pSTAT3 low and pSTAT3 intermediate n=5). eGFR was calculated using the CKiD U25 equation and expressed in ml/min per 1.73 m². Urine protein-to-creatinine ratio is in mg/g creatinine. Albumin is in mg/dl. CI, confidence interval; pSTAT3 hi, pSTAT3 high; STAT, signal transducer and activator of transcription; UPCR, urine protein/creatinine ratio.

To determine whether cytokine signaling drives STAT3 hyperactivity in FSGS rather than intrinsic STAT3 gain of function, multiplex immunoassay was performed on plasma samples from patients with FSGS, and patients' genomic data were examined for the presence of pathogenic STAT3 variants. There were no pathogenic STAT3 variants identified in this cohort. Overall, there was broad cytokine dysregulation in a subset of patients compared with controls (Figure 2A). Absolute levels of upstream cytokine regulators of the JAK/STAT pathway were measured, and IL2, IL4, IL5, IL6, IL10, IL12p40, and IL15 levels were significantly increased in patients compared with controls (Figure 2B). This suggests that broad inflammation is present in the patients and that it activates downstream kinases.

Figure 2.

Multiplex cytokine analysis demonstrates cytokine dysregulation in FSGS. (A) Multiplex cytokine analysis of patient plasma expressed as the log₂-transformed fold change over the mean of healthy controls (n=10). (B) Mean levels of cytokines involved in JAK/STAT signaling of patients (n=10) and controls (n=6) graphed on a log₁₀ scale. Error bars denote SEM. Significance was assessed using Welch t test; *P < 0.05 eGFR, UPC, and albumin after 1 year of follow-up.

Discussion

Although previous studies have linked the JAK/STAT pathway to various kidney diseases, few have studied the role of the JAK/STAT pathway in FSGS, and none have focused exclusively on pediatric primary FSGS. In summary, our study of ten pediatric patients with primary FSGS identified JAK/STAT dysregulation likely because of increased cytokine activators of this pathway. Differences in progression of disease defined by proteinuria, hypoalbuminemia, and decline in eGFR were associated with the degree of pSTAT3 hyperactivity in T cells.

In our cohort, basal pSTAT3 activity was significantly upregulated in most of the patients, whereas previous adult studies did not demonstrate increased unstimulated pSTAT3 activity in patients with FSGS compared with controls.²⁷ Differing analytic approaches or differences in the patients could explain these different results. Our approach of CyTOF followed by unsupervised clustering with machine learning algorithms allowed us to both analyze cell-type–specific pSTAT activity on the individual level and differentiate between subsets of patients on the basis of pSTAT activity, which is a strength of our study. Children with FSGS, the participants of our study, are more likely to have primary FSGS as opposed to secondary causes in adults, so differences in JAK/STAT pathway activity may be inherently different on that basis. Our baseline analysis included patients with a wide range of eGFR in variable remission and relapse status. On the basis of our observation of the risk of progression being associated with STAT3 hyperactivity, it is likely that timing of baseline studies is relevant when considering the association with clinical status. Our findings of JAK/STAT dysregulation in pediatric primary FSGS are in line with previous work on the role of the JAK/STAT pathway in various kidney diseases such as diabetic nephropathy,^13,15 ADPKD¹⁸, and HIV-associated nephropathy.¹⁹ Given that the JAK/STAT pathway is involved in a variety of cellular processes governing inflammation, immune responses, proliferation, and metabolism,⁶ this finding is perhaps unsurprising, because cellular injury and fibrosis are intrinsic to the pathophysiology of FSGS. One possible mechanism by which the JAK/STAT pathway mediates podocyte injury and fibrosis is through the APOL1 pathway, because several studies have suggested increased APOL1 signaling downstream of STAT1, STAT2, and STAT3 activation.^20,23 However, the specific mechanisms linking JAK/STAT dysregulation with the development and progression of FSGS warrants additional study. Although JAK/STAT genetic variants have been associated with various autoimmune conditions, we did not identify any pathogenic alleles in the JAK/STAT family in our cohort. In addition, none of the patients had the APOL1 high-risk genotype (G1/G1, G1/G2, or G2/G2), so differences in JAK/STAT signaling are likely attributed to differences in regulation instead of genetic variation in this cohort.

Currently, prognostic indicators do not exist for FSGS progression other than general disease severity markers such as kidney function, proteinuria, hypoalbuminemia, and responsiveness to existing treatments.²⁸ In this study, increased pSTAT3 levels did not correlate with differences in eGFR, proteinuria, or albumin at the time of enrollment. However, STAT3 phosphorylation distinguished patients with a progressively worsening trajectory from those with more stable disease. This correlation between pSTAT3 and clinical outcomes is most dominant in T cells compared with other cell types. This is a novel finding and differs from some previous studies of FSGS.²⁷ One limitation of the study is the lack of longitudinal follow-up of pSTAT3 levels, which could be beneficial in future studies. However, on the basis of baseline pSTAT3 levels alone, our findings suggest that pSTAT3 levels could be a novel prognostic marker and identify patients who could most benefit from targeted therapy.

We demonstrated that broad cytokine dysregulation underlies the JAK/STAT hyperactivity observed in FSGS. Despite many of these patients being on immune suppression, the abundance of cytokine activity implies a hyperinflammatory state compared with controls. Notably, members of the IL-2 family (including IL-2, IL-4, and IL-15) were elevated in patients compared with controls. IL-2 is a central immunomodulatory cytokine with dual roles in immune activation and immune tolerance, both promoting the proliferation of activated CD4⁺, CD8⁺, and NK cells and maintaining the function of regulatory T cells.²⁹ Experimental evidence demonstrates that podocytes express IL-2R, and IL-2 stimulation activates the JAK/STAT signaling pathway, specifically increasing STAT5 and JAK3 phosphorylation. This activation results in upregulation of proapoptotic markers in podocytes and leads to podocyte injury and loss of glomerular filtration barrier integrity.³⁰ The demonstration of IL-2 hyperactivity suggests a possible link between cytokine signaling and podocyte injury in our cohort. The observation of increased cytokines upstream of various JAK/STAT signaling pathways may lead us to consider broad cytokine inhibition via JAK inhibitors. Tofacitinib, a potent inhibitor of JAK1 and JAK3, has been US Food and Drug Administration approved for the treatment of rheumatoid arthritis, psoriatic arthritis, ulcerative colitis, and polyarticular juvenile idiopathic arthritis³¹ but has not been tested in nephrotic syndrome other than in our previously described case of JAK1 mutation.²⁴ Baricitinib, a JAK1/2 inhibitor, has been tested in diabetic kidney disease,¹⁵ and is currently undergoing a phase 2 clinical trial in APOL1-mediated FSGS (ClinicalTrials.gov identifier NCT05237388). To our knowledge, no studies have been performed using JAK inhibitors in primary FSGS involving children and adults. Alternatively, a more targeted approach to attenuating pSTAT3 would be the use of tocilizumab because IL-6 is a known activator of STAT3. Whether targeting IL-6 and the JAK/STAT pathway could potentially reduce proteinuria or prevent clinical progression of FSGS clinically remains to be shown in future prospective studies.

Supplementary Material

kidney360-7-260-s001.pdf ^{(1.4MB, pdf)}

kidney360-7-260-s002.pdf ^{(159.5KB, pdf)}

kidney360-7-260-s003.pdf ^{(172.5KB, pdf)}

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/KN9/B381.

Author Contributions

Conceptualization: Dusan Bogunovic, Louise Malle, Jeffrey Saland, Carol L. Shen.

Data curation: Sofija Buta, Michael Horesh, Jean Lim, Louise Malle, Marta Martin-Fernandez, Aum Patel, Ashley Richardson, Haylen Rosberger, Carol L. Shen.

Formal analysis: Dusan Bogunovic, Louise Malle, Carol L. Shen.

Funding acquisition: Dusan Bogunovic, Carol L. Shen.

Investigation: Dusan Bogunovic, Sofija Buta, Michael Horesh, Jean Lim, Marta Martin-Fernandez, Aum Patel, Ashley Richardson, Haylen Rosberger, Carol L. Shen.

Methodology: Dusan Bogunovic, Sofija Buta, Michael Horesh, Jean Lim, Louise Malle, Marta Martin-Fernandez, Aum Patel, Ashley Richardson, Haylen Rosberger, Jeffrey Saland, Carol L. Shen.

Project administration: Sofija Buta, Carol L. Shen.

Resources: Dusan Bogunovic, Ashley Richardson, Carol L. Shen.

Software: Jean Lim, Aum Patel, Haylen Rosberger.

Supervision: Dusan Bogunovic, Jeffrey Saland.

Visualization: Ashley Richardson, Carol L. Shen.

Writing – original draft: Carol L. Shen.

Writing – review & editing: Dusan Bogunovic, Sofija Buta, Michael Horesh, Jean Lim, Louise Malle, Marta Martin-Fernandez, Aum Patel, Ashley Richardson, Haylen Rosberger, Jeffrey Saland, Carol L. Shen.

Funding

C.L. Shen: Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, National Institues of Health Ruth L. Kirschstein National Research Service Award Institutional Research Training Grant (Parent T32, 5T32GM082773-14). D. Bogunovic: National Institutes of Health (R01AI148963).

Declarative Statements

This study includes clinical experimentation and received Institutional Review Board or Ethics Committee approval. All patients provided written informed consent. This study includes clinical experimentation and complies with the Declaration of Helsinki.

Data Sharing Statements

Original data generated for the study will be made available upon reasonable request to the corresponding author. Data Type: Health Care Data; Raw Data/Source Data; Software Executable Code; Research Protocols; Observational Data.

Supplemental Material

This article contains the following supplemental material online at http://links.lww.com/KN9/B382.

Supplemental Figure 1. Scatter plot with linear regression fit of relative pSTAT3 levels in T cell subtypes of patients versus median control (log₂ transformed), plotted against clinical outcomes.