JAK Inhibitor Blocks COVID-19-cytokine-induced JAK-STAT-APOL1 Signaling in Glomerular Cells And Podocytopathy in Human Kidney Organoids Ⅱ

Our results shift the current paradigm of interferon-induced APOL1-nephropathy. Several examples in which high interferon states caused collapsing glomerulopathy in carriers of high-risk APOL1 genotype led to a paradigm that privileged interferons as the chief second-hit triggers of APOL1-mediated glomerulopathy (31, 36, 37). This paradigm was further reinforced by the fact that interferon alpha, beta, and gamma induced APOL1 expression in cultured podocyte and endothelial cell lines (22). However, this archetype is challenged by the observation that interferons are not always elevated in the serum of patients with COVID-19 infection and COVAN, whereas other cytokines including IL-6, IL-1β, and IL-18 are increased (4, 17, 18). In the current study, we demonstrated that even in the absence of interferons, these non-interferon cytokines individually and collectively induced robust APOL1 expression in human podocytes and GECs while also highlighting a previously unappreciated synergism. By demonstrating that JAK-STAT signaling is the central mediator of these combined cytokine effects, our results provide a plausible explanation for how the COVID-19 cytokine storm drives APOL1 expression and the high incidence of collapsing glomerulopathy seen in patients with risk variant APOL1 and COVID-19 infection. These findings may have implications for other APOL1-mediated nephropathies.

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While our results show that COVID-19-induced cytokines are sufficient to induce APOL1 expression and cause the loss of kidney micro-organoid podocytes, they do not exclude the possibility that SARs-CoV-2 may also directly infect kidney cells as it was recently reported (38, 39). However, SARs-CoV-2 in human kidney tissue has only been demonstrated in autopsy specimens in which the confounding contribution of tissue autolysis could not be excluded (3, 4, 15, 16). Most reports from kidney biopsies of patients with COVAN have failed to detect the SARs-CoV-2 virus despite using sensitive methods (3, 4), including two of the nine cases in the current study which were tested by immunohistochemistry and in situ hybridization.

By demonstrating cytokine-induced podocytopathy, our kidney micro-organoid model diverged from the recently published kidney organoid model of APOL1-mediated kidney disease by Liu et al who generated kidney organoids from CRISPR-edited iPSCs of a non-African donor in which G0 APOL1 alleles were edited to G1 alleles but on a G0 genetic background. While interferon gamma-induced APOL1 expression in their organoids, it did not cause cytotoxicity (40). The lack of cytotoxicity in their kidney organoid model could explain the less toxic genetic background on which G1 mutations were superimposed. It is known that the cytotoxicity of APOL1 haplotype is affected by its genetic background (41). The kidney micro-organoid in the present study was generated from unedited iPSCs of an African American carrier of the G1G2 genotype. Preservation of the native genetic haplotype may have contributed to the APOL1-associated cytotoxicity seen in our kidney micro-organoids.

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Furthermore, in contrast to the upregulated APOL1 protein expression we report here, a recent study found no difference in APOL1 mRNA level in the kidney biopsy of one patient with COVAN compared to healthy controls (26). This disagreement in our results may be explained by the transient nature of APOL1 mRNA relative to protein, especially when biopsies were obtained several weeks after the initial diagnosis of COVID-19 infection when the acute effects of COVID-19-induced cytokine storm and the corresponding mRNA expression profile may have dissipated. It is conceivable that the farther one is from the COVID-19-induced cytokine storm, the weaker the acute phase reactants downstream of the cytokine receptor become, including phosphorylated STAT proteins and APOL1 mRNA. Our results suggest that the induced APOL1 protein persists beyond APOL1 mRNA and phosphorylated STATs.

This study has three major clinical implications. One, they underscore the need to genotype Black or Hispanic individuals found to have collapsing glomerulopathy in the context of active or recent COVID-19 infection. However, if kidney biopsy is not feasible or possible, APOL1 genotyping of Black or Hispanic COVID-19-infected individuals with new or worsening proteinuria and AKI is also likely to be high yield. Secondly, because multiple COVID-19-induced cytokines redundantly activate the JAK-STAT pathway to induce APOL1 expression, a therapeutic strategy based on selective removal or inhibition of any one cytokine is unlikely to be effective in preventing or treating COVAN. Currently, baricitinib is only authorized for use under an emergency use authorization for treatment of COVID-19 requiring supplemental oxygen. Therefore, its potential as a treatment for COVAN requires serious consideration, especially for Black and Hispanic carriers of high-risk APOL1 genotypes who have COVID-19 infection. Lastly, based on the evidence that interferon deficiency is associated with severe COVID-19 infection, it was proposed that interferon be administered as therapy for COVID-19. At the time of this writing, according to ClinicalTrials.gov, thirty-six clinical trials of interferon as therapy in COVID-19 are either ongoing or completed. In contrast to the rationale behind these clinical trials, our results caution against the administration of interferons as a treatment for COVID-19 infection in carriers of high-risk APOL1 genotype because interferons could upregulate expression of pathogenic APOL1 in the kidney and precipitate COVAN.

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Our study has some limitations. Our COVAN case series of nine biopsy-proven collapsing glomerulopathy cases is a relatively small sample size and may have underestimated the association between high-risk genotype and COVAN or been subject to sampling bias. The logistical challenge of obtaining kidney biopsies from patients with active COVID-19 infection in acute care setting limits the frequency of kidney biopsies in this population. The same factor is likely responsible for the fact that most of the biopsies in this study were performed several weeks to months after the initial diagnosis of COVID-19 infection. Analysis of kidney biopsies obtained closer to the infectious trigger may provide additional insights into early cellular phenotypes. Additionally, the current study does not identify who and when to treat COVAN with JAK inhibitors. Answers to these questions and the determination of the efficacy of JAK inhibition as treatment for COVAN will be the focus of future investigations. This work highlights the association of high-risk APOL1 genotype and the role of JAK-STAT-APOL1 signaling in the development of COVAN. As our understanding of the COVID-19 pandemic evolves, there is an urgent need to increase clinician and public health awareness about the renal complications of COVID-19. There is also an urgent need for COVAN therapy. Our study offers new data on JAK inhibitors as strong therapeutic candidates for APOL1-associated COVAN.

Methods

Antibodies and reagents.

Primary antibodies against the following proteins were used: APOL1 rabbit anti-human [Genentech, 3.1C1&3.7D6; Western blot [WB] 1:5000 (final concentration 0.05 ug/mL); Genentech, 5.17D12 [IHC] 1:4000 (final concentration 0.95 ug/mL) according to recent report (42)]; APOL1 mouse anti-human [Genentech, 4.17A5; Immunofluorescence [IF] 1:2000 (final concentration 2.13 ug/mL)]; GAPDH mouse anti-human (Santa Cruz Biotechnology, sc47724; WB 1:200); Vinculin mouse anti-human (Sigma-Aldrich, V9131; WB 1:200); NEPH1 mouse anti-human (Santa Cruz Biotechnology, sc373787; WB 1:300); WT1 rabbit anti-human (Abcam, ab89901; WB 1:1000); STAT1 mouse anti-human (Cell Signaling Technology [CST], 9176s; WB 1:1000); STAT2 rabbit anti-human (CST, 72604: WB 1:1000); STAT3 mouse anti-human (CST, 9139; WB 1:1000); PhosphorylatedSTAT1 (Y701) rabbit anti-human (CST, 9167; WB 1:1000); Phosphorylated-STAT2 (Y690) rabbit antihuman (CST, 88410; WB 1:1000); Phosphorylated-STAT3 (Y705) rabbit anti-human (CST, 9145; WB 1:2000); PODXL goat anti-human (R&D, AF1658; IF 1:500), E-Cadherin rabbit anti-human (Cell Signaling, 3195S; IF 1:200), NEPH1 mouse anti-human (Santa Cruz, sc-373787; IF 1:100). Secondary antibodies included goat anti-rabbit IgG, HRP-linked antibody (CST, 7074s; WB 1:1000); horse antimouse IgG, HRP-linked antibody (CST, 7076s; WB 1:1000); Alexa Fluor 488 conjugated donkey antimouse (Jackson ImmunoResearch, 715-546-150; IF 1:1000), Alexa Fluor 594 conjugated donkey antigoat (Jackson ImmunoResearch, 705-585-147; IF 1:1000), and Alexa Fluor 405 conjugated donkey anti-rabbit (Thermo Scientific, A48258; 1:000). For qRT-PCR, TaqMan Gene Expression Assays included APOL1 (Hs01066280_m1), GAPDH (Hs03929097_g1), and PECAM-1 (Hs00169777_m1). Kidney Immunohistochemistry staining for APOL1, synaptopodin, and CD31. Starting with formalin fixed paraffin embedded kidney biopsy slides, antigen retrieval was performed with (EDTA solution at pH 8.0) for 56 minutes at 100 °C. Primary antibodies to APOL1 (5.17D12, Genentech), synaptopodin (Progen Biotechnik, 61094), and CD31 (Cell Signaling, 3528s) were applied at 1:4000 (final concentration 0.95 ug/mL), 1:100 and 1:1600, respectively for 60 minutes at 36 °C. Ready-to-use HQ-conjugated secondary anti-rabbit multimers (760-4815) were incubated for 12 minutes at 36 °C. This was followed by the addition of anti-HQ HRP for 12 min. DAB (760-159) was incubated for 5 minutes at room temperature. The tissue section was counterstained with hematoxylin (760-2021) for 4 min at room temperature. Bluing reagent (760-2037) was added for 4 minutes at room temperature.

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Cytokine treatment of cultured cells.

Primary human glomerular endothelial cells, primary podocytes subcultured from human donor kidney, and organoid-derived podocytes were treated with 50ng/mL, 20ng/mL, or 10ng/mL concentration of the following cytokines: Recombinant Human CXCL9 (Biolegend, 578102); Recombinant Human CXCL10 (Biolegend, 573502); Recombinant Human CXCL13 (Biolegend, 573502); IL-1β (PeproTech, 200-01B); IL-15 (PeproTech, 200-15); IL-18 (Biolegend, 592102); IL-8 (Biolegend, 574202); Interferon (IFN) alpha 1 (Sigma-Aldrich, SRP4596); IFN beta (Peprotech, 300-02BC); IFN gamma (Sigma-Aldrich, I17001); IL-6 (Peprotech, 200-06); TNF-alpha (Peprotech, 300-01A); MCP-1 (CCL2) (Peprotech, 300-04); MIP-1alpha (CCL3) (Peprotech, 300-08); IL-10 (Peprotech, 200-10); IL-7 (Peprotech, 200-07); IL-2 (Peprotech, 200-02); G-CSF (Peprotech, 300- 23). A standardized cytokine concentration of 50ng/mL was pre-determined based on precedent from human cell treatments evaluating cytokine shock syndromes in COVID-19 infection (17). Follow-up experiments used concentrations of 20ng/mL and 10ng/mL. Subsequent treatments, including organoid and organoid-derived podocyte experiments, were performed using 10ng/mL. JAK 1/2 inhibitor, baricitinib (INCB028050) (Selleckchem, S2851), was used at 10uM final concentration in all experiments.

Primary human glomerular endothelial cell culture. The frozen stock of low-risk (G0G0) primary human glomerular endothelial cells were purchased from Celprogen (36066-05), thawed, and cultured in human glomerular endothelial primary cell culture complete media with serum, antibiotic-free (Celprogen, M36066-05SA) on plates coated with proprietary extracellular matrix (Celprogen, E36066-05-PD10 and E36066-05-12Well) in the humidified environment at 37°C and 5% CO2. Cells were passaged using 1X Trypsin EDTA (Celprogen, T1509-014). Cells were used for experiments between passages 2 to 4. Cells were validated by qRT-PCR showing enrichment in PECAM1 gene expression (endothelial cell marker, also known as CD31).

Glomerular isolation from donor human kidney and podocyte subculture. Human donor kidney was procured through the National Disease Research Interchange (NDRI). The donor APOL1 genotype was G0G1. Glomerular isolation was performed using the sieve method adapted from prior publication (43, 44). Briefly, working on ice in a sterile hood, the kidney was first decapsulated and cut in half mid-sagittally. The medulla was dissected away, leaving the cortex remaining. The cortex was then minced and passed sequentially through stainless steel mesh sieves (sizes 425µm, 250µm) and collected on top of a third sieve (150µm) while washing frequently with pre-cooled Phosphate Buffered Saline (PBS) with 1% BSA (without calcium and magnesium) (Endecotts, Sieves 100SIW.150, 100SIW.250, 100SIW.425). Glomeruli were collected, centrifuged, and resuspended in 5mL PBS. 10µL of the sample were stained with NucBlue Live ReadyProbes Reagent (Hoechst 33342) (Thermo Fisher Scientific, R37605) and visualized by light microscopy. Glomeruli were then incubated in digestion buffer [DMEM/F12, 1mg/mL each of (collagenase I, IV, and V), DNAse I (50 U/mL or 50 µg/mL)] at 37°C for 1hr to obtain a single cell suspension (Thermo Fisher Scientific, 10565018; StemCell Technologies, 07415m 07426, 07430; Sigma-Aldrich, 11284932001). DMEM/F12 with 10%FBS (R&D Systems, S10350H) was added to stop digestion and the sample was centrifuged at 450g minutes at 4°C. Isolated podocytes were subcultured in Advanced RPMI (Fischer Scientific, MT10040CV) with 10%FBS and 1%penicillin-streptomycin (Thermo Fisher Scientific, 15070063) on vitronectin coated plates (StemCell Technologies, 07004) at 37°C and 5% CO2. Subcultured cells were visible at ~5 days and were treated after 2 weeks in culture.

Protein extraction and Western blotting. All culture plates and samples were maintained at 4°C. A monolayer of cells was lysed and harvested with Cell Lysis Buffer (Cell Signaling, 9803) with complete mini protease inhibitor and phosphatase inhibitor (Sigma-Aldrich, 04693159001, 04906837001).

Samples were sonicated at level 4 for 10 seconds each, centrifuged at 12000rpm x5 min, the supernatant was collected in a new Eppendorf tube, and protein concentration was determined by BCA protein assay (Pierce, 23225). Protein lysates were diluted with 4x LaemmLi sample buffer 2-mercaptoethanol (BioRad, 1610747; Thermo Fisher, 21985023) and heated at 95-100°C for 5 minutes. Protein lysates were then separated using Criterion TGX stain-free gels (4-20%) and transferred using the Bio-Rad TransBlot Turbo transfer system. Transferred membranes were blocked for 1hr in 3% nonfat milk in Tris-buffered saline and incubated with specific primary antibodies overnight at 4°C. Subsequent day after standard washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for a minimum 1hr at room temp prior to imaging with Bio-Rad ChemiDoc MP Imaging System per manufacturer's instructions.

RNA extraction and qRT-PCR. RNA was isolated from cell monolayer using RLT lysis buffer and Qiagen RNEasy Mini Kit (74106) per the manufacturer's instructions. RNA was transcribed into cDNA using Invitrogen SuperScript IV Reverse Transcriptase reagents and protocol (Thermo Fisher, 18091050). qRT-PCR reactions were performed with Applied Biosystems TaqMan Fast Advanced Master Mix (Fischer Scientific, 44-445-57) and Gene Expression Assays using QuantStudio 6 Flex System (Thermo Fisher Scientific) using ∆∆Ct method with GAPDH as reference gene.

Genotyping. DNA was extracted from FFPE tissue blocks using the QIAamp DNA FFPE Tissue Kit (Qiagen, 56404) per the manufacturer's protocol. Genotyping was performed using Applied Biosystems Taqman allelic discrimination assays for G1 SNP (p.Ser342Gly) and G2 polymorphism (p.Asn388_Tyr389del) using QuantStudio 6 Flex System. This assay has 100% analytic specificity and an analytic sensitivity (limit of detection) of 1.0ng DNA for the detection of APOL1 risk variants in DNA extracted from peripheral blood monocytes. Assays were previously validated by comparing results to direct sequencing (Sanger sequencing). Genotyping quality control measures included the use of technical replicates, 100% matching of positive controls of each genotype, and negative controls.

Derivation of G1G2 patient iPSC kidney micro-organoid. Kidney micro-organoids were derived per the published protocol (27), with some modifications. Briefly, induced human pluripotent stem cells (iPSC) were dissociated into single cells using TrypLE Select and seeded onto vitronectin-coated plates in StemFlex media (Thermo Fisher Scientific, A3349401) with 10uM Rho kinase inhibitor (Tocris Bioscience, 1254) at a density of 11,000-14,000 cells/cm2 and cultured in humidified environment at 37°C and 5% CO2. Cells were then transitioned to TeSR-E6 media (Stemcell Technologies, 05946) with 8 µM CHIR99021 (Tocris Bioscience, 4423) for 4 days. From day 5 to day 7, cells were treated with 200 ng/mL FGF9, 1 µg/mL heparin and 1 µM CHIR99021. On day 7, cells were washed with PBS and dissociated with TrypLE. Dissociated cells were then washed with plain DMEM and centrifuged at 300xg for 5 minutes. Cell pellet was resuspended in Stage1 media [TeSR-E6 containing 200 ng/mL FGF9, 1 µg/mL heparin, 1 µM CHIR99021, 0.1% PVA, 10 µM Rho kinase inhibitor (Tocris Bioscience)] and transferred to a 24-well AggreWell 400 plate (Stemcell, 34411) at approximately 1.2 million cells/well. The plate was then centrifuged at 100xg for 3 minutes and incubated for 48 hours at 37°C and 5% CO2 in a standard cell culture incubator. After 48 hours (day7+2), organoids from the AggreWell were transferred to a 6-well low attachment plate with Stage2 media [TeSR-E6 containing 200 ng/mL FGF9, 1 µg/mL heparin, 1 µM CHIR99021, 0.1% PVA] on orbital shaker inside cell incubator for another 72 hours. From day 7+5 onwards, all organoids were refreshed with Stage 3 media [TeSR-E6 containing 0.1% PVA] on alternative days until used for experiments.

Podocyte isolation from micro-organoid. Isolation of glomeruli from kidney organoids was adapted from a previously described protocol (45). Briefly, groups of iPSC-derived kidney organoids with an initial starting cell number of 1.2x106 iPSCs were dissociated by incubation with TrypLE select (Thermo Fisher) for 5 min at 37 °C. Gentle mixing using a 1 mL pipette was applied every 2-3 min to aid dissociation. Per group, a single 70 µm cell strainer (PluriSelect) was placed onto a 50 mL tube (Falcon) and the mesh was hydrated with PBS. The cell solution was added to the using a 1 mL pipette, allowing flow-through of the solution by gravity. Using the plunger from a 1 mL sterile syringe, the remaining cell solution captured on the strainer was gently pushed through the mesh. The strainer was washed with PBS and discarded, keeping the cell flow through. The cell flow-through was then pipetted onto a pre-hydrated 40 µm cell strainer (Pluriselect) and placed onto a fresh 50 mL tube (Falcon) allowing single cells to flow through by gravity and washing the sieve extensively with PBS to remove any remaining single cells. The largest glomeruli were then collected from the 40 µm cell strainer by inverting the sieve onto a fresh 50 mL tube and washed using PBS to flush out the captured glomeruli. This process was repeated using flow-through from 40 µm process using the final 30 µm cell strainer (PluriSelect) to collect the smaller glomeruli. Isolated glomeruli from IPSC-derived kidney organoids were cultured on vitronectin-coated plates with advanced RPMI 1640 containing 10% FBS in a standard cell culture incubator at 37 °C plus 5% CO2. The media were refreshed every other day until cells were used for experiments.

Immunofluorescence staining, micro-organoid. Kidney micro-organoids were washed with PBS and fixed in 4% PFA in PBS for 30-45 min on ice. Fixed organoids were then washed three times with PBS, and incubated in 30% sucrose in PBS overnight at 4°C. Organoids were embedded in OCT and subjected to 10 µm cryosectioning with a Laica Cryostat. Organoid cryosections were washed three times with PBS and then blocked with blocking buffer 1 (1% fish gelatin, 2% donkey serum, 0.3% Triton X-100 in PBS) for one hour at room temperature. Next, cryosections were incubated with primary antibodies in blocking buffer 1 overnight at 4°C. Cryosections were washed three times with PBS. After washing, cryosections were incubated with secondary antibodies in blocking buffer 1 for 2 hours at room temperature. After five washes with PBS, the cryosections were mounted with a Prolong Glass antifade mounting solution (Thermo Scientific, P36980). Fluorescent images were generated using an ECHO microscope.

Immunofluorescence staining, podocyte. Podocyte cultures were washed with PBS and fixed in 4% PFA in PBS for 10-15 min at room temperature. After fixation, cells were washed three times with PBS and blocked in blocking buffer 2 (1% fisher gelatin, 2% donkey serum, 0.1% saponin in PBS) for 30-60 min. Cells were incubated with primary antibodies in blocking buffer 2 for 2 hours at room temperature (or overnight at 4°C). Cells were washed three times with blocking buffer 2, and incubated with secondary antibodies in blocking buffer 2 for 1 hour at room temperature. After three washes with blocking buffer 2 and one final wash with PBS, the cells were mounted with a Drop-n-Stain EverBrite mounting medium (Biotium, 23008). Fluorescent images were captured using an ECHO microscope.

Viability Testing. For cell viability and ATP measurements, organoid-derived podocytes were plated onto vitronectin-coated 96-well plate in 100uL media (StemCell Technologies, 07004; Corning, CLS3603) and cultured in Advanced RPMI+10%FBS+1%PS (Fischer Scientific, MT10040CV; Thermo Fisher Scientific, 15070063) in humidified environment at 37°C and 5% CO2. Organoid-derived podocytes were simultaneously plated onto 6-well plate to be used for RNA extraction for qPCR and onto a 24-well plate to be used for immunohistochemistry (IHC). Cells were treated at ~80% confluency with six conditions [control, IFNG (10ng/mL), IFNG (10ng/mL) + baricitinib (final concentration 10uM), All Cytokines (10ng/mL), and All Cytokines (10ng/mL) + baricitinib (final concentration 10uM)]. Media was changed at Q48hrs and cells were evaluated at 96hrs. 96-well plate was processed using Promega cell viability and ATP assays as further mentioned below; the 6-well plate was processed for RNA extraction, cDNA synthesis, and qRT-PCR; and 24-well plate was fixed in 4% PFA for IHC. CellTiterFluorTM Cell Viability Assay (Promega, G6080) and CellTiter-Glo® 2.0 Assay (Promega, G9241) were performed using standard protocol instructions provided by the manufacturer. The assays were multiplexed per protocol. Fluorescence (non-lytic protease assay) and luminescence (lytic ATP assay) were measured using a SpectramaxM3 fluorometer. The difference in measures was determined by Student's unpaired t-test.

Statistics. All data are presented as mean ± SD. GraphPad Prism 8.3.1 software was used for data analysis. Cytokine conditions inducing >1.5 fold APOL1 transcript compared to media-treated control were analyzed for significance using an unpaired t-test with Holm-Sidak correction for multiple comparisons. The P-values reported are the adjusted p-values. Significance was set at p<0.05.

Study approval. This study was approved by the Institutional Review Board (IRB) of Duke University, North Carolina. Patient informed consent was not required by the IRB because the case portion of this study was a retrospective review of clinical and archived pathologic material only. Human kidney for glomerular isolation and podocyte subculture was procured through the National Disease Research Interchange (NDRI); this human tissue research was also pre-approved through Duke's IRB. NDRI requires all tissue source sites to obtain informed consent from the tissue donor or surrogate.

Author Contributions SEN, GL, SD, KS, and DS performed experiments and edited manuscripts. AW and DT performed and interpreted histopathology, and IHC, and edited the manuscript. GH contributed essential reagents and edited the manuscript. SEN and OAO designed the study, analyzed and interpreted data, designed figures, and wrote and edited the manuscript.

Acknowledgments This work was supported by 1DP2DK124891-01, 1R01MD016401-01, and the Whitehead Scholar Award (OAO); SEN was supported by Duke Nephrology T32 Training Grant (5T32DK007731-24). We thank Suzie Scales (Genentech) for the generous gift of APOL1-specific antibodies. We thank the Research Histology Lab at Duke BRPC and Steven R. Colon at PhotoPath, Duke Department of Pathology.

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