Hypoxia hits APOL1 in the kidney

Abstract

Individuals of African ancestry carrying two pathogenic variants of apolipoprotein 1 (APOL1) have a substantially increased risk for developing chronic kidney disease. The course of APOL1 nephropathy is extremely heterogeneous and shaped by systemic factors such as a response to interferon. However, additional environmental factors operating in this second-hit model have been less well defined. Here, we reveal that stabilization of hypoxia-inducible transcription factors (HIF) by hypoxia or HIF prolyl hydroxylase inhibitors activates transcription of APOL1 in podocytes and tubular cells. An active regulatory DNA-element upstream of APOL1 that interacted with HIF was identified. This enhancer was accessible preferentially in kidney cells. Importantly, upregulation of APOL1 by HIF was additive to the effects of interferon. Furthermore, HIF stimulated expression of APOL1 in tubular cells derived from the urine of an individual carrying a risk variant for kidney disease. Thus, hypoxic insults may serve as important modulators of APOL1 nephropathy. Individuals of African ancestry carrying two pathogenic variants of apolipoprotein 1 (APOL1) have a substantially increased risk for developing chronic kidney disease. The course of APOL1 nephropathy is extremely heterogeneous and shaped by systemic factors such as a response to interferon. However, additional environmental factors operating in this second-hit model have been less well defined. Here, we reveal that stabilization of hypoxia-inducible transcription factors (HIF) by hypoxia or HIF prolyl hydroxylase inhibitors activates transcription of APOL1 in podocytes and tubular cells. An active regulatory DNA-element upstream of APOL1 that interacted with HIF was identified. This enhancer was accessible preferentially in kidney cells. Importantly, upregulation of APOL1 by HIF was additive to the effects of interferon. Furthermore, HIF stimulated expression of APOL1 in tubular cells derived from the urine of an individual carrying a risk variant for kidney disease. Thus, hypoxic insults may serve as important modulators of APOL1 nephropathy. Lay SummaryGenetic variants in the gene apolipoprotein 1 (APOL1) can lead to chronic kidney disease. Other factors, such as autoimmune diseases or viral infections, influence the course of kidney disease in patients with risk variants. These factors are incompletely understood. Here, we found that low oxygen levels (hypoxia) and drugs that stabilize hypoxia-inducible transcription factors (HIFs) activate transcription of APOL1 in kidney cells. This regulation was specific for kidney cells and was additive to effects of interferon, another inducer of APOL1. In addition, HIF stimulated the expression of risk variants of APOL1 in tubular cells that were collected from the urine of a patient. We conclude that hypoxic conditions may serve as important modulators of APOL1-associated kidney disease. Genetic variants in the gene apolipoprotein 1 (APOL1) can lead to chronic kidney disease. Other factors, such as autoimmune diseases or viral infections, influence the course of kidney disease in patients with risk variants. These factors are incompletely understood. Here, we found that low oxygen levels (hypoxia) and drugs that stabilize hypoxia-inducible transcription factors (HIFs) activate transcription of APOL1 in kidney cells. This regulation was specific for kidney cells and was additive to effects of interferon, another inducer of APOL1. In addition, HIF stimulated the expression of risk variants of APOL1 in tubular cells that were collected from the urine of a patient. We conclude that hypoxic conditions may serve as important modulators of APOL1-associated kidney disease. The presence of 2 alleles of the coding variants G1 and G2 in the apolipoprotein L1 (APOL1) gene increases the risk of developing chronic kidney disease dramatically, with odds ratios ranging from 2.6 to 29, depending on the underlying condition.1Limou S. Nelson G.W. Kopp J.B. et al.APOL1 kidney risk alleles: population genetics and disease associations.Adv Chronic Kidney Dis. 2014; 21: 426-433Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar For example, APOL1 nephropathy manifests as focal segmental glomerular sclerosis or HIV-associated nephrosclerosis in patients with sub-Saharan African ancestry. In these individuals, the frequency of carrying 2 risk alleles is ≈13%.2Daneshpajouhnejad P. Kopp J.B. Winkler C.A. et al.The evolving story of apolipoprotein L1 nephropathy: the end of the beginning.Nat Rev Nephrol. 2022; 18: 307-320Crossref PubMed Scopus (18) Google Scholar Mechanistically, APOL1 risk variants are supposed to exert a gain of dysfunction of the APOL1 protein, which affects molecular processes, such as mitochondrial function, inflammasome activation, endoplasmic reticulum stress, autophagy, and ion transport.2Daneshpajouhnejad P. Kopp J.B. Winkler C.A. et al.The evolving story of apolipoprotein L1 nephropathy: the end of the beginning.Nat Rev Nephrol. 2022; 18: 307-320Crossref PubMed Scopus (18) Google Scholar Thus, any stimulus augmenting the concentration of the APOL1 risk variants will have significant impact on the course of kidney disease. In this respect, high levels of interferons caused by either viral infection or therapeutic application (e.g., in patients with hepatitis C) led to development of collapsing focal segmental glomerular sclerosis in APOL1 risk carriers.3Nichols B. Jog P. Lee J.H. et al.Innate immunity pathways regulate the nephropathy gene apolipoprotein L1.Kidney Int. 2015; 87: 332-342Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar Interestingly, only a minority of risk allele carriers develop APOL1 nephropathy. This supports the hypothesis of a 2-hit model requiring the presence of APOL1 risk alleles and contribution of environmental or systemic factors, such as viral infection, to initiate and promote APOL1 nephropathy. Apart from the interferon response, there is little evidence so far for the involvement of other factors influencing APOL1 expression. The risk of developing APOL1 nephropathy persists in kidney allografts from APOL1 risk variant–positive donors.4Reeves-Daniel A.M. DePalma J.A. Bleyer A.J. et al.The APOL1 gene and allograft survival after kidney transplantation.Am J Transplant. 2011; 11: 1025-1030Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar This indicates that the deleterious effects of APOL1 risk variants are generated within the cells of the kidney and, thus, any factors operating on APOL1 expression in renal cells will modify the risk of developing kidney disease. In the kidney, podocytes, endothelial cells, and proximal tubular cells have been identified to express APOL1.2Daneshpajouhnejad P. Kopp J.B. Winkler C.A. et al.The evolving story of apolipoprotein L1 nephropathy: the end of the beginning.Nat Rev Nephrol. 2022; 18: 307-320Crossref PubMed Scopus (18) Google Scholar In many kidney diseases, including acute kidney injury, inflammation, and fibrosis, these cells are exposed to hypoxia, which leads to stabilization of hypoxia-inducible factors (HIFs).5Schodel J. Ratcliffe P.J. Mechanisms of hypoxia signalling: new implications for nephrology.Nat Rev Nephrol. 2019; 15: 641-659Crossref PubMed Scopus (181) Google Scholar For example, HIF stabilization specifically in podocytes causes nephritis in mice, indicating a prominent role of HIF in this compartment.6Ding M. Cui S. Li C. et al.Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice.Nat Med. 2006; 12: 1081-1087Crossref PubMed Scopus (177) Google Scholar Recently, HIF stabilizers have been approved for therapy of renal anemia in patients with chronic kidney disease.7Maxwell P.H. Eckardt K.U. HIF prolyl hydroxylase inhibitors for the treatment of renal anaemia and beyond.Nat Rev Nephrol. 2016; 12: 157-168Crossref PubMed Scopus (203) Google Scholar These compounds were developed to stabilize HIF-2α in interstitial cells of the kidney and to induce the HIF target gene erythropoietin. Here, we explore the effects of hypoxia and HIF stabilization on the regulation of APOL1 expression in renal cells. The human podocyte cell line was a gift from M. Saleem and cultured in supplemented Roswell Park Memorial Institute medium. Proliferating podocytes were cultured under 33 °C; for differentiation, subconfluent dishes were transferred to 37 °C and cultured for 10 days. Human urinary primary tubular cells (PTCs) were isolated from a patient with a G0/G1 APOL1 genotype following a published protocol.8Zhou T. Benda C. Dunzinger S. et al.Generation of human induced pluripotent stem cells from urine samples.Nat Protoc. 2012; 7: 2080-2089Crossref PubMed Scopus (438) Google Scholar HEK293T cells expressing APOL1 G0, G1, and G2 variants lacking the signal peptide under the control of a doxycycline-inducible promoter were cultured in standard Dulbecco’s modified Eagle’s medium.9Granado D. Muller D. Krausel V. et al.Intracellular APOL1 risk variants cause cytotoxicity accompanied by energy depletion.J Am Soc Nephrol. 2017; 28: 3227-3238Crossref PubMed Scopus (64) Google Scholar Cells were lysed in urea/sodium dodecylsulfate buffer, and proteins were resolved by sodium dodecylsulfate–polyacrylamide gel electrophoresis. Proteins were detected using an HIF-1α antibody (Cay10006421; Cayman Chemicals), an HIF-2α antibody (AF2997; R&D Systems), an APOL1 antibody (Ab108315; Abcam), and a β-actin antibody (A3854; Sigma Aldrich). Horseradish peroxidase–conjugated secondary antibodies were used as applicable (Dako, Agilent Technologies). Assay for transposase-accessible chromatin (ATAC) experiments were performed as previously described.10Protze J. Naas S. Kruger R. et al.The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression.J Biol Chem. 2022; 298101699Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar A total of 60,000 cells were directly subjected to the Omni-ATAC protocol, as described by Corces et al.11Corces M.R. Trevino A.E. Hamilton E.G. et al.An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues.Nat Methods. 2017; 14: 959-962Crossref PubMed Scopus (1020) Google Scholar Chromatin immunoprecipitation (ChIP) experiments were performed using the ChIP-IT High Sensitivity kit (Active motif). For immunoprecipitations, 20 μg of chromatin and 3–6 μl of antibodies directed against HIF-1α (Cay10006421; Cayman Chemicals), HIF-1β (NB100-110; Novus Biologicals), HIF-2α (PM9; kind gift from Professor Sir Peter Ratcliffe, Oxford), or H3K27ac (ab4729; Abcam) were used. To test whether hypoxia controls expression of APOL1, we performed transcriptomic analysis of podocytes cultured under hypoxic (16 hours, 1% O2) or control conditions using RNA sequencing. Strikingly, differential gene expression analysis revealed that according to adjusted P values, APOL1 is the second most significantly induced gene under hypoxic conditions in these cells (Figure 1a). Exposure of podocytes to the HIF-hydroxylase inhibitor dimethyloxalylglycine (DMOG) also increased APOL1 mRNA significantly, indicating involvement of HIF in APOL1 transcriptional regulation (Supplementary Figure S1). Increased mRNA levels also translated into higher protein levels of APOL1 (Figure 1b). Similar results were obtained in a series of primary tubular cells isolated from nondiseased regions of tumor nephrectomy specimens, indicating a conserved regulation in a broader spectrum of renal cells (Supplementary Figure S2). To confirm the involvement of HIF, we transfected siRNA against HIF subunits (HIF-1α and HIF-2α) into podocytes and measured expression of APOL1 (Figure 1c; Supplementary Table S1). Induction of APOL1 protein by DMOG was reduced especially in HIF-1α–depleted podocytes (Figure 1c). Similar results were obtained in podocytes targeting HIF using Clustered regularly interspaced palindromic repeats/Cas9 (CRISPR/Cas9) protein together with HIF-subunit specific guide RNAs and in tubular cells using siRNA against HIF (Supplementary Figure S3). To evaluate the overlap of HIF and APOL1 mRNA in different renal cell types, we reanalyzed published single-cell RNA-sequencing (scRNA) data from nondiseased kidneys.12Zhang Y. Narayanan S.P. Mannan R. et al.Single-cell analyses of renal cell cancers reveal insights into tumor microenvironment, cell of origin, and therapy response.Proc Natl Acad Sci U S A. 2021; 118e2103240118Google Scholar This analysis confirmed a high degree of overlap of HIF and APOL1 mRNA, especially in podocytes, vasculature, and, to a lesser extent, tubular cells, which provides a prerequisite for the observed regulation of APOL1 expression by hypoxia in cells of the human kidney (Figure 1d). Recently, analysis of acute kidney injury in humans by scRNA-seq revealed remarkable transcriptomic changes, especially in tubular cells of the injured kidneys.13Hinze C. Kocks C. Leiz J. et al.Single-cell transcriptomics reveals common epithelial response patterns in human acute kidney injury.Genome Med. 2022; 14: 103Crossref PubMed Scopus (15) Google Scholar Hypoxia signaling was among the most significant upregulated pathways in this compartment. We reanalyzed the available data and determined significantly increased levels of APOL1 mRNA in tubular cells from injured kidneys compared with control kidneys, indicating that the aforementioned regulation of APOL1 by hypoxia occurs in vivo (Supplementary Figure S4). To further explore the role of HIF in regulating APOL1 expression, we generated HIF ChIP-sequencing data from podocytes exposed to DMOG and observed HIF-DNA interactions of both HIF α-subunits and the β-subunit ≈3 kb upstream of the APOL1 promoter (Figure 1e). The identified putative regulatory DNA element displayed marked accessibility and activity, as determined by ATAC sequencing and H3K27ac ChIP sequencing, respectively. Interestingly, when comparing ATAC-sequencing data from podocytes with DNAse hypersensitive sites defined in 733 biosamples,14Meuleman W. Muratov A. Rynes E. et al.Index and biological spectrum of human DNase I hypersensitive sites.Nature. 2020; 584: 244-251Crossref PubMed Scopus (115) Google Scholar we noted open chromatin preferentially in samples from the renal cluster of these samples, suggesting the existence of a kidney-associated enhancer at this position (Figure 1e). Thus, our data indicate that HIF regulates APOL1 mRNA expression in podocytes and tubular cells from a kidney-associated enhancer. APOL1 expression in the kidney is triggered by inflammatory stimuli, such as interferon gamma. To test whether HIF signaling interferes with the interferon pathway on APOL1 expression, we exposed podocytes and tubular cells to DMOG, interferon gamma, or a combination of both (Figure 2a and b ; Supplementary Figure S5). Strikingly, a combination of both stimuli induced APOL1 mRNA and protein expression to higher levels than each stimulus alone. This indicates that HIF and interferon gamma regulate expression of APOL1 independent from each other in renal cells. To further corroborate this finding, we introduced the sequence containing the hypoxia-responsive element of the HIF-binding enhancer into a reporter plasmid. On exposure of the transfected HEK293T cells to DMOG, we measured increased activity of the reporter. This effect was unchanged on interferon gamma suggesting that interferon gamma from the HIF-binding kidney-associated enhancer (Figure effects of APOL1 on kidney are by the 2 APOL1 risk We identified individual for the variant G1 and isolated tubular cells from the urine urinary these cells increased APOL1 mRNA expression on HIF stabilization by DMOG (Figure The allele for in the on HIF indicating that HIF expression from both alleles (Figure HIF stabilizers have been for to patients with renal To evaluate an effect of these compounds on APOL1 we exposed human urinary from the G0/G1 individual to a of these and detected increased levels of APOL1 mRNA and protein in cells (Figure and In these we in the of the inhibitors in HIF-1α and in APOL1 For example, and led to of APOL1 mRNA also in course experiments in these cells, HIF-1α protein and increased APOL1 protein which with an on the effects of HIF stabilizers (Supplementary Figure et and of HIF prolyl hydroxylase inhibitors in 2017; PubMed Google Scholar the of to and 10 in experiments in human urinary and podocytes, effects on HIF stabilization and APOL1 protein were to the effects of higher of (Supplementary and The in HIF-1α protein levels the increases in APOL1 mRNA may to additional transcriptional or of APOL1 regulation caused by In this respect, we observed higher levels of APOL1 mRNA when cells with the inhibitor DMOG, which prolyl hydroxylase proteins and an HIF hydroxylase that HIF transcriptional activity by at of In line with a role of in regulating APOL1 expression, a combination of the inhibitor and the inhibitor led to increased APOL1 mRNA in when compared with (Supplementary Figure Thus, APOL1 to an HIF-1α target gene that is to To gain insights on whether increased levels of APOL1 risk variants and stabilization of HIF have an impact on cell we to an available HEK293T cell model in which APOL1 variants lacking the signal peptide can by a doxycycline-inducible D. Muller D. Krausel V. et al.Intracellular APOL1 risk variants cause cytotoxicity accompanied by energy depletion.J Am Soc Nephrol. 2017; 28: 3227-3238Crossref PubMed Scopus (64) Google Scholar In these cells, APOL1 expression is by HIF, which for an of effects of HIF stabilization and APOL1 risk variant (Supplementary Figure led to protein levels of G0, G1, and G2 variants in the cells (Supplementary Figure in a expression of G1 and G2 variants reduced cell (Figure D. Muller D. Krausel V. et al.Intracellular APOL1 risk variants cause cytotoxicity accompanied by energy depletion.J Am Soc Nephrol. 2017; 28: 3227-3238Crossref PubMed Scopus (64) Google Scholar Strikingly, exposure of the cells to the HIF stabilizers and further cell (Figure The effect of the HIF stabilizers was most prominent in cells and observed in control cells (Figure In to the regulatory role of HIF in APOL1 expression, this that of both stimuli (e.g., in acute kidney injury of patients with a high inflammatory can have effects on renal cell our data that hypoxia and HIF stabilization expression of APOL1 and pathogenic variants in renal cells and that the presence of HIF together with APOL1 risk variants effects on cell survival (Figure with the involvement of HIF in many kidney diseases, our are with a model in which hypoxic insults (e.g., observed in acute kidney of kidney disease by augmenting expression of APOL1 risk variants. In this respect, a among patients with African ancestry in the revealed that development and of disease acute kidney injury with the presence of 2 APOL1 risk A.M. et al.APOL1 risk acute kidney injury, and in with African ancestry with from the Med. 2022; PubMed Scopus Google Scholar the primary insults in this disease have been viral infection and inflammation, is to that a hypoxic in the injured kidneys expression of the pathogenic variants and of kidney disease. chronic hypoxia may influence expression of APOL1 variants. In this respect, is that in patients with cell a of chronic hypoxia, APOL1-associated is R. C. et of APOL1 G1 and G2 variants in cell disease kidney is the J 2017; PubMed Scopus Google Scholar data indicating reduced cell on HIF stabilization and expression of APOL1 risk variants to additive deleterious effects of both signaling of mitochondrial D. Muller D. Krausel V. et al.Intracellular APOL1 risk variants cause cytotoxicity accompanied by energy depletion.J Am Soc Nephrol. 2017; 28: 3227-3238Crossref PubMed Scopus (64) Google Scholar Thus, further of the of HIF and APOL1 in renal cells is In the 733 data the renal cluster is the most prominent accessible chromatin at the APOL1 W. Muratov A. Rynes E. et al.Index and biological spectrum of human DNase I hypersensitive sites.Nature. 2020; 584: 244-251Crossref PubMed Scopus (115) Google Scholar However, when we other cell in the for chromatin activity at this we noted that cells, a cell also evidence for an active enhancer at this We used available HIF ChIP-sequencing and transcriptomic data and confirmed that APOL1 is also by HIF in cells (Supplementary Figure J.A. M. et of the and transcription factors in 2019; PubMed Scopus Google Scholar The substantially to levels of APOL1, to additional of the HIF pathway in regulating APOL1 effects of both on glomerular disease have been described in For example, HIF-2α a progressive glomerulonephritis of the HIF target gene M. Cui S. Li C. et al.Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice.Nat Med. 2006; 12: 1081-1087Crossref PubMed Scopus (177) Google Scholar of HIF-1α the development of in the podocyte B. T. et and regulates expression interactions with Int. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar express APOL1, this indicates that HIF a broader response of However, of APOL1 risk variants causes in mice, the effect of these variants on kidney P. J. Park et expression of human APOL1 risk variants in podocytes kidney disease in mice.Nat Med. 2017; PubMed Scopus Google Scholar the role of HIF in human podocytes is is at levels and is of regulating APOL1 expression in vivo in B. et expression of hypoxia-inducible and glomerular Am Soc Nephrol. 14: PubMed Scopus Google Scholar We also expression of HIF-2α in isolated primary tubular cells. HIF-2α been detected at levels in tubular cells of the kidney in and humans in C. S. et of hypoxia-inducible and in hypoxic and Am Soc Nephrol. PubMed Scopus Google T. M. et tubular expression and causes and 2012; PubMed Scopus Google Scholar The for this is by different of the used to HIF-2α or in the expression of the isolated cells. However, transcriptional regulation of APOL1 to on HIF-1α in tubular cells. The in to hypoxia, a of HIF stabilizers regulate APOL1 expression in renal cells may of for APOL1 nephropathy patients with kidney our that the treatment of patients with HIF stabilizers to levels may also APOL1 expression and kidney However, in our of APOL1 transcription is on the additional of which is in human kidneys and to APOL1 expression in J. D. B. et HIF the expression of hypoxia-inducible in podocytes and tubular Int. Full Text Full Text PDF PubMed Scopus Google Scholar A of our is that experiments risk variant primary cells were in cells from only The data from signal so far that indicates that to is reduced in patients on HIF However, in these the of patients carrying APOL1 risk alleles to a significant signal (e.g., of patients with African ancestry in the in Chronic Kidney a et for the treatment of anemia in patients J Med. 2021; PubMed Scopus Google Scholar We following on glomerular in patients carrying APOL1 risk variants are on treatment with HIF the that other stimuli, such as may expression of APOL1 or and cell survival further the of these in patients at from and from the other We and for This was by the for and at the and the to and to is by the to was by the was by the and the On Kidney with Supplementary Supplementary Methods. Supplementary Table used in this Supplementary Figure of RNA-sequencing data from podocytes after dimethyloxalylglycine (DMOG) Supplementary Figure APOL1 RNA and protein expression in primary tubular cells (PTCs) on hypoxic Supplementary Figure RNA and protein expression of APOL1 in primary tubular cells (PTCs) after hypoxia-inducible (HIF) Supplementary Figure APOL1 RNA expression in experiments from kidneys of controls and patients with acute kidney injury Supplementary Figure RNA and protein expression of APOL1 in primary tubular cells (PTCs) after hypoxia-inducible (HIF) stabilization and interferon Supplementary Figure course experiments of APOL1 expression using different inhibitors in primary tubular cells Supplementary Figure APOL1 expression in experiments using different inhibitors in primary tubular cells Supplementary Figure course experiments of APOL1 expression using different inhibitors in Supplementary Figure APOL1 RNA expression in primary tubular cells (PTCs) after hypoxia-inducible (HIF) stabilization and Supplementary Figure of APOL1 expression in APOL1 HEK293T cells. Supplementary Figure APOL1 expression and hypoxia-inducible (HIF) chromatin immunoprecipitation at the APOL1 in cells.

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