KidneyGPS: a user-friendly web application to help prioritize kidney function genes and variants based on evidence from genome-wide association studies.

BACKGROUND: Genome-wide association studies (GWAS) have identified hundreds of genetic loci associated with kidney function. By combining these findings with post-GWAS information (e.g., statistical fine-mapping to identify independent association signals and to narrow down signals to causal variants; or different sources of annotation data), new hypotheses regarding physiology and disease aetiology can be obtained. These hypotheses need to be tested in laboratory experiments, for example, to identify new therapeutic targets. For this purpose, the evidence obtained from GWAS and post-GWAS analyses must be processed and presented in a way that they are easily accessible to kidney researchers without specific GWAS expertise. MAIN: Here we present KidneyGPS, a user-friendly web-application that combines genetic variant association for estimated glomerular filtration rate (eGFR) from the Chronic Kidney Disease Genetics consortium with annotation of (i) genetic variants with functional or regulatory effects ("SNP-to-gene" mapping), (ii) genes with kidney phenotypes in mice or human ("gene-to-phenotype"), and (iii) drugability of genes (to support re-purposing). KidneyGPS adopts a comprehensive approach summarizing evidence for all 5906 genes in the 424 GWAS loci for eGFR identified previously and the 35,885 variants in the 99% credible sets of 594 independent signals. KidneyGPS enables user-friendly access to the abundance of information by search functions for genes, variants, and regions. KidneyGPS also provides a function ("GPS tab") to generate lists of genes with specific characteristics thus enabling customizable Gene Prioritisation (GPS). These specific characteristics can be as broad as any gene in the 424 loci with a known kidney phenotype in mice or human; or they can be highly focussed on genes mapping to genetic variants or signals with particularly with high statistical support. KidneyGPS is implemented with RShiny in a modularized fashion to facilitate update of input data ( https://kidneygps.ur.de/gps/ ). CONCLUSION: With the focus on kidney function related evidence, KidneyGPS fills a gap between large general platforms for accessing GWAS and post-GWAS results and the specific needs of the kidney research community. This makes KidneyGPS an important platform for kidney researchers to help translate in silico research results into in vitro or in vivo research.

Hypoxia and renal fibrosis.

Renal fibrosis is the final stage of most progressive kidney diseases. Chronic kidney disease (CKD) is associated with high comorbidity and mortality. Thus, preventing fibrosis and thereby preserving kidney function increases the quality of life and prolongs the survival of patients with CKD. Many processes such as inflammation or metabolic stress modulate the progression of kidney fibrosis. Hypoxia has also been implicated in the pathogenesis of renal fibrosis, and oxygen sensing in the kidney is of outstanding importance for the body. The dysregulation of oxygen sensing in the diseased kidney is best exemplified by the loss of stimulation of erythropoietin production from interstitial cells in the fibrotic kidney despite anemia. Furthermore, hypoxia is present in acute or chronic kidney diseases and may affect all cell types present in the kidney including tubular and glomerular cells as well as resident immune cells. Pro- and antifibrotic effects of the transcription factors hypoxia-inducible factors 1 and 2 have been described in a plethora of animal models of acute and chronic kidney diseases, but recent advances in sequencing technologies now allow for novel and deeper insights into the role of hypoxia and its cell type-specific effects on the progression of renal fibrosis, especially in humans. Here, we review existing literature on how hypoxia impacts the development and progression of renal fibrosis.

The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression.

Evolution of clear cell renal cell carcinoma is guided by dysregulation of hypoxia-inducible transcription factor (HIF) pathways following loss of the von Hippel-Lindau tumor suppressor protein. Renal cell carcinoma (RCC)-associated polymorphisms influence HIF-DNA interactions at enhancers of important oncogenes thereby modulating the risk of developing renal cancer. A strong signal of genome-wide association with RCC was determined for the single nucleotide polymorphism (SNP) rs4903064, located on chr14q.24.2 within an intron of DPF3, encoding for Double PHD Fingers 3, a member of chromatin remodeling complexes; however, it is unclear how the risk allele operates in renal cells. In this study, we used tissue specimens and primary renal cells from a large cohort of RCC patients to examine the function of this polymorphism. In clear cell renal cell carcinoma tissue, isolated tumor cells as well as in primary renal tubular cells, in which HIF was stabilized, we determined genotype-specific increases of DPF3 mRNA levels and identified that the risk SNP resides in an active enhancer region, creating a novel HIF-binding motif. We then confirmed allele-specific HIF binding to this locus using chromatin immunoprecipitation of HIF subunits. Consequentially, HIF-mediated DPF3 regulation was dependent on the presence of the risk allele. Finally, we show that DPF3 deletion in proximal tubular cells retarded cell growth, indicating potential roles for DPF3 in cell proliferation. Our analyses suggest that the HIF pathway differentially operates on a SNP-induced hypoxia-response element at 14q24.2, thereby affecting DPF3 expression, which increases the risk of developing renal cancer.

Hypoxia hits APOL1 in the kidney.

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.

Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis.

Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans. The chimeric protein comprises a calpain domain and a unique circularly permutated globin domain. ADGB expression levels are most abundant in mammalian testis, but its cell-type-specific expression, regulation, and function have remained unexplored. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that-in addition to the testes-ADGB is prominently expressed in the female reproductive tract, lungs, and brain, specifically being associated with cell types forming motile cilia. Correlation analysis suggested coregulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. Investigating the transcriptional regulation of the ADGB gene, we characterized its promoter using epigenomic datasets, exogenous promoter-dependent luciferase assays, and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays revealed that FOXJ1 indeed substantially enhanced luciferase activity driven by the ADGB promoter. ChIP assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. FOXJ1 overexpression significantly increased endogenous ADGB mRNA levels in HEK293 and MCF-7 cells. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. The complex transcriptional regulation of the ADGB locus was illustrated by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicated an ADGB-dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB in mammals.

Optimal translational termination requires C4 lysyl hydroxylation of eRF1.

Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation.

Hypoxia drives glucose transporter 3 expression through hypoxia-inducible transcription factor (HIF)-mediated induction of the long noncoding RNA NICI.

Hypoxia-inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long noncoding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long noncoding RNA Noncoding Intergenic Co-Induced transcript (NICI) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knockdown or knockout of NICI attenuated hypoxic induction of SLC2A3, indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR-mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than posttranscriptional mechanisms because knockout of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the von Hippel-Lindau (VHL) tumor suppressor and is highly expressed in clear cell renal cell carcinoma (ccRCC), where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy.

Molecular diagnosis of kidney transplant failure based on urine.

In light of the organ shortage, there is a great responsibility to assess postmortal organs for which procurement has been consented and to increase the life span of transplanted organs. The former responsibility has moved many centers to accept extended criteria organs. The latter responsibility requires an exact diagnosis and, if possible, omission of the harmful influence on the transplant. We report the course of a kidney transplant that showed a steady decline of function over a decade, displaying numerous cysts of different sizes. Clinical workup excluded the most frequent causes of chronic transplant failure. The filed allocation documents mentioned the donor's disease of oral-facial-digital syndrome, a rare ciliopathy, which can also affect the kidney. Molecular diagnosis was performed by culturing donor tubular cells from the recipient´s urine more than 10 years after transplantation. Next-generation panel sequencing with DNA from tubular urinary cells revealed a novel truncating mutation in OFD1, which sufficiently explains the features of the kidney transplants, also found in the second kidney allograft. Despite this severe donor disease, lifesaving transplantation with good long-term outcome was enabled for 5 recipients.

Distal and proximal hypoxia response elements cooperate to regulate organ-specific erythropoietin gene expression.

While it is well-established that distal hypoxia response elements (HREs) regulate hypoxia-inducible factor (HIF) target genes such as erythropoietin (Epo), an interplay between multiple distal and proximal (promoter) HREs has not been described so far. Hepatic Epo expression is regulated by a HRE located downstream of the EPO gene, but this 3' HRE is dispensable for renal EPO gene expression. We previously identified a 5' HRE and could show that both HREs direct exogenous reporter gene expression. Here, we show that whereas in hepatic cells the 3' but not the 5' HRE is required, in neuronal cells both the 5' and 3' HREs contribute to endogenous Epo induction. Moreover, two novel putative HREs were identified in the EPO promoter. In hepatoma cells HIF interacted mainly with the distal 3' HRE, but in neuronal cells HIF most strongly bound the promoter, to a lesser extent the 3' HRE, and not at all the 5' HRE. Interestingly, mutation of either of the two distal HREs abrogated HIF binding to the 3' and promoter HREs. These results suggest that a canonical functional HRE can recruit multiple, not necessarily HIF, transcription factors to mediate HIF binding to different distant HREs in an organ-specific manner.

Multiple renal cancer susceptibility polymorphisms modulate the HIF pathway.

Un-physiological activation of hypoxia inducible factor (HIF) is an early event in most renal cell cancers (RCC) following inactivation of the von Hippel-Lindau tumor suppressor. Despite intense study, how this impinges on cancer development is incompletely understood. To test for the impact of genetic signals on this pathway, we aligned human RCC-susceptibility polymorphisms with genome-wide assays of HIF-binding and observed highly significant overlap. Allele-specific assays of HIF binding, chromatin conformation and gene expression together with eQTL analyses in human tumors were applied to mechanistic analysis of one such overlapping site at chromosome 12p12.1. This defined a novel stage-specific mechanism in which the risk polymorphism, rs12814794, directly creates a new HIF-binding site that mediates HIF-1α isoform specific upregulation of its target BHLHE41. The alignment of multiple sites in the HIF cis-acting apparatus with RCC-susceptibility polymorphisms strongly supports a causal model in which minor variation in this pathway exerts significant effects on RCC development.

Now a Nobel gas: oxygen.

The recent bestowal of the Nobel Prize 2019 in Physiology or Medicine to Gregg L. Semenza, Sir Peter J. Ratcliffe, and William G. Kaelin Jr. celebrates a series of remarkable discoveries that span from the physiological research question on how oxygen deficiency (hypoxia) induces the red blood cell forming hormone erythropoietin (Epo) to the first clinical application of a novel family of Epo-inducing drugs to treat patients suffering from renal anemia. This review looks back at the most important findings made by the three Nobel laureates, highlights current research trends, and sheds an eye on future perspectives of hypoxia research, including emerging and potential clinical applications.

Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases.

The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/ß-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping.

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/ß DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the -82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the -82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the -82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia.

Extensive regulation of the non-coding transcriptome by hypoxia: role of HIF in releasing paused RNApol2.

Hypoxia is central to both ischaemic and neoplastic diseases. However, the non-coding transcriptional response to hypoxia is largely uncharacterized. We undertook integrated genomic analyses of both non-coding and coding transcripts using massively parallel sequencing and interfaced this data with pan-genomic analyses of hypoxia-inducible factor (HIF) and RNApol2 binding in hypoxic cells. These analyses revealed that all classes of RNA are profoundly regulated by hypoxia and implicated HIF as a major direct regulator of both the non-coding and coding transcriptome, acting predominantly through release of pre-bound promoter-paused RNApol2. These findings indicate that the transcriptional response to hypoxia is substantially more extensive than previously considered.

P2Y2R is a direct target of HIF-1α and mediates secretion-dependent cyst growth of renal cyst-forming epithelial cells.

Polycystic kidney diseases are characterized by numerous renal cysts that continuously enlarge resulting in compression of intact nephrons and tissue hypoxia. Recently, we have shown that hypoxia-inducible factor (HIF)-1α promotes secretion-dependent cyst expansion, presumably by transcriptional regulation of proteins that are involved in calcium-activated chloride secretion. Here, we report that HIF-1α directly activates expression of the purinergic receptor P2Y2R in human primary renal tubular cells. In addition, we found that P2Y2R is highly expressed in cyst-lining cells of human ADPKD kidneys as well as PKD1 orthologous mouse kidneys. Knockdown of P2Y2R in renal collecting duct cells inhibited calcium-dependent chloride secretion in Ussing chamber analyses. In line with these findings, knockdown of P2Y2R retarded cyst expansion in vitro and prevented ATP- and HIF-1α-dependent cyst growth. In conclusion, P2Y2R mediates ATP-dependent cyst growth and is transcriptionally regulated by HIF-1α. These findings provide further mechanistic evidence on how hypoxia promotes cyst growth.

Pan-genomic binding of hypoxia-inducible transcription factors.

Hypoxia-inducible transcription factors (HIFs) mediate the cellular response to hypoxia. HIF-DNA binding triggers a transcriptional program that acts to both restore oxygen homeostasis and adapt cells to low oxygen availability. In this context, HIF is centrally involved in many physiologic and pathophysiological processes such as development, high altitude adaptation, ischemic disease, inflammation, and cancer. The recent development of chromatin immunoprecipitation coupled to genome-wide DNA sequence analysis allows the position and extent of HIF binding to DNA to be characterized across the entire genome and correlated with genetic, epigenetic, and transcriptional analyses. This review summarizes recent pan-genomic analyses of HIF binding and HIF-dependent transcriptional regulation.

Selective stabilization of HIF-1α in renal tubular cells by 2-oxoglutarate analogues.

The role of proximal versus distal tubular injury in the pathogenesis of acute kidney injury (AKI) is debatable. Inhibition of prolyl hydroxylases that regulate the degradation of hypoxia-inducible transcription factors (HIFs) is a promising therapeutic approach to optimize energy preservation under hypoxia and has successfully been applied to protect kidney structure and function in AKI models. Presently used prolyl hydroxylase inhibitors are lipophilic 2-oxoglutarate analogues (2OGAs) that are widely taken up in cells of most organs. Given the selective expression of organic anion transporters (OATs) in renal proximal tubular cells, we hypothesized that hydrophilic 2OGAs can specifically target proximal tubular cells. We found that cellular hydrophilic 2OGAs uptake depended on OATs and largely confined to the kidney, where it resulted in activation of HIF target genes only in proximal tubular cells. When applied in ischemia-reperfusion experiments, systemically active 2OGA preserved kidney structure and function, but OAT1-transported 2OGA was not protective, suggesting that HIF stabilization in distal tubular rather than proximal tubular cells and/or nontubular cells mediates protective effects. This study provides proof of concept for selective drug targeting of proximal tubular cells on the basis of specific transporters, gives insights into the role of different nephron segments in AKI pathophysiology, and may offer options for long-term HIF stabilization in proximal tubules without confounding effects of erythropoietin induction in peritubular cells and unwarranted extrarenal effects.

High-resolution genome-wide mapping of HIF-binding sites by ChIP-seq.

Hypoxia-inducible factor (HIF) regulates the major transcriptional cascade central to the response of all mammalian cells to alterations in oxygen tension. Expression arrays indicate that many hundreds of genes are regulated by this pathway, controlling diverse processes that in turn orchestrate both oxygen delivery and utilization. However, the extent to which HIF exerts direct versus indirect control over gene expression together with the factors dictating the range of HIF-regulated genes remains unclear. Using chromatin immunoprecipitation linked to high throughput sequencing, we identify HIF-binding sites across the genome, independently of gene architecture. Using gene set enrichment analysis, we demonstrate robust associations with the regulation of gene expression by HIF, indicating that these sites operate over long genomic intervals. Analysis of HIF-binding motifs demonstrates sequence preferences outside of the core RCGTG-binding motif but does not reveal any additional absolute sequence requirements. Across the entire genome, only a small proportion of these potential binding sites are bound by HIF, although occupancy of potential sites was enhanced approximately 20-fold at normoxic DNAse1 hypersensitivity sites (irrespective of distance from promoters), suggesting that epigenetic regulation of chromatin may have an important role in defining the response to hypoxia.

CRISPR Activator Approaches to Study Endogenous Androglobin Gene Regulation.

Androglobin (ADGB), the most recently identified member of the mammalian globin family, is a chimeric protein with an unusual, embedded globin domain that is circularly permutated and exhibits hallmarks of a hexacoordinated heme-binding scheme. Whereas abundant expression of ADGB was initially found to be mainly restricted to cells in the postmeiotic stages of spermatogenesis, more recent RNA-Seq-based expression analysis data revealed that ADGB is detectable in cells carrying motile cilia or flagella. This very tight regulation of ADGB gene expression urges the need for alternative techniques to study endogenous expression in classical mammalian cell models, which do not express ADGB. We describe here the use of CRISPR activation (CRISPRa) technology to induce endogenous ADGB gene expression in HEK293T, MCF-7, and HeLa cells from its promoter and illustrate how this method can be employed to validate putative regulatory DNA elements of ADGB in promoter and enhancer regions.