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Presents the abstract "Functional Epigenomics Identifies Hepatocyte Growth Factor Activator Inhibitor Type 2/SPINT2 as a Frequently Inactivated Tumour Suppressor in Papillary and Clear Cell Renal Cell Carcinoma," by Mark Morris, D. Gentle, M. Abdulrahman, E. N. Maina, K. Gupta, R. E. Banks, M. S. Wiesener, T. Kishida, Y. Masahiro, B. Teh, F. Latif, and E. R. Maher.

Objective: Hypoxia-inducible transcription factor (HIF) mediates cellular and systemic adaptations to an insufficient oxygen supply, stimulating the transcription of, e.g., glycolytic enzymes, angiogenic factors and erythropoietin (EPO). HIF is a heterodimer which consists of an oxygen-regulated β subunit and a constitutively expressed α subunit. The two alternative HIFα subunits, HIF-1α and HIF-2α, are differentially expressed in vivo. Overexpression experiments in vitro suggested a partial functional redundancy. Methods: Here, we used RNA interference in various human cell lines to determine the relative contribution of HIF-1α versus HIF-2α to the hypoxic gene induction. Results: We found that in HeLa and Hep3B cells the hypoxic induction of most HIF target genes was responsive to HIF-1α knockdown, but not to HIF-2α knock-down. The same applied for the activation of reporter genes driven by hypoxia responsive elements (HREs). Surprisingly, EPO was the only gene investigated which showed responsiveness only to HIF-2α knock-down, as observed in Hep3B and neuroblastoma Kelly cells. In contrast to the EPO-HRE reporter, the complete EPO enhancer displayed dependency on HIF-2α regulation, indicating that cis-acting elements other than the HRE contribute to the HIF-2α effect. We also found that overexpression of the HIFα subunits led to a loss of target gene specificity. Conclusions: In conclusion, EPO is the only HIF-2α target gene identified so far. The fact that renal peritubular fibroblasts, the major source of EPO in the adult, express HIF-2α but not HIF-1α protein, supports our finding. The hypoxic induction of the EPO gene seems to require the interaction of HIF-2α with other DNA-binding factors. [ABSTRACT FROM AUTHOR]

Objective: Erythropoietin (EPO), the hypoxia-inducible stimulator of erythropoiesis, elicits its function through binding to a specific receptor (EPO-R). The expression of EPO-R is not restricted to erythoid progenitor cells but has also been shown in non hematopoetic tissues such as the vasculature, brain, heart, kidney and several cancer cells. In addition, EPO expression has been found in various tissues apart from liver and kidneys. Different experimental data suggest that binding of EPO to its receptor outside the bone marrow may have protective effects under conditions of tissue hypoxia, possibly through inhibition of apoptosis. However, little is known about the regulation of these paracrine systems. In view of the evidence that EPO-R activation occurs under hypoxic conditions, we tested whether the expression of the EPO-R itself is also regulated by hypoxia. Methods: Using RNase Protection Assay we studied the expression and regulation of EPO-R mRNA in different cell lines known to express EPO-R. Functional data were obtained in reporter gene assays using luciferase constructs of the EPO-R promoter which were transiently transfected into different cell lines and analysed for oxygendependent regulation. Furthermore we performed IHC to study EPO-R protein expression. Results: Exposure of the human renal cell carcinoma cell lines CAKI and RCC4 as well as the rodent neuroblastoma cell line PC12W to hypoxia and the iron chelator dipyridyl, which mimicks hypoxia, for 16 hours resulted in a 2-3 fold increase in EPO-R mRNA expression. Interestingly, this was not seen in a number of other cell lines, where expression of EPO-R was abundant at baseline but not inducible by hypoxia. Inhibition of transcription by actinomycin D showed that increased expression of EPO-R mRNA occurs at the transcriptional level and not through increased mRNA stability during hypoxia. Transiently transfected cells with luciferase reporter genes containing fragments of the human and mouse EPO-R promoter confirmed that EPO-R gene transcription is stimulated by hypoxia and iron chelation. IHC on tissue culture cell lines showed hypoxia-inducible expression also at the protein levels. Conclusions: Our data show that EPO-R is not only induced by hypoxia but also by related stimuli, known to induce the hypoxiainducible transcription factor HIF. This suggests that HIF might be involved in the regulation of the EPO-R gene. In association with the well studied oxygen dependent regulation and lately described protective effects of EPO outside the bone marrow hypoxic upregulation of its receptor would be a sensible and relevant additive mechanism to maintain tolerance against hypoxia and prevent cell injury. [ABSTRACT FROM AUTHOR]

The VHL TSG regulates proteosomal destruction of the a subunits of HIF-1 and HIF-2 such that VHL inactivation results in overexpression of HIF hypoxia-inducible target genes such as carbonic anhydrase 9 (CA9) and VEGF. However it is not clear whether HIF-1/HIF-2 is oncogenic Recently we showed that pVHL regulates Cyclin D1 (CCND1) expression. CCND1 is overexpressed in many RCC, but it is unclear whether pVHL regulates CCND1 via a HIF-dependent mechanism. Sporadic and familial RCC tumours were analysed to investigate the relationship between CCND1, HIF-la/CA9 expression and VHL inactivation. Overexpression of both CCND1 and HIF-la was common in clear cell (CC) RCC (23/30) but not in non-clear cell RCC (0/5, P=0.0024). All CCRCC with VHL mutations demonstrated overexpression of HIF-la, and in most cases, CCND1. However, HIF-1a and CCND1 overexpression also occurred in CCRCC without VHL inactivation. To further investigate mechanisms of CCND1 dysregulation in RCC immunostaining of CA9 and CCND1 in VHL and non-VHL familial RCC was done. Overexpression of CA9 and CCND1 was detected in all VHL RCC (n=12) but in only a small subset of non-VHL RCC. Thus in RCC with VHL inactivation upregulation of HIF-1/CA9 and CCND1 are closely associated and there was little evidence that specific VHL mutations might cause CCND1 and not HIF-1 dysregulation. Immunostaining for CA9 and CCND1 may provide a sensitive method for identifying RCC with VHL inactivation and be useful for prognostic studies. [ABSTRACT FROM AUTHOR]

Background: The mammalian target of rapamycin inhibitor (mTORi) therapy after kidney transplantation is solely monitored pharmacokinetically, not necessarily reflecting PI3K-Akt-mTOR pathway blockade efficacy leading to potential under-or overimmunosuppression., Methods: In this cross-sectional study, phosphoflow cytometry was used to determine the efficacy of mTOR inhibition in peripheral T- and B-lymphocyte subsets by assessing p70S6 kinase (p70S6K) phosphorylation in renal transplant recipients upon treatment with a combination of either mTORi and calcineurin inhibitors (n = 18), or mTORi with mycophenolic acid (n = 9). Nine dialysis patients with end-stage renal disease and 17 healthy age-matched volunteers served as controls., Results: mTORi treatment reduced p70S6K phosphorylation in CD4 + , CD8 + T, and CD19 + B cells compared with healthy controls (HCs). Subpopulation analysis of CD4 + T cells and CD19 + B cells revealed a significant reduction of p70S6K phosphorylation in CD4 + CD45RA - CD25 - Th cells ( P  < 0.05), CD24 hi CD38 hi transitional B cells ( P  < 0.001), CD24 + CD38 - memory B cells ( P  < 0.001), and CD24 int CD38 int -naive B cells ( P  < 0.05) upon mTORi treatment, whereas CD4 + CD45RA - CD25 ++ CD127 - regulatory T cells and CD24 - CD38 hi plasmablasts were not affected. Compared with mTORi + mycophenolic acid therapy, mTORi + calcineurin inhibitor treatment exhibited an even stronger inhibition of p70S6K phosphorylation in CD4 + CD45RA - CD25 - Th cells and CD8 + T cells. However, trough levels of mTORi did not correlate with p70S6K phosphorylation., Conclusions: mTORi selectively inhibited p70S6K phosphorylation in select lymphocyte subtypes. Assessing p70S6K phosphorylation by phosphoflow cytometry may serve as an approach to understand cell subset specific effects of mTORi providing detailed pharmacodynamic information for individualizing immunosuppression., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Background: We previously reported excellent efficacy and improved safety aspects of rapid steroid withdrawal (RSWD) in the randomized controlled 1-year "Harmony" trial with 587 predominantly deceased-donor kidney transplant recipients randomized either to basiliximab or rabbit antithymocyte globulin induction therapy and compared with standard immunosuppressive therapy consisting of basiliximab, low tacrolimus once daily, mycophenolate mofetil and corticosteroids., Methods: The 5-year post-trial follow-up (FU) data were obtained in an observational manner at a 3- and a 5-year visit only for those Harmony patients who consented to participate and covered clinical events that occurred from the second year onwards., Results: Biopsy-proven acute rejection and death-censored graft loss rates remained low and independent of RSWD. Rapid steroid withdrawal was an independent positive factor for patient survival (adjusted hazard ratio 0.554, 95% confidence interval 0.314-0.976; P = .041).The reduced incidence of post-transplantation diabetes mellitus in RSWD patients during the original 1-year study period was not compensated by later incidences during FU. Incidences of other important outcome parameters such as opportunistic infections, malignancies, cardiovascular morbidity/risk factors, donor-specific antibody formation or kidney function did not differ during FU period., Conclusions: With all the limitations of a post-trial FU study, the Harmony FU data confirm excellent efficacy and beneficial safety aspects of RSWD under modern immunosuppressive therapy over the course of 5 years after kidney transplantation in an immunologically low-risk, elderly population of Caucasian kidney transplant recipients. Trial registration: Clinical trial registration number: Investigator Initiated Trial (NCT00724022, FU study DRKS00005786)., (© The Author(s) 2023. Published by Oxford University Press on behalf of the ERA.)

The interplay between genetic and environmental factors influences the course of chronic kidney disease (CKD). In this context, genetic alterations in the kidney disease gene MUC1 (Mucin1) predispose to the development of CKD. These variations comprise the polymorphism rs4072037, which alters splicing of MUC1 mRNA, the length of a region with variable number of tandem repeats (VNTR), and rare autosomal-dominant inherited dominant-negative mutations in or 5' to the VNTR that causes autosomal dominant tubulointerstitial kidney disease (ADTKD- MUC1 ). As hypoxia plays a pivotal role in states of acute and chronic kidney injury, we explored the effects of hypoxia-inducible transcription factors (HIF) on the expression of MUC1 and its pathogenic variants in isolated primary human renal tubular cells. We defined a HIF-binding DNA regulatory element in the promoter-proximal region of MUC1 from which hypoxia or treatment with HIF stabilizers, which were recently approved for an anti-anemic therapy in CKD patients, increased levels of wild-type MUC1 and the disease-associated variants. Thus, application of these compounds might exert unfavorable effects in patients carrying MUC1 risk variants., (© 2023 Naas et al.)

Hereditary chronic kidney disease (CKD) appears to be more frequent than the clinical perception. Exome sequencing (ES) studies in CKD cohorts could identify pathogenic variants in ~10% of individuals. Tubulointerstitial kidney diseases, showing no typical clinical/histologic finding but tubulointerstitial fibrosis, are particularly difficult to diagnose. We used a targeted panel (29 genes) and MUC1-SNaPshot to sequence 271 DNAs, selected in defined disease entities and age cutoffs from 5217 individuals in the German Chronic Kidney Disease cohort. We identified 33 pathogenic variants. Of these 27 (81.8%) were in COL4A3/4/5, the largest group being 15 COL4A5 variants with nine unrelated individuals carrying c.1871G>A, p.(Gly624Asp). We found three cysteine variants in UMOD, a novel missense and a novel splice variant in HNF1B and the homoplastic MTTF variant m.616T>C. Copy-number analysis identified a heterozygous COL4A5 deletion, and a HNF1B duplication/deletion, respectively. Overall, pathogenic variants were present in 12.5% (34/271) and variants of unknown significance in 9.6% (26/271) of selected individuals. Bioinformatic predictions paired with gold standard diagnostics for MUC1 (SNaPshot) could not identify the typical cytosine duplication ("c.428dupC") in any individual, implying that ADTKD-MUC1 is rare. Our study shows that >10% of selected individuals carry disease-causing variants in genes partly associated with tubulointerstitial kidney diseases. COL4A3/4/5 genes constitute the largest fraction, implying they are regularly overlooked using clinical Alport syndrome criteria and displaying the existence of phenocopies. We identified variants easily missed by some ES pipelines. The clinical filtering criteria applied enriched for an underlying genetic disorder., (© 2022. The Author(s).)

The kidney-specific gene UMOD encodes for uromodulin, the most abundant protein excreted in normal urine. Rare large-effect variants in UMOD cause autosomal dominant tubulointerstitial kidney disease (ADTKD), while common low-impact variants strongly associate with kidney function and the risk of chronic kidney disease (CKD) in the general population. It is unknown whether intermediate-effect variants in UMOD contribute to CKD. Here, candidate intermediate-effect UMOD variants were identified using large-population and ADTKD cohorts. Biological and phenotypical effects were investigated using cell models, in silico simulations, patient samples, and international databases and biobanks. Eight UMOD missense variants reported in ADTKD are present in the Genome Aggregation Database (gnomAD), with minor allele frequency (MAF) ranging from 10 -5 to 10 -3 . Among them, the missense variant p.Thr62Pro is detected in ∼1/1,000 individuals of European ancestry, shows incomplete penetrance but a high genetic load in familial clusters of CKD, and is associated with kidney failure in the 100,000 Genomes Project (odds ratio [OR] = 3.99 [1.84 to 8.98]) and the UK Biobank (OR = 4.12 [1.32 to 12.85). Compared with canonical ADTKD mutations, the p.Thr62Pro carriers displayed reduced disease severity, with slower progression of CKD and an intermediate reduction of urinary uromodulin levels, in line with an intermediate trafficking defect in vitro and modest induction of endoplasmic reticulum (ER) stress. Identification of an intermediate-effect UMOD variant completes the spectrum of UMOD -associated kidney diseases and provides insights into the mechanisms of ADTKD and the genetic architecture of CKD.

The human genome contains more than one million tandem repeats (TRs), DNA sequences containing multiple approximate copies of a motif repeated contiguously. TRs account for significant genetic variation, with 50 + diseases attributed to changes in motif number. A few diseases have been to be caused by small indels in variable number tandem repeats (VNTRs) including poly-cystic kidney disease type 1 (MCKD1) and monogenic type 1 diabetes. However, small indels in VNTRs are largely unexplored mainly due to the long and complex structure of VNTRs with multiple motifs. We developed a method, code-adVNTR, that utilizes multi-motif hidden Markov models to detect both, motif count variation and small indels, within VNTRs. In simulated data, code-adVNTR outperformed GATK-HaplotypeCaller in calling small indels within large VNTRs. We used code-adVNTR to characterize coding VNTRs in the 1000 genomes data identifying many population-specific variants, and to reliably call MUC1 mutations for MCKD1., Competing Interests: V.B. is a co-founder, serves on the scientific advisory board, and has equity interest in Boundless Bio, inc. (BB) and Abterra (Ab), and receives income from DP and Ab. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies., (© 2022 The Author(s).)

Timely recognition and treatment of acute kidney graft rejection is important to prevent premature graft failure. A predefined urinary marker set for acute T cell-mediated rejection (TCMR) containing 14 peptides was tested for this purpose in a multicenter in-place validation study., Methods: Three hundred twenty-nine prospectively collected and 306 archived urine samples from 11 transplant centers in Germany, France, and Belgium were examined. Samples were taken immediately before a biopsy, performed for graft dysfunction within the first transplant year. Primary outcomes were sensitivity and specificity of the marker set for the diagnosis of biopsy-proven acute TCMR, with prespecified thresholds of 83% for sensitivity and 70% for specificity., Results: Eighty-two patients (13%) had acute TCMR grade I-III. In relation to the biopsy diagnosis of TCMR, the sensitivity of the urine test was 0.66 (95% confidence interval, 0.56-0.76) and the specificity 0.47 (95% confidence interval, 0.43-0.51), with an area under the curve (AUC) of 0.60. The different TCMR grades I-III were not reflected by the marker set, and borderline TCMR was not specifically detected. Secondary independent masked assessment of biopsies consented by 2 pathologists revealed an interobserver kappa value of 0.49 for diagnosing TCMR, compared with the local center's diagnosis. Using this consensus diagnosis, the AUC of the urine test was 0.63 (sensitivity 0.73, specificity 0.45). Post hoc optimization of the marker set improved the diagnostic performance in the study cohort (AUC 0.67) and in an independent patient cohort (AUC 0.69)., Conclusions: This study illustrates the difficulty of proteomics-based diagnosis of TCMR and highlights the need for rigorous independent in-place validation and optimization of diagnostic biomarkers., Competing Interests: J.M. is an employee of Mosaiques Diagnostics GmbH. The other authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

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., Competing Interests: Conflict of interest The authors declare no competing financial interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: 17q12 microdeletions containing HNF1B and intragenic variants within this gene are associated with variable developmental, endocrine, and renal anomalies, often already noted prenatally as hyperechogenic/cystic kidneys. Here, we describe prenatal and postnatal phenotypes of seven individuals with HNF1B aberrations and compare their clinical and genetic data to those of previous studies., Methods: Prenatal sequencing and postnatal chromosomal microarray analysis were performed in seven individuals with renal and/or neurodevelopmental phenotypes. We evaluated HNF1B-related clinical features from 82 studies and reclassified 192 reported intragenic HNF1B variants., Results: In a prenatal case, we identified a novel in-frame deletion p.(Gly239del) within the HNF1B DNA-binding domain, a mutational hot spot as demonstrated by spatial clustering analysis and high computational prediction scores. The six postnatally diagnosed individuals harbored 17q12 microdeletions. Literature screening revealed variable reporting of HNF1B-associated clinical traits. Overall, both mutation groups showed a high phenotypic heterogeneity. The reclassification of all previously reported intragenic HNF1B variants provided an up-to-date overview of the mutational spectrum., Conclusions: We highlight the value of prenatal HNF1B screening in renal developmental diseases. Standardized clinical reporting and systematic classification of HNF1B variants are necessary for a more accurate risk quantification of prenatal and postnatal clinical features, improving genetic counseling and prenatal decision making., (© 2019 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Background: Several subunits of the SWI/SNF chromatin remodeling complex are implicated in both cancer and neurodevelopmental disorders (NDD). Though there is no clinical evidence for an increased tumor risk in individuals with NDDs due to germline mutations in most of these genes so far, this has been repeatedly proposed and discussed. A young woman with NDD due to a de novo mutation in ARID1B now presented with a large renal (> 19 cm in diameter) and multiple hepatic angiomyolipomas (AMLs) but no other signs of tuberous sclerosis complex., Methods: We analyzed tumor and healthy tissue samples with exome and panel sequencing., Results: Additionally to the previously known, germline ARID1B variant we identified a post-zygotic truncating TSC2 variant in both renal and hepatic AMLs but not in any of the healthy tissues. We did not detect any further, obvious tumor driver events. The identification of a passenger variant in SIPA1L3 in both AMLs points to a common clonal origin. Metastasis of the renal AML into the liver is unlikely on the basis of discordant histopathological features. Our findings therefore point to very low-grade mosaicism for the TSC2 variant, possibly in a yet unknown mesenchymal precursor cell that expanded clonally during tumor development. A possible contribution of the germline ARID1B variant to the tumorigenesis remains unclear but cannot be excluded given the absence of any other evident tumor drivers in the AMLs., Conclusion: This unique case highlights the blurred line between tumor genetics and post-zygotic events that can complicate exact molecular diagnoses in patients with rare manifestations. It also demonstrates the relevance of multiple disorders in a single individual, the challenges of detecting low-grade mosaicisms, and the importance of proper diagnosis for treatment and surveillance.

Factor H related-protein 5 (CFHR5) is a surface-acting complement activator and variations in the CFHR5 gene are linked to CFHR glomerulonephritis. In this study, we show that FHR5 binds to laminin-521, the major constituent of the glomerular basement membrane, and to mesangial laminin-211. Furthermore, we identify malondialdehyde-acetaldehyde (MAA) epitopes, which are exposed on the surface of human necrotic cells ( Homo sapiens ), as new FHR5 ligands. Using a set of novel deletion fragments, we show that FHR5 binds to laminin-521, MAA epitopes, heparin, and human necrotic cells (HUVECs) via the middle region [short consensus repeats (SCRs) 5-7]. In contrast, surface-bound FHR5 contacts C3b via the C-terminal region (SCRs8-9). Thus, FHR5 uses separate domains for C3b binding and cell surface interaction. MAA epitopes serve as a complement-activating surface by recruiting FHR5. The complement activator FHR5 and the complement inhibitor factor H both bind to oxidation-specific MAA epitopes and FHR5 competes with factor H for binding. The C3 glomerulopathy-associated FHR2 1-2 -FHR5 hybrid protein is more potent in MAA epitope binding and activation compared with wild-type FHR5. The implications of these results for pathology of CFHR glomerulonephritis are discussed. In conclusion, we identify laminins and oxidation-specific MAA epitopes as novel FHR5 ligands and show that the surface-binding site of FHR5 (SCRs5-7) is separated from the C3b binding site (SCRs8-9). Furthermore, FHR5 competes with factor H for binding to MAA epitopes and activates complement on these modified structures., (Copyright © 2018 by The American Association of Immunologists, Inc.)