Timely Monitoring of SARS-CoV-2 RNA Fragments in Wastewater Shows the Emergence of JN.1 (BA.2.86.1.1, Clade 23I) in Berlin, Germany.

The importance of COVID-19 surveillance from wastewater continues to grow since case-based surveillance in the general population has been scaled back world-wide. In Berlin, Germany, quantitative and genomic wastewater monitoring for SARS-CoV-2 is performed in three wastewater treatment plants (WWTP) covering 84% of the population since December 2021. The SARS-CoV-2 Omicron sublineage JN.1 (B.2.86.1.1), was first identified from wastewater on 22 October 2023 and rapidly became the dominant sublineage. This change was accompanied by a parallel and still ongoing increase in the notification-based 7-day-hospitalization incidence of COVID-19 and COVID-19 ICU utilization, indicating increasing COVID-19 activity in the (hospital-prone) population and a higher strain on the healthcare system. In retrospect, unique mutations of JN.1 could be identified in wastewater as early as September 2023 but were of unknown relevance at the time. The timely detection of new sublineages in wastewater therefore depends on the availability of new sequences from GISAID and updates to Pango lineage definitions and Nextclade. We show that genomic wastewater surveillance provides timely public health evidence on a regional level, complementing the existing indicators.

SARS-CoV-2 infection dynamics revealed by wastewater sequencing analysis and deconvolution.

The use of RNA sequencing from wastewater samples is a valuable way for estimating infection dynamics and circulating lineages of SARS-CoV-2. This approach is independent from testing individuals and can therefore become the key tool to monitor this and potentially other viruses. However, it is equally important to develop easily accessible and scalable tools which can highlight critical changes in infection rates and dynamics over time across different locations given sequencing data from wastewater. Here, we provide an analysis of lineage dynamics in Berlin and New York City using wastewater sequencing and present PiGx SARS-CoV-2, a highly reproducible computational analysis pipeline with comprehensive reports. This end-to-end pipeline includes all steps from raw data to shareable reports, additional taxonomic analysis, deconvolution and geospatial time series analyses. Using simulated datasets (in silico generated and spiked-in samples) we could demonstrate the accuracy of our pipeline calculating proportions of Variants of Concern (VOC) from environmental as well as pre-mixed samples (spiked-in). By applying our pipeline on a dataset of wastewater samples from Berlin between February 2021 and January 2022, we could reconstruct the emergence of B.1.1.7(alpha) in February/March 2021 and the replacement dynamics from B.1.617.2 (delta) to BA.1 and BA.2 (omicron) during the winter of 2021/2022. Using data from very-short-reads generated in an industrial scale setting, we could see even higher accuracy in our deconvolution. Lastly, using a targeted sequencing dataset from New York City (receptor-binding-domain (RBD) only), we could reproduce the results recovering the proportions of the so-called cryptic lineages shown in the original study. Overall our study provides an in-depth analysis reconstructing virus lineage dynamics from wastewater. While applying our tool on a wide range of different datasets (from different types of wastewater sample locations and sequenced with different methods), we show that PiGx SARS-CoV-2 can be used to identify new mutations and detect any emerging new lineages in a highly automated and scalable way. Our approach can support efforts to establish continuous monitoring and early-warning projects for detecting SARS-CoV-2 or any other pathogen.

Biallelic Expression of Mucin-1 in Autosomal Dominant Tubulointerstitial Kidney Disease: Implications for Nongenetic Disease Recognition.

BACKGROUND: Providing the correct diagnosis for patients with tubulointerstitial kidney disease and secondary degenerative disorders, such as hypertension, remains a challenge. The autosomal dominant tubulointerstitial kidney disease (ADTKD) subtype caused by MUC1 mutations (ADTKD-MUC1) is particularly difficult to diagnose, because the mutational hotspot is a complex repeat domain, inaccessible with routine sequencing techniques. Here, we further evaluated SNaPshot minisequencing as a technique for diagnosing ADTKD-MUC1 and assessed immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique. METHODS: We re-evaluated detection of MUC1 mutations by targeted repeat enrichment and SNaPshot minisequencing by haplotype reconstruction via microsatellite analysis in three independent ADTKD-MUC1 families. Additionally, we generated rabbit polyclonal antibodies against MUC1-fs and evaluated immunodetection of wild-type and mutated allele products in human kidney biopsy specimens. RESULTS: The detection of MUC1 mutations by SNaPshot minisequencing was robust. Immunostaining with our MUC1-fs antibodies and an MUC1 antibody showed that both proteins are readily detectable in human ADTKD-MUC1 kidneys, with mucin 1 localized to the apical membrane and MUC1-fs abundantly distributed throughout the cytoplasm. Notably, immunohistochemical analysis of MUC1-fs expression in clinical kidney samples facilitated reliable prediction of the disease status of individual patients. CONCLUSIONS: Diagnosing ADTKD-MUC1 by molecular genetics is possible, but it is technically demanding and labor intensive. However, immunohistochemistry on kidney biopsy specimens is feasible for nongenetic diagnosis of ADTKD-MUC1 and therefore, a valid method to select families for further diagnostics. Our data are compatible with the hypothesis that specific molecular effects of MUC1-fs underlie the pathogenesis of this disease.

Hypoxia inhibits nephrogenesis through paracrine Vegfa despite the ability to enhance tubulogenesis.

Reduced nephron number predisposes to hypertension and kidney disease. Interaction of the branching ureteric bud and surrounding mesenchymal cells determines nephron number. Since oxygen supply may be critical for intrauterine development, we tested whether hypoxia and hypoxia-inducible factor-1α (HIF-1α) influence nephrogenesis. We found that HIF-1α is required for branching of MDCK cells. In addition, culture of metanephric mouse kidneys with ureteric bud cell-specific stabilization or knockout of HIF-1α revealed a positive impact of HIF-1α on nephrogenesis. In contrast, widespread stabilization of HIF-1α in metanephric kidneys through hypoxia or HIF stabilizers impaired nephrogenesis, and pharmacological HIF inhibition enhanced nephrogenesis. Several lines of evidence suggest an inhibitory effect through the hypoxia response of mesenchymal cells. HIF-1α was expressed in mesenchymal cells during nephrogenesis. Expression of the anti-branching factors Bmp4 and Vegfa, secreted by mesenchymal cells, was increased upon HIF stabilization. The conditioned medium from hypoxic metanephric kidneys inhibited MDCK branching, which was partially rescued by Vegfa antibodies. Thus, the effect of HIF-1α on nephrogenesis appears context dependent. While HIF-1α in the ureteric bud is of importance for proper branching morphogenesis, the net effect of hypoxia-induced HIF activation in the embryonic kidney appears to be mesenchymal cell-dependent inhibition of ureter branching.

Renal fibrosis is the common feature of autosomal dominant tubulointerstitial kidney diseases caused by mutations in mucin 1 or uromodulin.

For decades, ill-defined autosomal dominant renal diseases have been reported, which originate from tubular cells and lead to tubular atrophy and interstitial fibrosis. These diseases are clinically indistinguishable, but caused by mutations in at least four different genes: UMOD, HNF1B, REN, and, as recently described, MUC1. Affected family members show renal fibrosis in the biopsy and gradually declining renal function, with renal failure usually occurring between the third and sixth decade of life. Here we describe 10 families and define eligibility criteria to consider this type of inherited disease, as well as propose a practicable approach for diagnosis. In contrast to what the frequently used term 'Medullary Cystic Kidney Disease' implies, development of (medullary) cysts is neither an early nor a typical feature, as determined by MRI. In addition to Sanger and gene panel sequencing of the four genes, we established SNaPshot minisequencing for the predescribed cytosine duplication within a distinct repeat region of MUC1 causing a frameshift. A mutation was found in 7 of 9 families (3 in UMOD and 4 in MUC1), with one indeterminate (UMOD p.T62P). On the basis of clinical and pathological characteristics we propose the term 'Autosomal Dominant Tubulointerstitial Kidney Disease' as an improved terminology. This should enhance recognition and correct diagnosis of affected individuals, facilitate genetic counseling, and stimulate research into the underlying pathophysiology.

Hypoxia regulates the sperm associated antigen 4 (SPAG4) via HIF, which is expressed in renal clear cell carcinoma and promotes migration and invasion in vitro.

Hypoxia leads to the upregulation of a variety of genes mediated largely via the hypoxia inducible transcription factor (HIF). Prominent HIF-regulated target genes such as the vascular endothelial growth factor (VEGF), the glucose transporter 1 (Glut-1), or erythropoietin (EPO) help to assure survival of cells and organisms in a low oxygenated environment. Here, we are the first to report the hypoxic regulation of the sperm associated antigen 4 (SPAG4). SPAG4 is a member of the cancer testis (CT) gene family and to date little is known about its physiological function or its involvement in tumor biology. A number of CT family candidate genes are therefore currently being investigated as potential cancer markers, due to their predominant testicular expression pattern. We analyzed RNA and protein expression by RNAse protection assay, immunofluorescent as well as immunohistological stainings. To evaluate the influence of SPAG4 on migration and invasion capabilities, siRNA knockdown as well as transient overexpression was performed prior to scratch or invasion assay analysis. The hypoxic regulation of SPAG4 is clearly mediated in a HIF-1 and VHL dependent manner. We furthermore show upregulation of SPAG4 expression in human renal clear cell carcinoma (RCC) and co-localization within the nucleolus in physiological human testis tissue. SPAG4 knockdown reduces the invasion capability of RCC cells in vitro and overexpression leads to enhancement of tumor cell migration. Together, SPAG4 could possibly play a role in the invasion capability and growth of renal tumors and could represent an interesting target for clinical intervention.

Renal uptake of the antiapoptotic protein survivin is mediated by megalin at the apical membrane of the proximal tubule.

The inhibitor of apoptosis protein survivin is a bifunctional molecule that regulates cellular division and survival. We have previously shown that survivin protein can be found at high concentrations in the adult kidney, particularly in the proximal tubules. Here, survivin is localized primarily at the apical membrane, a pattern that may indicate absorption of the protein. Several proteins in primary urine are internalized by megalin, an endocytosis receptor, which is in principle found in the same localization as survivin. Immunolabeling for survivin in different species confirmed survivin signal localizing to the apical membrane of the proximal tubule. Immunoelectron microscopy also showed apical localization of survivin in human kidneys. Furthermore, in polarized human primary tubular cells endogenous as well as external recombinant survivin is stored in the apical region of the cells. Costaining of survivin and megalin by immunohistochemistry and immunoelectron microscopy confirmed colocalization. Finally, by surface plasmon resonance we were able to demonstrate that survivin binds megalin and cubilin and that megalin knockout mice lose survivin through the urine. Survivin accumulates at the apical membrane of the renal tubule by reuptake, which is achieved by the endocytic receptor megalin, collaborating with cubilin. For this to occur, survivin will have to circulate in the blood and be filtered into the primary urine. It is not known at this stage what the functional role of tubular survivin is. However, a small number of experimental and clinical reports implicate that renal survivin is important for functional integrity of the kidney.

Renal tubular HIF-2α expression requires VHL inactivation and causes fibrosis and cysts.

The Hypoxia-inducible transcription Factor (HIF) represents an important adaptive mechanism under hypoxia, whereas sustained activation may also have deleterious effects. HIF activity is determined by the oxygen regulated α-subunits HIF-1α or HIF-2α. Both are regulated by oxygen dependent degradation, which is controlled by the tumor suppressor "von Hippel-Lindau" (VHL), the gatekeeper of renal tubular growth control. HIF appears to play a particular role for the kidney, where renal EPO production, organ preservation from ischemia-reperfusion injury and renal tumorigenesis are prominent examples. Whereas HIF-1α is inducible in physiological renal mouse, rat and human tubular epithelia, HIF-2α is never detected in these cells, in any species. In contrast, distinct early lesions of biallelic VHL inactivation in kidneys of the hereditary VHL syndrome show strong HIF-2α expression. Furthermore, knockout of VHL in the mouse tubular apparatus enables HIF-2α expression. Continuous transgenic expression of HIF-2α by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to multiple renal cysts. In conclusion, VHL appears to specifically repress HIF-2α in renal epithelia. Unphysiological expression of HIF-2α in tubular epithelia has deleterious effects. Our data are compatible with dedifferentiation of renal epithelial cells by sustained HIF-2α expression. However, HIF-2α overexpression alone is insufficient to induce tumors. Thus, our data bear implications for renal tumorigenesis, epithelial differentiation and renal repair mechanisms.

The GTPase RAB20 is a HIF target with mitochondrial localization mediating apoptosis in hypoxia.

Hypoxia is a common pathogenic stress, which requires adaptive activation of the Hypoxia-inducible transcription factor (HIF). In concert transcriptional HIF targets enhance oxygen availability and simultaneously reduce oxygen demand, enabling survival in a hypoxic microenvironment. Here, we describe the characterization of a new HIF-1 target gene, Rab20, which is a member of the Rab family of small GTP-binding proteins, regulating intracellular trafficking and vesicle formation. Rab20 is directly regulated by HIF-1, resulting in rapid upregulation of Rab20 mRNA as well as protein under hypoxia. Furthermore, exogenous as well as endogenous Rab20 protein colocalizes with mitochondria. Knockdown studies reveal that Rab20 is involved in hypoxia induced apoptosis. Since mitochondria play a key role in the control of cell death, we suggest that regulating mitochondrial homeostasis in hypoxia is a key function of Rab20. Furthermore, our study implicates that cellular transport pathways play a role in oxygen homeostasis. Hypoxia-induced Rab20 may influence tissue homeostasis and repair during and after hypoxic stress.

Hypoxia-inducible protein 2 is a novel lipid droplet protein and a specific target gene of hypoxia-inducible factor-1.

Hypoxia-inducible protein 2 (HIG2) has been implicated in canonical Wnt signaling, both as target and activator. The potential link between hypoxia and an oncogenic signaling pathway might play a pivotal role in renal clear-cell carcinoma characterized by constitutive activation of hypoxia-inducible factors (HIFs), and hence prompted us to analyze HIG2 regulation and function in detail. HIG2 was up-regulated by hypoxia and HIF inducers in all cell types and mouse organs investigated and abundantly expressed in renal clear-cell carcinomas. Promoter analyses, gel shifts, and siRNA studies revealed that HIG2 is a direct and specific target of HIF-1, but not responsive to HIF-2. Surprisingly, HIG2 was not secreted, and HIG2 overexpression neither stimulated proliferation nor activated Wnt signaling. Instead, we show that HIG2 decorates the hemimembrane of lipid droplets, whose number and size increase on hypoxic inhibition of fatty acid ß-oxidation, and colocalizes with the lipid droplet proteins adipophilin and TIP47. Normoxic overexpression of HIG2 was sufficient to increase neutral lipid deposition in HeLa cells and stimulated cytokine expression. HIG2 could be detected in atherosclerotic arteries and fatty liver disease, suggesting that this ubiquitously inducible HIF-1 target gene may play an important functional role in diseases associated with pathological lipid accumulation.

HIF-1 or HIF-2 induction is sufficient to achieve cell cycle arrest in NIH3T3 mouse fibroblasts independent from hypoxia.

Hypoxia is a severe stress which induces physiological and molecular adaptations, where the latter is dominated by the Hypoxia-inducible transcription Factor (HIF). A well described response on cellular level upon exposure to hypoxia is a reversible cell cycle arrest, which probably renders the cells more resistant to the difficult environment. The individual roles of hypoxia itself and of the isoforms HIF-1alpha and HIF-2alpha in cell cycle regulation are poorly understood and discussed controversially. In order to characterize the isolated effect of both HIFalpha isoforms on the cell cycle we generated tetracycline inducible, HIF-1alpha and -2alpha expressing NIH3T3 cells. The cDNAs for HIFalpha were mutated to generate stable and active HIF under normoxia. Upon activation of both HIFalpha subunits, the total number of living cells was reduced and long-term stimulation of HIF led to complete loss of transgene expression, implicating a strong negative selection pressure. Equally, colony forming activity was reduced by activation of both HIFalpha subunits. Cell cycle analyses showed that HIF activation resulted in a prominent cell cycle arrest in G(1)-phase, similarly to the hypoxic effect. Both, HIF-1alpha and HIF-2alpha were able to induce the expression of the cyclin-dependent kinase inhibitor p27 on reporter gene and protein level. Our study shows that HIF-1 and HIF-2 can individually arrest the cell cycle independent from hypoxia. These findings have implications for the resistance of tumor cells to the environment and treatment, but also for physiological cells. Importantly, recent approaches to stabilize HIFalpha in normoxia could have deleterious effects on proliferating tissues.

The specific contribution of hypoxia-inducible factor-2alpha to hypoxic gene expression in vitro is limited and modulated by cell type-specific and exogenous factors.

Cellular integrity in hypoxia is dependent on molecular adaptations dominated by the heterodimeric transcription factor hypoxia-inducible factor (HIF). The HIF complex contains one of two alternative oxygen-regulated alpha-subunits considered to play distinct roles in the hypoxia response. Although HIF-2alpha may be more important in tumour biology and erythropoiesis, the spectrum of individual target genes is still insufficiently characterized. We therefore performed an Affymetrix gene array on Hep3B cells stimulated with a hypoxia-mimetic and transfected with either HIF-1alpha or HIF-2alpha siRNA. 271 transcripts were found to be induced HIF-dependently, including most previously identified HIF targets and a number of novel genes. Most were influenced by HIF-1alpha knock-down, whereas a smaller number were regulated by HIF-2alpha. Validation of a selection of genes by RNase protection confirmed the hypoxic regulation and HIF-1alpha- or HIF-2alpha-dependency in most cases, with the latter showing a lower amplitude. Many HIF-2alpha targets also responded to HIF-1alpha knock-down. Interestingly, regulation by HIF-2alpha was markedly influenced not only by cell type, but also by cell culture conditions, features that were not shared with HIF-1alpha-regulated genes. Thus, HIF-2alpha effects are modulated by a number of intrinsic and extrinsic factors which may be most relevant in tumour cells.

The tumor gene survivin is highly expressed in adult renal tubular cells: implications for a pathophysiological role in the kidney.

The inhibitor of apoptosis protein survivin is of critical importance for regulation of cellular division and survival. Published data point to a restricted function of survivin in embryonic development and cancer; thus survivin has been broadly proposed as an ideal molecular target for specific anti-cancer therapy. In contrast to this paradigm, we report here broad expression of survivin in adult differentiated tissues, as demonstrated at the mRNA and protein levels. Focusing on the kidney, survivin is strongly expressed in proximal tubuli, particularly at the apical membrane, which can be verified in rat, mouse, and human kidneys. In the latter, survivin expression seems to be even stronger in proximal tubuli than in adjacent cancerous tissue. Primary and immortalized human renal tubular cells also showed high levels of survivin protein expression, and RNA interference resulted in a partial G(2)/M arrest of the cell cycle and increased rate of apoptosis. In conclusion, survivin may be of importance for renal pathophysiology and pathology. The predominant apical expression of survivin may indicate a further, yet unknown, function. Interventional strategies to inhibit survivin's function in malignancy need to be carefully (re)evaluated for renal side effects, as well as for other possible organ dysfunctions.