Five-year single-center analysis of cytomegalovirus viremia in kidney transplant recipients and possible implication for novel prophylactic therapy approaches.

BACKGROUND: Cytomegalovirus (CMV) infections are a common complication after kidney transplantation (KTx) and negatively affecting patient outcome. Valganciclovir (VGC) prophylaxis is often limited by drug-induced side effects and dose reduction due to decline in kidney function. METHOD: In the present study, episodes of CMV viremia in the first year after KTx in a cohort of 316 recipients were analyzed retrospectively to identify risk factors linked to persistent infections. RESULTS: In the studied cohort, 18.7% of patients showed a high-risk (HR) constellation (D+/R-) for CMV infections. CMV viremia affected 22% of our cohort, with HR patients being the most affected cohort (44.1%). Within this group, most viremic events (65.3%) occurred while patients were still on prophylactic therapy, showing significantly higher viral loads and a longer duration compared to seropositive recipients. CONCLUSION: The analysis at hand revealed that detection of viremia under ongoing antiviral prophylaxis bears an increased risk for sustained viral replication and antiviral drug resistance in HR patients. We identified low estimated glomerular filtration rate (eGFR) and lower dose VGC prophylaxis post-KTx as a risk factor for breakthrough infections in HR patients in our single center cohort. These patients might benefit from a closer CMV monitoring or novel prophylactic agents as letermovir.

Protein half-life determines expression of proteostatic networks in podocyte differentiation.

Podocytes are highly specialized, epithelial, postmitotic cells, which maintain the renal filtration barrier. When adapting to considerable metabolic and mechanical stress, podocytes need to accurately maintain their proteome. Immortalized podocyte cell lines are a widely used model for studying podocyte biology in health and disease in vitro. In this study, we performed a comprehensive proteomic analysis of the cultured human podocyte proteome in both proliferative and differentiated conditions at a depth of >7000 proteins. Similar to mouse podocytes, human podocyte differentiation involved a shift in proteostasis: undifferentiated podocytes have high expression of proteasomal proteins, whereas differentiated podocytes have high expression of lysosomal proteins. Additional analyses with pulsed stable-isotope labeling by amino acids in cell culture and protein degradation assays determined protein dynamics and half-lives. These studies unraveled a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal, and membrane proteins were stabilized, particularly in differentiated podocytes. Importantly, protein half-lives strongly contributed to protein abundance in each state. These data suggest that regulation of protein turnover of particular cellular functions determines podocyte differentiation, a paradigm involving mitophagy and, potentially, of importance in conditions of increased podocyte stress and damage.-Schroeter, C. B., Koehler, S., Kann, M., Schermer, B., Benzing, T., Brinkkoetter, P. T., Rinschen, M. M. Protein half-life determines expression of proteostatic networks in podocyte differentiation.

A Single-Cell Transcriptome Atlas of the Mouse Glomerulus.

Background Three different cell types constitute the glomerular filter: mesangial cells, endothelial cells, and podocytes. However, to what extent cellular heterogeneity exists within healthy glomerular cell populations remains unknown.Methods We used nanodroplet-based highly parallel transcriptional profiling to characterize the cellular content of purified wild-type mouse glomeruli.Results Unsupervised clustering of nearly 13,000 single-cell transcriptomes identified the three known glomerular cell types. We provide a comprehensive online atlas of gene expression in glomerular cells that can be queried and visualized using an interactive and freely available database. Novel marker genes for all glomerular cell types were identified and supported by immunohistochemistry images obtained from the Human Protein Atlas. Subclustering of endothelial cells revealed a subset of endothelium that expressed marker genes related to endothelial proliferation. By comparison, the podocyte population appeared more homogeneous but contained three smaller, previously unknown subpopulations.Conclusions Our study comprehensively characterized gene expression in individual glomerular cells and sets the stage for the dissection of glomerular function at the single-cell level in health and disease.

Single-nephron proteomes connect morphology and function in proteinuric kidney disease.

In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney diseases are frequent and assorted diseases affecting a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins while genetic ablation of LAMP1-correlated lysosomal proteases could ameliorate glomerular damage in vivo. Furthermore, proteomic analyses of individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases revealed increased abundance of lysosomal proteins, in combination with a decreased abundance of mutated gene products. Thus, altered protein homeostasis (proteostasis) is a conserved key mechanism in proteinuric kidney diseases. Moreover, our technology can capture intra-individual variability in diseases of the kidney and other tissues at a sub-biopsy scale.

WT1 targets Gas1 to maintain nephron progenitor cells by modulating FGF signals.

Development of the metanephric kidney depends on tightly regulated interplay between self-renewal and differentiation of a nephron progenitor cell (NPC) pool. Several key factors required for the survival of NPCs have been identified, including fibroblast growth factor (FGF) signaling and the transcription factor Wilms' tumor suppressor 1 (WT1). Here, we present evidence that WT1 modulates FGF signaling by activating the expression of growth arrest-specific 1 (Gas1), a novel WT1 target gene and novel modulator of FGF signaling. We show that WT1 directly binds to a conserved DNA binding motif within the Gas1 promoter and activates Gas1 mRNA transcription in NPCs. We confirm that WT1 is required for Gas1 expression in kidneys in vivo. Loss of function of GAS1 in vivo results in hypoplastic kidneys with reduced nephron mass due to premature depletion of NPCs. Although kidney development in Gas1 knockout mice progresses normally until E15.5, NPCs show decreased rates of proliferation at this stage and are depleted as of E17.5. Lastly, we show that Gas1 is selectively required for FGF-stimulated AKT signaling in vitro. In summary, our data suggest a model in which WT1 modulates receptor tyrosine kinase signaling in NPCs by directing the expression of Gas1.

N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton.

Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. We applied a novel proteomics technology that enables proteome-wide identification, mapping, and quantification of protein N-termini to comprehensively characterize cleaved podocyte proteins in the glomerulus in vivo We found evidence that defined proteolytic cleavage results in various proteoforms of important podocyte proteins, including those of podocin, nephrin, neph1, α-actinin-4, and vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease action during podocyte injury in vitro, including diminished proteolysis of α-actinin-4. Differentially regulated protease substrates comprised cytoskeletal proteins as well as intermediate filaments. Determination of preferential protease motifs during podocyte damage indicated activation of caspase proteases and inhibition of arginine-specific proteases. Several proteolytic processes were clearly site-specific, were conserved across species, and could be confirmed by differential migration behavior of protein fragments in gel electrophoresis. Some of the proteolytic changes discovered in vitro also occurred in two in vivo models of podocyte damage (WT1 heterozygous knockout mice and puromycin aminonucleoside-treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and podocyte cytoskeleton are regulated targets of proteolytic modification, which is altered upon podocyte damage.

Quantitative deep mapping of the cultured podocyte proteome uncovers shifts in proteostatic mechanisms during differentiation.

The renal filtration barrier is maintained by the renal podocyte, an epithelial postmitotic cell. Immortalized mouse podocyte cell lines-both in the differentiated and undifferentiated state-are widely utilized tools to estimate podocyte injury and cytoskeletal rearrangement processes in vitro. Here, we mapped the cultured podocyte proteome at a depth of more than 8,800 proteins and quantified 7,240 proteins. Copy numbers of proteins mutated in forms of hereditary nephrotic syndrome or focal segmental glomerulosclerosis (FSGS) were assessed. We found that cultured podocytes express abundant copy numbers of endogenous receptors, such as tyrosine kinase membrane receptors, the G protein-coupled receptor (GPCR), NPR3 (ANP receptor), and several poorly characterized GPCRs. The data set was correlated with deep mapping mRNA sequencing ("mRNAseq") data from the native mouse podocyte, the native mouse podocyte proteome and staining intensities from the human protein atlas. The generated data set was similar to these previously published resources, but several native and high-abundant podocyte-specific proteins were not identified in the data set. Notably, this data set detected general perturbations in proteostatic mechanisms as a dominant alteration during podocyte differentiation, with high proteasome activity in the undifferentiated state and markedly increased expression of lysosomal proteins in the differentiated state. Phosphoproteomics analysis of mouse podocytes at a resolution of more than 3,000 sites suggested a preference of phosphorylation of actin filament-associated proteins in the differentiated state. The data set obtained here provides a resource and provides the means for deep mapping of the native podocyte proteome and phosphoproteome in a similar manner.

Genome-Wide Analysis of Wilms' Tumor 1-Controlled Gene Expression in Podocytes Reveals Key Regulatory Mechanisms.

The transcription factor Wilms' tumor suppressor 1 (WT1) is key to podocyte development and viability; however, WT1 transcriptional networks in podocytes remain elusive. We provide a comprehensive analysis of the genome-wide WT1 transcriptional network in podocytes in vivo using chromatin immunoprecipitation followed by sequencing (ChIPseq) and RNA sequencing techniques. Our data show a specific role for WT1 in regulating the podocyte-specific transcriptome through binding to both promoters and enhancers of target genes. Furthermore, we inferred a podocyte transcription factor network consisting of WT1, LMX1B, TCF21, Fox-class and TEAD family transcription factors, and MAFB that uses tissue-specific enhancers to control podocyte gene expression. In addition to previously described WT1-dependent target genes, ChIPseq identified novel WT1-dependent signaling systems. These targets included components of the Hippo signaling system, underscoring the power of genome-wide transcriptional-network analyses. Together, our data elucidate a comprehensive gene regulatory network in podocytes suggesting that WT1 gene regulatory function and podocyte cell-type specification can best be understood in the context of transcription factor-regulatory element network interplay.

The transcription factor Sry-related HMG box-4 (SOX4) is required for normal renal development in vivo.

BACKGROUND: The DNA-binding transcription factor Wilms' Tumor Suppressor-1 (WT1) plays an essential role in nephron progenitor differentiation during renal development. We previously used Wt1 chromatin-immunoprecipitation coupled to microarray (ChIP-chip) to identify novel Wt1 target genes that may regulate nephrogenesis in vivo. We discovered that all three members of the SoxC subfamily, namely, Sox4, Sox11, and Sox12, are bound by Wt1 in mouse embryonic kidneys in vivo. SoxC genes play master roles in determining neuronal and mesenchymal progenitor cell fate in a multitude of developmental processes, but their function in the developing kidney is largely unknown. RESULTS: Here we show that all three SoxC genes are expressed in the nephrogenic lineages during renal development. Conditional ablation of Sox4 in nephron progenitors and their cellular descendants (Sox4(nephron-) mice) results in a significant reduction in nephron endowment. By postnatal day (P)7, Sox4(nephron-) renal corpuscles exhibit reduced numbers of Wt1+ podocytes together with loss of expression of the slit diaphragm protein nephrin. Sox4(nephron-) mice develop early-onset proteinacious glomerular injury within 2 weeks of birth progressing to end-stage renal failure within 5-9 months. CONCLUSIONS: Collectively, our results demonstrate an essential requirement of Sox4 for normal renal development in vivo.

Gene regulatory networks in disease and ageing.

The precise control of gene expression is required for the maintenance of cellular homeostasis and proper cellular function, and the declining control of gene expression with age is considered a major contributor to age-associated changes in cellular physiology and disease. The coordination of gene expression can be represented through models of the molecular interactions that govern gene expression levels, so-called gene regulatory networks. Gene regulatory networks can represent interactions that occur through signal transduction, those that involve regulatory transcription factors, or statistical models of gene-gene relationships based on the premise that certain sets of genes tend to be coexpressed across a range of conditions and cell types. Advances in experimental and computational technologies have enabled the inference of these networks on an unprecedented scale and at unprecedented precision. Here, we delineate different types of gene regulatory networks and their cell-biological interpretation. We describe methods for inferring such networks from large-scale, multi-omics datasets and present applications that have aided our understanding of cellular ageing and disease mechanisms.

First experiences with machine learning predictions of accelerated declining eGFR slope of living kidney donors 3 years after donation.

BACKGROUND: Living kidney donors are screened pre-donation to estimate the risk of end-stage kidney disease (ESKD). We evaluate Machine Learning (ML) to predict the progression of kidney function deterioration over time using the estimated GFR (eGFR) slope as the target variable. METHODS: We included 238 living kidney donors who underwent donor nephrectomy. We divided the dataset based on the eGFR slope in the third follow-up year, resulting in 185 donors with an average eGFR slope and 53 donors with an accelerated declining eGFR-slope. We trained three Machine Learning-models (Random Forest [RF], Extreme Gradient Boosting [XG], Support Vector Machine [SVM]) and Logistic Regression (LR) for predictions. Predefined data subsets served for training to explore whether parameters of an ESKD risk score alone suffice or additional clinical and time-zero biopsy parameters enhance predictions. Machine learning-driven feature selection identified the best predictive parameters. RESULTS: None of the four models classified the eGFR slope with an AUC greater than 0.6 or an F1 score surpassing 0.41 despite training on different data subsets. Following machine learning-driven feature selection and subsequent retraining on these selected features, random forest and extreme gradient boosting outperformed other models, achieving an AUC of 0.66 and an F1 score of 0.44. After feature selection, two predictive donor attributes consistently appeared in all models: smoking-related features and glomerulitis of the Banff Lesion Score. CONCLUSIONS: Training machine learning-models with distinct predefined data subsets yielded unsatisfactory results. However, the efficacy of random forest and extreme gradient boosting improved when trained exclusively with machine learning-driven selected features, suggesting that the quality, rather than the quantity, of features is crucial for machine learning-model performance. This study offers insights into the application of emerging machine learning-techniques for the screening of living kidney donors.

Impact of first-line use of caplacizumab on treatment outcomes in immune thrombotic thrombocytopenic purpura.

BACKGROUND: The von Willebrand factor-directed nanobody caplacizumab has greatly changed the treatment of immune thrombotic thrombocytopenic purpura (iTTP) in recent years. Data from randomized controlled trials established efficacy and safety. OBJECTIVES: This study aims to address open questions regarding patient selection, tailoring of therapy duration, obstacles in prescribing caplacizumab in iTTP, effect on adjunct treatment, and outcomes in the real-world setting. METHODS: We report retrospective, observational cohorts of 113 iTTP episodes treated with caplacizumab and 119 historical control episodes treated without caplacizumab. We aggregated data from the caplacizumab phase II/III trials and real-world data from France, the United Kingdom, Germany, and Austria (846 episodes, 396 treated with caplacizumab, and 450 historical controls). RESULTS: Caplacizumab was efficacious in iTTP, independent of the timing of therapy initiation, but curtailed the time of active iTTP only when used in the first-line therapy within 72 hours after diagnosis and until at least partial ADAMTS13-activity remission. Aggregated data from multiple study populations showed that caplacizumab use resulted in significant absolute risk reduction of 2.87% for iTTP-related mortality (number needed to treat 35) and a relative risk reduction of 59%. CONCLUSION: Caplacizumab should be used in first line and until ADAMTS13-remission, lowers iTTP-related mortality and refractoriness, and decreases the number of daily plasma exchange and hospital stay. This trial is registered at www. CLINICALTRIALS: gov as #NCT04985318.

Constitutive activation of the mTOR signaling pathway within the normal glomerulus.

Agents that target the activity of the mammalian target of rapamycin (mTOR) kinase in humans are associated with proteinuria. However, the mechanisms underlying mTOR activity and signaling within the kidney are poorly understood. In this study, we developed a sensitive immunofluorescence technique for the evaluation of activated pmTOR and its associated signals in situ. While we find that pmTOR is rarely expressed in normal non-renal tissues, we consistently find intense expression in glomeruli within normal mouse and human kidneys. Using double staining, we find that the expression of pmTOR co-localizes with nephrin in podocytes and expression appears minimal within other cell types in the glomerulus. In addition, we found that pmTOR was expressed on occasional renal tubular cells within mouse and human kidney specimens. We also evaluated mTOR signaling in magnetic bead-isolated glomeruli from normal mice and, by Western blot analysis, we confirmed function of the pathway in glomerular cells vs. interstitial cells. Furthermore, we found that the activity of the pathway as well as the expression of VEGF, a target of mTOR-induced signaling, were reduced within glomeruli of mice following treatment with rapamycin. Collectively, these findings demonstrate that the mTOR signaling pathway is constitutively hyperactive within podocytes. We suggest that pmTOR signaling functions to regulate glomerular homeostasis in part via the inducible expression of VEGF.

[Update 2021: COVID-19 from the perspective of nephrology]. / Update 2021: COVID-19 aus Sicht der Nephrologie.

Increasing insight into the clinical phenotype and mechanisms of SARS-CoV-2 infections and COVID-19 has identified damage of the kidneys as a key player in the course of the disease. This manuscript updates our previous summary with current knowledge on kidney involvement in COVID-19 and chronic kidney disease as a risk factor for severe COVID-19, as well as recommendations regarding SARS-CoV-2 vaccination for patients suffering from chronic kidney disease and after organ transplantation, respectively. populations, SARS-CoV-2 vaccination is strongly recommended for all patients suffering from chronic kidney disease and after kidney transplantation.

Monitoring of hepatitis E virus RNA during treatment for chronic hepatitis E virus infection after renal transplantation.

BACKGROUND: Recently, chronic hepatitis E virus (HEV) infections gained increasing attention as a possible cause for elevated liver enzymes of unknown origin and liver cirrhosis in solid organ transplant recipients. Reduction of immunosuppressive therapy and/or use of antiviral drug ribavirin have been established as possible treatment strategies. METHODS: The efficacy of dose reduction of mycophenolic acid (MPA) and ribavirin therapy was retrospectively analyzed in eight renal transplant patients of our outpatient clinic who were diagnosed with HEV infection by detection of specific antibodies (immunoglobulin M and immunoglobulin G) and/or positive RNA in blood and stool. In four patients serial HEV viral loads in blood were measured. RESULTS: Only one patient reached HEV clearance after reduction of immunosuppressive therapy (predominantly reduction of MPA daily dose) alone, whereas six patients were treated with ribavirin after reduction of immunosuppressive therapy due to persistent virus replication. Four of six patients reached HEV clearance after 3 months of ribavirin therapy. HEV clearance was observed after 34-42 days. Two patients, both treated with rituximab within the last 12 months before diagnosis of HEV infection, needed prolonged ribavirin therapy due to persistent viral replication. CONCLUSION: Reduction of daily dose of MPA therapy alone in transplant patients with chronic HEV infection may not be sufficient to control viral replication. HEV clearance under ribavirin therapy shows interindividual variability. Therefore, serial viral monitoring may be useful to personalize treatment duration. Rituximab therapy is a risk factor for complicated-to-treat chronic HEV infection.

Combined Therapy with Intravenous Immunoglobulins, Letermovir and (Val-)Ganciclovir in Complicated Courses of CMV-Infection in Transplant Recipients.

The treatment options for cytomegalovirus (CMV) infections in immunosuppressed patients are limited, mainly consisting of (val-)ganciclovir (VGC/GCV) as the first-line treatment. We report on three transplant recipients, one stem cell transplant (allo-HSCT) patient and two kidney transplant (KTx) recipients, with prolonged CMV viremia treated with a combined therapy based on letermovir (LMV), CMV-specific intravenous immunoglobulins (IVIg), and VGC/GCV, which led to the sustained control of CMV viremia in all patients.

Hand-Assisted Retroperitoneoscopic Donor Nephrectomy Compared to Anterior Approach Open Donor Nephrectomy: Improved Long-Term Physical Component Score in Health-Related Quality of Life in Living Kidney Donors.

BACKGROUND: Health-related quality of life (HRQL), fatigue, anxiety, and depression are crucial for the living kidney donor (LKD). Follow-up data for HRQL of LKDs comparing surgical techniques, especially regarding hand-assisted retroperitoneoscopic donor nephrectomy (HARP), are sparse. The aim of this study was to evaluate the influence of abdominal wall trauma minimized by HARP in comparison to open anterior approach donor nephrectomy (AA) on HRQL and additional psychosocial aspects of LKDs during the long-term follow-up. MATERIAL AND METHODS: This is a cross-sectional study comparing psychosocial aspects of LKD between HARP and AA. RESULTS: This study included 100 LKDs (68 HARP, 28 AA, and 4 were excluded secondary to incomplete data). The time to follow-up was 22.6 ± 11.7 (HARP) vs 58.7 ± 13.9 (AA) months (P < .005). Complications ≥3a° due to Clavien-Dindo classification was 0% in both groups. There were higher scores in all physical aspects for HARP donors vs AA donors at that time (physical function: 89.8 ± 14.6 vs 80.0 ± 19.9, P = .008, and the physical component score: 53.9 ± 7.6 vs 48.6 ± 8.5, P = .006). One year later (follow-up time + 12 months), HRQL for HARP donors was still higher. Mental items showed no significant differences. HARP donors showed better physical scores compared to the age-matched nondonor population (AA donors had lower scores). Neither the Multidimensional Fatigue Inventory-20 (MFI-20) or the Hospital Anxiety and Depression Scale (HADS) showed any differences between the 2 groups. Fatigue scores were higher for HARP and for AA compared to the age-matched population. CONCLUSIONS: LKDs undergoing HARP showed better physical performance as part of HRQL in the long-term follow-up.

CALINCA-A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease.

Diseases of the renal filtration unit-the glomerulus-are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

[COVID-19: The Perspective Of Nephrology]. / COVID-19 aus Sicht des Nephrologen.

Increasing insight into the clinical phenotype and mechanisms of SARS-CoV-2 infections and COVID-19 has identified damage of the kidneys as a key player in the course of the disease. This manuscript summarizes the current knowledge on direct viral infection of kidney tissue, proteinuria and acute kidney injury in COVID-19, and management of patients on chronic dialysis as well as after kidney transplantation. Direct infection of podocytes and proximal tubular cells by SARS-CoV-2 has been confirmed and results in proteinuria and hematuria at an early stage of COVID-19. In this context, any kidney affection is a predictor of worse outcomes among COVID-19 patients irrespective of the initial presentation and increases the risk of acute kidney injury. Specific therapies for kidney damage and acute kidney injury within COVID-19 that could be generally recommended are currently lacking. Patients on chronic hemodialysis in particular are at risk for contracting SARS-CoV-2 infections as indicated by outbreaks and super-spreading events in hemodialysis facilities. Immunosuppressive therapy after kidney transplantation needs to be adapted upon diagnosis of COVID-19 depending on the severity of the initial presentation.