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Abstract In recent years functional multiphoton (MP) imaging of vital mouse tissues and stimulation emission depletion (STED) imaging of optically cleared tissues allowed new insights into kidney biology. Here, we present a novel workflow where MP imaging of calcium signals can be combined with super-resolved STED imaging for morphological analysis of the slit diaphragm (SD) within the same glomerulus. Mice expressing the calcium indicator GCaMP3 in podocytes served as healthy controls or were challenged with two different doses of nephrotoxic serum (NTS). NTS induced glomerular damage in a dose dependent manner measured by shortening of SD length. In acute kidney slices (AKS) intracellular calcium levels increased upon disease but showed a high variation between glomeruli. We could not find a clear correlation between intracellular calcium levels and SD length in the same glomerulus. Remarkably, analysis of the SD morphology of glomeruli selected during MP calcium imaging revealed a higher percentage of completely disrupted SD architecture than estimated by STED imaging alone. Our novel co-imaging protocol is applicable to a broad range of research questions. It can be used with different tissues and is compatible with diverse reporters and target proteins.

Abstract Podocytes form the kidney filtration barrier and continuously adjust to external stimuli to preserve their integrity even in the presence of inflammation. It was suggested that canonical toll-like receptor signaling, mediated by the adaptor protein MYD88, plays a crucial role in initiating inflammatory responses in glomerulonephritis (GN). We explored the influence of podocyte-intrinsic MYD88 by challenging wild-type (WT) and podocyte-specific Myd88 knockout (MyD88pko) mice, with a model of experimental GN (nephrotoxic nephritis, NTN). Next-generation sequencing revealed a robust upregulation of inflammatory pathways and changes in cytoskeletal and cell adhesion proteins in sorted podocytes from WT mice during disease. Unchallenged MyD88pko mice were healthy and showed no proteinuria, normal kidney function and lacked morphological changes. During NTN, MyD88pko exhibited a transient increase in proteinuria in comparison to littermates, while histological damage, podocyte ultrastructure in STED imaging and frequencies of infiltrating immune cells by flow cytometry were unchanged. MYD88-deficiency led to subtle changes in the podocyte transcriptome, without a significant impact on the overall podocyte response to inflammation, presumably through MYD88-independent signaling pathways. In conclusion, our study reveals a comprehensive analysis of podocyte adaptation to an inflammatory environment on the transcriptome level, while MYD88-deficiency had only limited impact on the course of GN suggesting additional signaling through MYD88-independent signaling.

Idiopathic inflammatory myopathy (IIM) summarizes rare, systemic autoimmune conditions primarily characterized by inflammatory damage to the skeletal muscle. Although primary damage occurs to the muscle, these IIM-related conditions involve other organs, including the skin, lungs, upper gastrointestinal tract, joints, and heart. While many patients have an adequate response to immunosuppressive treatment, some patients develop rapidly progressive and treatment-resistant life-threatening courses. Treatment-resistant IIM is challenging for the treating physician and requires interdisciplinary and individualized treatment approaches. Extracorporeal therapy is one option for rescue therapy, with immunoadsorption (IA) having proven more effective than plasma exchange regarding the removal of circulating antibodies. Despite its efficacy and desirable safety profile, the clinical value of IA use in IIM is understudied with no controlled trials reported. Here, we present a review of the current knowledge regarding the management of treatment-resistant IIM and the cases of three patients with treatment-resistant IIM (two with dermatomyositis and one with immune-mediated necrotizing myopathy) who have successfully been treated with IA. All patients responded well to the therapy and experienced no IA-related complications. Taken together, we found IA to be a safe and effective treatment option in treatment-resistant IIM.

Abstract The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocin Δexon5 ) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocinwildtype and podocinΔexon5. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes’ foot process morphology. Mice homozygous for podocin Δexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes’ slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocin Δexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels. The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.

Abstract Background Solute carrier family 13 member 5 (SLC13A5) is a Na+-coupled citrate co-transporter that mediates entry of extracellular citrate into the cytosol. SLC13A5 inhibition has been proposed as a target for reducing progression of kidney disease. The aim of this study was to leverage the Mendelian randomization paradigm to gain insight into the effects of SLC13A5 inhibition in humans, towards prioritizing and informing clinical development efforts. Methods The primary Mendelian randomization analyses investigated the effect of SLC13A5 inhibition on measures of kidney function, including creatinine and cystatin C-based measures of estimated glomerular filtration rate (creatinine-eGFR and cystatin C-eGFR), blood urea nitrogen (BUN), urine albumin-creatinine ratio (uACR), and risk of chronic kidney disease and microalbuminuria. Secondary analyses included a paired plasma and urine metabolome-wide association study, investigation of secondary traits related to SLC13A5 biology, a phenome-wide association study (PheWAS), and a proteome-wide association study. All analyses were compared to the effect of genetically predicted plasma citrate levels using variants selected from across the genome, and statistical sensitivity analyses robust to the inclusion of pleiotropic variants were also performed. Data were obtained from large-scale genetic consortia and biobanks, with sample sizes ranging from 5023 to 1,320,016 individuals. Results We found evidence of associations between genetically proxied SLC13A5 inhibition and higher creatinine-eGFR (p = 0.002), cystatin C-eGFR (p = 0.005), and lower BUN (p = 3 × 10−4). Statistical sensitivity analyses robust to the inclusion of pleiotropic variants suggested that these effects may be a consequence of higher plasma citrate levels. There was no strong evidence of associations of genetically proxied SLC13A5 inhibition with uACR or risk of CKD or microalbuminuria. Secondary analyses identified evidence of associations with higher plasma calcium levels (p = 6 × 10−13) and lower fasting glucose (p = 0.02). PheWAS did not identify any safety concerns. Conclusions This Mendelian randomization analysis provides human-centric insight to guide clinical development of an SLC13A5 inhibitor. We identify plasma calcium and citrate as biologically plausible biomarkers of target engagement, and plasma citrate as a potential biomarker of mechanism of action. Our human genetic evidence corroborates evidence from various animal models to support effects of SLC13A5 inhibition on improving kidney function.

Objective: Older patients with nonvalvular atrial fibrillation (AF) are at high risk for frailty and geriatric syndromes (GSs), which modulate their individual prognosis and are therefore relevant for further management. Because few studies have evaluated the geriatric profile of older AF patients, this secondary analysis aims to further characterize the patterns of GSs and geriatric resources (GRs) in AF patients and their association with anticoagulation use. Methods: Data from 362 hospitalized patients aged 65 years and older with AF (n = 181, 77.8 ± 5.8 years, 38% female) and without AF (non-AF [NAF]; n = 181, 77.5 ± 5.9 years, 40% female) admitted to an internal medicine and nephrology ward of a large university hospital in Germany were included. All patients underwent usual care plus a comprehensive geriatric assessment (CGA) including calculation of the Multidimensional Prognostic Index (MPI) and collection of 17 GSs and 10 GRs. Patients were followed up by telephone 6 and 12 months after discharge to collect data on their health status. Results: The mean MPI score of 0.47 indicated an average risk of poor outcome, and patients with AF had a significantly higher MPI than those without AF (p = 0.040). After adjustment for chronological age, biological sex, Cumulative Illness Rating Scale (CIRS) for relevant chronic diagnoses and MPI as a proxy for biological age, AF patients had significantly more mnestic resources (63.5% vs. 33.1%, p < 0.001), a tendency for less age-appropriate living conditions (56.4% vs. 72.9%, p = 0.051) and more sensory impairment (78.5% vs. 52.5%, p < 0.001) than NAF patients. They also had a higher number of GSs (p = 0.046). AF patients on oral anticoagulants (OACs, n = 91) had less age-appropriate living conditions (48.4% vs. 64.4%, p < 0.05) and mnestic resources (36.3% vs. 54.4%, p < 0.01), but more emotional resources (80.2% vs. 65.6%, p < 0.05) and chronic pain (56% vs. 40%, p < 0.05) than patients without OACs (n = 90). Overall, mortality at 1 year was increased in patients with a higher MPI (p < 0.009, adjusted for age, sex and CIRS), with a diagnosis of AF (p = 0.007, adjusted for age, sex, CIRS and MPI), with of male sex (p = 0.008, adjusted for age, CIRS and MPI) and those with AF and treated with hemodialysis (p = 0.022, compared to AF patients without dialysis treatment). Conclusions: Patients with AF and patients with AF and OACs show differences in their multidimensional frailty degree as well as GR and GS profiles compared to patients without AF or with AF not treated with OACs. Mortality after 1 year is increased in AF patients with a higher MPI and dialysis, independently from OAC use and overall burden of chronic disease as assessed per CIRS. GRs and GSs, especially age-appropriate living conditions, emotional resources, sensory impairment and chronic pain, can be considered as factors that may modify the individual impact of frailty, underscoring the relevance of these parameters in the management of older patients.

Background: Sarcopenia is associated with adverse health outcomes. Understanding the association between sarcopenia, multidimensional frailty, and prognosis is essential for improving patient care. The aim of this study was to assess the prevalence and prognostic signature of sarcopenia in an acute hospital setting co-led by internists and geriatricians. Methods: Sarcopenia was assessed by applying the European Working Group on Sarcopenia in Older People (EWGSOP2) algorithm, including the SARC-F score, handgrip strength, bioelectrical impedance analysis (BIA), and Timed Up and Go (TUG) test, to 97 older multimorbid inpatients (76.5 ± 6.8 years, 55% women). The patients underwent a Comprehensive Geriatric Assessment (CGA) including an evaluation of Geriatric Syndromes (GSs) and Resources (GR) and prognosis calculation using the CGA-based Multidimensional Prognostic Index (MPI), European Quality of life—5 Dimensions (EQ-5D-5L) scale, Rosenberg Self-Esteem Scale (RSES), and Geriatric Depression Scale (GDS). Information on survival and rehospitalizations was collected 1, 3, and 6 months after discharge. Results: Sarcopenia was present in 63% (95% CI: 54–72%) of patients and categorized as probable (31%), confirmed (13%), and severe sarcopenia (18%). Sarcopenic patients showed significantly higher median MPI-values (p < 0.001), more GSs (p = 0.033), fewer GR (p = 0.003), lower EQ-5D-5L scores (p < 0.001), and lower RSES scores (p = 0.025) than non-sarcopenic patients. Six months after discharge, being sarcopenic at baseline was predictive of falls (p = 0.027) and quality of life (p = 0.043), independent of age, gender, and MPI. Conclusions: Sarcopenia is highly prevalent in older hospitalized multimorbid patients and is associated with poorer prognosis, mood, and quality of life up to 6 months after discharge, independent of age, sex, and MPI as surrogate markers of biological age.

Abstract Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are glomerulopathies associated with nephrotic syndrome. Primary forms of these diseases are treated with various regimes of immunosuppression. Frequently relapsing or glucocorticoid-dependent courses remain challenging. Here, a B-cell-depleting strategy with rituximab represents a salvage option although data are sparse in the adult population. In particular, there is limited evidence on the efficacy of restoring remission after initial successful treatment with rituximab and whether patients benefit from an individualized, relapse-based approach. We identified 13 patients who received multiple therapies with rituximab from the FOrMe-registry (NCT03949972), a nationwide registry for MCD and FSGS in Germany, or from the University Hospital of Cologne. Disease status, changes in serum creatinine, proteinuria, and time to relapse were evaluated. Relapse-free survival was compared to the patients’ previous therapy regimens. Through all treatment cycles, an improvement of disease activity was shown leading to a complete remission in 72% and partial remission in 26% after 3 ( $$p

Abstract Cilia are sensory organelles that project from the surface of almost all cells. Nephronophthisis (NPH) and NPH-related ciliopathies are degenerative genetic diseases caused by mutation of cilia-associated genes. These kidney disorders are characterized by progressive loss of functional tubular epithelial cells which is associated with inflammation, progressive fibrosis, and cyst formation, ultimately leading to end-stage renal disease. However, disease mechanisms remain poorly understood. Here, we show that targeted deletion of cilia in renal epithelial cells enhanced susceptibility to necroptotic cell death under inflammatory conditions. Treatment of non-ciliated cells with tumor necrosis factor (TNF) α and the SMAC mimetic birinapant resulted in Ripk1-dependent cell death, while viability of ciliated cells was almost not affected. Cell death could be enhanced and shifted toward necroptosis by the caspase inhibitor emricasan, which could be blocked by inhibitors of Ripk1 and Ripk3. Moreover, combined treatment of ciliated and non-ciliated cells with TNFα and cycloheximide induced a cell death response that could be partially rescued with emricasan in ciliated cells. In contrast, non-ciliated cells responded with pronounced cell death that was blocked by necroptosis inhibitors. Consistently, combined treatment with interferon-γ and emricasan induced cell death only in non-ciliated cells. Mechanistically, enhanced necroptosis induced by loss of cilia could be explained by induction of Ripk3 and increased abundance of autophagy components, including p62 and LC3 associated with the Ripk1/Ripk3 necrosome. Genetic ablation of cilia in renal tubular epithelial cells in mice resulted in TUNEL positivity and increased expression of Ripk3 in kidney tissue. Moreover, loss of Nphp1, the most frequent cause of NPH, further increased susceptibility to necroptosis in non-ciliated epithelial cells, suggesting that necroptosis might contribute to the pathogenesis of the disease. Together, these data provide a link between cilia-related signaling and cell death responses and shed new light on the disease pathogenesis of NPH-related ciliopathies.

Kidney metabolism in disease is important but not well understood. Here, using isotope-guided metabolomics, the authors show that lysine’s metabolic activity conveys kidney protection in hypertension through accelerated metabolism and physiological effects on tubular function.

Background Acute kidney injury (AKI) is a major risk factor for chronic kidney disease and increased mortality. Until now, no compelling preventive or therapeutic strategies have been identified. Dietary interventions have been proven highly effective in organ protection from ischemia reperfusion injury in mice and restricting dietary intake of sulfur‐containing amino acids (SAA) seems to be instrumental in this regard. The UNICORN trial aimed to evaluate the protective impact of restricting SAA intake before cardiac surgery on incidence of AKI. Methods and Results In this single‐center, randomized, controlled, double‐blind trial, 115 patients were assigned to a SAA‐reduced formula diet (LowS group) or a regular formula diet (control group) in a 1:1 ratio for 7 days before scheduled cardiac surgery. The primary end point was incidence of AKI within 72 hours after surgery, secondary end points included increase of serum creatinine at 24, 48, and 72 hours as well as safety parameters. Quantitative variables were analyzed with nonparametric methods, while categorical variables were evaluated by means of Chi‐square or Fisher test. SAA intake in the group with SAA reduced formula diet was successfully reduced by 77% (group with SAA reduced formula diet, 7.37[6.40–7.80] mg/kg per day versus control group, 32.33 [28.92–33.60] mg/kg per day, P

Abstract 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.

The molecular identity of renal glomerular cells is poorly characterized and rodent glomerulopathy models translate poorly to humans. Here, the authors show molecular signatures of glomerulus-associated cells using single cell RNA sequencing and highlight differences between mouse and human cells.

Reactive oxygen species (ROS), which excessively arise in diabetes and systemic inflammatory diseases, modify cellular lipids and cellular lipid composition leading to altered biophysical properties of cellular membranes. The impact of lipid peroxidation on transmembrane signaling routes is not yet well studied. The canonical transient receptor potential channel 6 (TRPC6) is implicated in the pathogenesis of several forms of glomerular diseases. TRPC6 is sensitive to membrane stretch and relies on a distinct lipid environment. This study investigates the effect of oxidative alterations to plasma membrane lipids on TRPC6 activity and the function of the glomerular filter. Knockout of the anti-oxidative, lipid modifying enzyme paraoxonase 2 (PON2) leads to altered biophysical properties of glomerular epithelial cells, which are called podocytes. Cortical stiffness, quantified by atomic force microscopy, was largely increased in PON2-deficient cultured podocytes. PON2 deficiency markedly enhanced TRPC6 channel currents and channel recovery. Treatment with the amphiphilic substance capsazepine in micromolar doses reduced cortical stiffness and abrogated TRPC6 conductance. In in vivo studies, capsazepine reduced the glomerular phenotype in the model of adriamycin-induced nephropathy in PON2 knockout mice and wildtype littermates. In diabetic AKITA mice, the progression of albuminuria and diabetic kidney disease was delayed. In summary, we provide evidence that the modification of membrane characteristics affects TRPC6 signaling. These results could spur future research to investigate modification of the direct lipid environment of TRPC6 as a future therapeutic strategy in glomerular disease.

Summary: The cellular response to hypoxia is crucial to organismal survival, and hypoxia-inducible factors (HIF) are the key mediators of this response. HIF-signaling is central to many human diseases and mediates longevity in the nematode. Despite the rapidly increasing knowledge on RNA-binding proteins (RBPs), little is known about their contribution to hypoxia-induced cellular adaptation. We used RNA interactome capture (RIC) in wild-type Caenorhabditis elegans and vhl-1 loss-of-function mutants to fill this gap. This approach identifies more than 1,300 nematode RBPs, 270 of which can be considered novel RBPs. Interestingly, loss of vhl-1 modulates the RBPome. This difference is not primarily explained by protein abundance suggesting differential RNA-binding. Taken together, our study provides a global view on the nematode RBPome and proteome as well as their modulation by HIF-signaling. The resulting RBP atlas is also provided as an interactive online data mining tool (http://shiny.cecad.uni-koeln.de:3838/celegans_rbpome). : Biological Sciences; Molecular Biology; Molecular Interaction; Molecular Network; Integrative Aspects of Cell Biology; Proteomics Subject Areas: Biological Sciences, Molecular Biology, Molecular Interaction, Molecular Network, Integrative Aspects of Cell Biology, Proteomics

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is a serious hazard for hemodialysis (HD) patients and kidney transplant (KTX) recipients as they suffer from an impaired immune response to SARS-CoV-2 vaccination. In addition, a definition of SARS-CoV-2 IgG titer that indicates a sufficient immune response, especially against new omicron variants, is urgently needed. In the present study, the immune response to either a third or a fourth dose of a mRNA vaccine was investigated in 309 dialysis and 36 KTX patients. SARS-CoV-2 IgG titer thresholds indicating neutralizing activity against wild type (WT) and the omicron variant BA.1 were quantified. After four vaccine doses, a high-neutralizing activity against WT was evidenced in HD patients, whereas the neutralizing rate against BA.1 was significant lower. Concerning KTX recipients, humoral and cellular immune responses after a third vaccination were still highly impaired. This calls for modified omicron-targeting vaccines.

Diabetes and inflammatory diseases are associated with an altered cellular lipid composition due to lipid peroxidation. The pathogenic potential of these lipid alterations in glomerular kidney diseases remains largely obscure as suitable cell culture and animal models are lacking. In glomerular disease, a loss of terminally differentiated glomerular epithelial cells called podocytes refers to irreversible damage. Podocytes are characterized by a complex ramified cellular architecture and highly active transmembrane signaling. Alterations in lipid composition in states of disease have been described in podocytes but the pathophysiologic mechanisms mediating podocyte damage are unclear. In this study, we employ a genetic deletion of the anti-oxidative, lipid-modifying paraoxonase 2 enzyme (PON2) as a model to study altered cellular lipid composition and its effects on cellular signaling in glomerular disease. PON2 deficiency reproduces features of an altered lipid composition of glomerular disease, characterized by an increase in ceramides and cholesterol. PON2 knockout mice are more susceptible to glomerular damage in models of aggravated oxidative stress such as adriamycin-induced nephropathy. Voltage clamp experiments in cultured podocytes reveal a largely increased TRPC6 conductance after a membrane stretch in PON2 deficiency. Correspondingly, a concomitant knockout of TRPC6 and PON2 partially rescues the aggravated glomerular phenotype of a PON2 knockout in the adriamycin model. This study establishes PON2 deficiency as a model to investigate the pathophysiologic mechanisms of podocyte dysfunction related to alterations in the lipid composition, as seen in diabetic and inflammatory glomerular disease. Expanding the knowledge on these routes and options of intervention could lead to novel treatment strategies for glomerular disease.

Older persons often loose independence during hospitalization. This analysis aimed at retrospectively evaluating the effects of a pilot individualized multidimensional intervention (IMI) on the comprehensive geriatric assessment (CGA)-based prognosis of older multimorbid patients in an acute internal medicine setting. Records from 72 patients aged 65 years and above who received the IMI were compared to those from 403 patients who received standard of care (SOC). All patients had undergone the CGA-based Multidimensional Prognostic Index (MPI) calculation on admission and at discharge. Patients were divided into three risk groups according to MPI score: Low-risk (MPI-1, 0-0.33), medium-risk (MPI-2, 0.34-0.66) and high-risk (MPI-3, 0.67-1). From admission to discharge, IMI patients showed significant improvements in their MPI score (P=0.014) and subdomains compared to SOC. This was particularly evident in MPI-2 and MPI-3 as well as in patients with poorer functions on MPI admission subdomains. An early geriatric intervention during hospitalization for disease-specific treatments in internal medicine settings improves overall individual prognosis in older multimorbid patients. Prospective randomized studies are needed to confirm these preliminary retrospective observations.

Abstract Background Transplant failure requires the consideration of numerous potential causes including rejection, acute tubular necrosis, infection, and recurrence of the original kidney disease. Kidney biopsy is generally required to approach these differential diagnoses. However, the histopathological findings on their own do not always lead to a definite diagnosis. Consequently, it is crucial to integrate them with clinical findings and patient history when discussing histopathological patterns of injury. The histopathologic finding of a membranoproliferative glomerulonephritis (MPGN) is one of the most challenging constellations since it does not refer to a specific disease entity but rather reflects a pattern of injury that is the result of many different causes. Whilst MPGN is occasionally classified as immune complex mediated, careful evaluation usually reveals an underlying disorder such as chronic infection, plasma cell dyscrasia, complement disorders, and autoimmune disease. Case presentation We describe the case of a 43-year-old woman who was referred to us because of a slowly rising serum creatinine 4 years after kidney transplantation. As in the native kidney, the biopsy revealed an MPGN pattern of injury. The cause of this finding had not been established prior to transplantation leading to a classification as idiopathic MPGN in the past. Further workup at the time of presentation and allograft failure revealed chronic infection of a ventriculoatrial shunt as the most probable cause. Conclusion This case underlines the fact that MPGN is not a disease but a histopathological description. Consequently, the causative disorder needs to be identified to avoid kidney failure and recurrence after transplantation.

Abstract Background Podocyte infolding glomerulopathy (PIG) is a rare histopathologic finding with global infolding of the podocytes into the glomerular basement membrane (GBM), accompanied by microstructures underneath. Described in 2002 for the first time, PIG was proposed as a new pathological entity in 2008 based on the largest case series so far. Yet all of the described cases derive from Asian countries. We report a case from Germany fulfilling the diagnostic criteria of PIG. Considering the scarcity of data on this entity especially in Western countries, collecting cases like ours and multicentric meta-analyses will be crucial to obtain a better understanding of PIG, its causes, clinical course and potential treatment options. Case presentation A 56-year-old Caucasian woman with a history of rheumatoid arthritis (RA), no other comorbidities and no known renal disease was admitted to the hospital with acute kidney injury (AKI) and nephrotic syndrome. Physical examination was unremarkable except for anasarca. Renal ultrasound revealed no abnormalities. Laboratory and urine analyses were consistent with the nephrotic syndrome and renal failure. Serological studies regarding ANA, ANCA, anti-PLA2R autoantibodies, complement, virus infections, immunofixation and quantitative light chain analysis were unremarkable. A renal biopsy was performed. Light microscopic examination showed flattened tubular epithelium consistent with acute tubular damage, no infiltrates and unremarkable glomeruli except diffuse and global holes in the GBM (Fig. 1a) and negative staining for immunoglobulin heavy-chains, light-chains and complement split products. Electron microscopy revealed a rare correlate for these holes: global peculiar infolding of podocyte cytoplasm into the GBM. Most of these infoldings were accompanied by condensation of the GBM underneath. No such condensation or electron dense deposits were found without these infoldings or outside the GBM. Conclusion Here we report the first case of PIG outside of Asia. Since there are only few reports about this specific finding, we feel there is a need to share information in an attempt to accumulate knowledge about this possible new entity and potential treatment options.

The negative impact of chronic kidney disease (CKD) on health status and quality of life in older patients has been well documented. However, data on frailty trajectories and long-term outcomes of older CKD patients undergoing structured Comprehensive Geriatric Assessment (CGA) with multidimensional frailty evaluation are sparse. Here, we analysed records from 375 CKD patients admitted to our university hospital (mean age 77.5 (SD 6.1) years, 36% female) who had undergone a CGA-based calculation of the frailty score with the multidimensional prognostic index (MPI) as well as follow-up evaluations at 3, 6 and 12 months after discharge. Based on the MPI score at admission, 21% of the patients were frail and 56% were prefrail. MPI values were significantly associated with KDIGO CKD stages (p = 0.003) and rehospitalisation after 6 months (p = 0.027) and mortality at 3, 6 and 12 months (p = 0.001), independent of chronological age. Kidney transplant recipients (KTR) showed a significantly lower frailty compared to patients with renal replacement therapy (RRT, p = 0.028). The association between frailty and mortality after 12 months appeared particularly strong for KTR (mean MPI 0.43 KTR vs. 0.52 RRT, p < 0.001) and for patients with hypoalbuminemia (p < 0.001). Interestingly, RRT was per se not significantly associated with mortality during follow up. However, compared to patients on RRT those with KTR had a significantly lower grade of care (p = 0.031) and lower rehospitalisation rates at 12 months (p = 0.010). The present analysis shows that the large majority of older CKD inpatients are prefrail or frail and that the risk for CKD-related adverse outcomes on the long term can be accurately stratified by CGA-based instruments. Further studies are needed to explore the prognostic and frailty-related signature of laboratory biomarkers in CKD.

BackgroundRapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose.MethodsTo avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK).ResultsWhilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK.ConclusionIn summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.

Cell cilia: Protein crucial for function identified A protein involved in building and maintaining thin protrusions from cell surfaces called cilia is implicated in “ciliopathies”, diseases in which ciliary function is disrupted. These include polycystic kidney disease and disorders collectively known as ciliary dyskinesias. “Primary cilia” perform sensory functions, detecting external chemical and physical signals and initiating responses within cells. In addition, “motile cilia” beat rhythmically to move fluids surrounding cells. Researchers in Germany and the Netherlands, led by Bernhard Schermer and Max C. Liebau at the University of Cologne, studied a protein called Ruvbl1, known to interact with DNA and other proteins. The researchers found it is crucial for the functioning of both types of cilia. Deleting the gene for Ruvbl1 in mice caused kidney failure and a build-up of fluid in the brain known as hydrocephalus. The research could help understand and ultimately treat ciliopathies.

Summary: Damage to and loss of glomerular podocytes has been identified as the culprit lesion in progressive kidney diseases. Here, we combine mass spectrometry-based proteomics with mRNA sequencing, bioinformatics, and hypothesis-driven studies to provide a comprehensive and quantitative map of mammalian podocytes that identifies unanticipated signaling pathways. Comparison of the in vivo datasets with proteomics data from podocyte cell cultures showed a limited value of available cell culture models. Moreover, in vivo stable isotope labeling by amino acids uncovered surprisingly rapid synthesis of mitochondrial proteins under steady-state conditions that was perturbed under autophagy-deficient, disease-susceptible conditions. Integration of acquired omics dimensions suggested FARP1 as a candidate essential for podocyte function, which could be substantiated by genetic analysis in humans and knockdown experiments in zebrafish. This work exemplifies how the integration of multi-omics datasets can identify a framework of cell-type-specific features relevant for organ health and disease. : The podocyte forms the most outer and essential part of the renal filter and restricts the passage of proteins from blood to urine. Rinschen et al. combine deep proteomic and transcriptomic data with protein dynamics from native mouse podocytes to reveal insights into podocyte biology and to identify candidate disease genes. Keywords: end-stage renal disease, systems biology, proteinuria, focal segmental glomerulosclerosis, pulse SILAC, metabolism, slit diaphragm, hereditary nephrotic syndrome, kinase, proteostasis

Dialysis patients and kidney transplant (KTX) recipients suffer from an impaired immune system and show a decreased response to the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) vaccination. We performed a retrospective analysis of 1505 serological SARS-CoV-2 measurements obtained from 887 dialysis patients and 86 KTX recipients. The results were separated by patient subgroups (dialysis/KTX) as well as SARS-CoV-2 status. The latter criterion included SARS-CoV-2-naïve patients with or without COVID-19 vaccination and convalescent patients receiving a booster shot. Serologies of 27 vaccinated healthy individuals served as the reference group. Vaccine-induced cellular immune response was quantified by an interferon-γ release assay in 32 KTX recipients. We determined seroconversion rates of 92.6%, 93.4%, and 71.4% in dialysis patients vaccinated with either BNT162b2, mRNA-1273, or AZD1222, respectively. Vaccination-induced anti-SARS-CoV-2 antibody titers were lower in dialysis patients compared to healthy individuals, and vaccination with mRNA-1273 induced higher titers than BNT162b2. The initial seroconversion rate was 39.5% in KTX recipients vaccinated with BNT162b2. A linear regression model identified medication with mycophenolate-mofetil/mycophenolic acid as an independent risk factor for missing seroconversion. Within a cohort of 32 KTX recipients, cellular and humoral immune reactivity to SARS-CoV-2 was detectable in three patients only. Conclusively, vaccine-induced seroconversion rates were similar in dialysis patients compared to healthy individuals but were strongly impaired in KTX recipients. Anti-SARS-CoV-2 IgG titers elicited by double active immunization were significantly lower in both cohorts compared to healthy individuals, and immune responses to vaccination vanished quickly.

Cyclin-dependent kinase 5 (Cdk5) is expressed in terminally differentiated cells, where it drives development, morphogenesis, and survival. Temporal and spatial kinase activity is regulated by specific activators of Cdk5, dependent on the cell type and environmental factors. In the kidney, Cdk5 is exclusively expressed in terminally differentiated glomerular epithelial cells called podocytes. In glomerular disease, signaling mechanisms via Cdk5 have been addressed by single or combined conventional knockout of known specific activators of Cdk5. A protective, anti-apoptotic role has been ascribed to Cdk5 but not a developmental phenotype, as in terminally differentiated neurons. The effector kinase itself has never been addressed in animal models of glomerular disease. In the present study, conditional and inducible knockout models of Cdk5 were analyzed to investigate the role of Cdk5 in podocyte development and glomerular disease. While mice with podocyte-specific knockout of Cdk5 had no developmental defects and regular lifespan, loss of Cdk5 in podocytes increased susceptibility to glomerular damage in the nephrotoxic nephritis model. Glomerular damage was associated with reduced anti-apoptotic signals in Cdk5-deficient mice. In summary, Cdk5 acts primarily as master regulator of podocyte survival during glomerular disease and—in contrast to neurons—does not impact on glomerular development or maintenance.

Acute kidney injury (AKI) is a frequent and critical complication in the clinical setting. In rodents, AKI can be effectively prevented through caloric restriction (CR), which has also been shown to increase lifespan in many species. In Caenorhabditis elegans (C. elegans), longevity studies revealed that a marked CR-induced reduction of endocannabinoids may be a key mechanism. Thus, we hypothesized that regulation of endocannabinoids, particularly arachidonoyl ethanolamide (AEA), might also play a role in CR-mediated protection from renal ischemia-reperfusion injury (IRI) in mammals including humans. In male C57Bl6J mice, CR significantly reduced renal IRI and led to a significant decrease of AEA. Supplementation of AEA to near-normal serum concentrations by repetitive intraperitoneal administration in CR mice, however, did not abrogate the protective effect of CR. We also analyzed serum samples taken before and after CR from patients of three different pilot trials of dietary interventions. In contrast to mice and C. elegans, we detected an increase of AEA. We conclude that endocannabinoid levels in mice are modulated by CR, but CR-mediated renal protection does not depend on this effect. Moreover, our results indicate that modulation of endocannabinoids by CR in humans may differ fundamentally from the effects in animal models.

Abstract Background Renal cell carcinoma is among the most prevalent malignancies. It is generally sporadic. However, genetic studies of rare familial forms have led to the identification of mutations in causative genes such as VHL and FLCN. Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé syndrome, a rare tumor syndrome which is characterized by the combination of renal cell carcinoma, pneumothorax and skin tumors. Methods Using Sanger sequencing we identify a heterozygous splice-site mutation in FLCN in lymphocyte DNA of a patient suffering from renal cell carcinoma. Furthermore, both tumor DNA and DNA from a metastasis are analyzed regarding this mutation. The pathogenic effect of the sequence alteration is confirmed by minigene assays and the biochemical consequences on the protein are examined using TALEN-mediated transgenesis in cultured cells. Results Here we describe an FLCN mutation in a 55-year-old patient who presented himself with progressive weight loss, bilateral kidney cysts and renal tumors. He and members of his family had a history of recurrent pneumothorax during the last few decades. Histology after tumor nephrectomy showed a mixed kidney cancer consisting of elements of a chromophobe renal cell carcinoma and dedifferentiated small cell carcinoma component. Subsequent FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. We demonstrate skipping of exon 11 to be the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. Interestingly, the truncated protein was still expressed both in cell culture and in tumor tissue, though it was strongly destabilized and its subcellular localization differed from wild-type FLCN. Both, altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation. Conclusions Identification of disease-causing mutations in BHD syndrome requires the analysis of intronic sequences. However, biochemical validation of the consecutive alterations of the resulting protein is especially important in these cases. Functional characterization of the disease-causing mutations in BHD syndrome may guide further research for the development of novel diagnostic and therapeutic strategies.

We describe the case of 50-year-old female patient who presented with severe gastrointestinal symptoms and progressive weight loss of unknown origin. Shortly after admission, she developed an acute flare of thrombotic thrombocytopaenic purpura (TTP) that had to be treated by plasma exchange therapy and rituximab administration. While the signs of TTP subsided, the gastrointestinal symptoms worsened with abdominal cramps, massive gastric retention, malnourishment and a stenosis due to extensive inflammation and wall thickening of the small bowel. Extensive diagnostic efforts yielded no specific cause, so the patient—based on the histopathological findings—was diagnosed with idiopathic non-granulomatous ulcerative jejunoileitis. Following a highly complicated clinical course over several months, successful remission of the inflammatory activity and recovery of the patient could be obtained by TNF-alpha blockade.

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.

Recently, the European Medicines Agency approved the use of the vasopressin V2 receptor antagonist tolvaptan to slow the progression of cyst development and renal insufficiency of autosomal dominant polycystic kidney disease (ADPKD) in adult patients with chronic kidney disease stages 1–3 at initiation of treatment with evidence of rapidly progressing disease. In this paper, on behalf of the ERA-EDTA Working Groups of Inherited Kidney Disorders and European Renal Best Practice, we aim to provide guidance for making the decision as to which ADPKD patients to treat with tolvaptan. The present position statement includes a series of recommendations resulting in a hierarchical decision algorithm that encompasses a sequence of risk-factor assessments in a descending order of reliability. By examining the best-validated markers first, we aim to identify ADPKD patients who have documented rapid disease progression or are likely to have rapid disease progression. We believe that this procedure offers the best opportunity to select patients who are most likely to benefit from tolvaptan, thus improving the benefit-to-risk ratio and cost-effectiveness of this treatment. It is important to emphasize that the decision to initiate treatment requires the consideration of many factors besides eligibility, such as contraindications, potential adverse events, as well as patient motivation and lifestyle factors, and requires shared decision-making with the patient.

Cystic kidney diseases affect patients of all age groups with the onset spanning from prenatal disease to late adulthood. Autosomal-dominant polycystic kidney disease (ADPKD) is by far the most common renal cystic disease. However, there are various cystic kidney diseases, the onset of which occurs at different times in life and depends on the type of the disease and the causative genes involved. When genetic kidney diseases are discussed in the adult setting this view is usually limited on autosomal-dominant kidney disease, the most frequent genetic disorder causing adult onset ESRD. Other diseases—such as autosomal-recessive polycystic kidney disease—are often being viewed as a disorder only important in pediatric nephrology. However, more recent data has revealed that, despite clear age peaks of onset for each disorder, all of them can also show highly variable phenotypes with classical adult onset genetic diseases being of importance in pediatrics and vice versa. Furthermore, the affected children need to be seen by adult nephrologists in the long term after transition, requiring knowledge on the underlying pediatric disease, potential extrarenal manifestations, and genetic counseling. Consequently, the view on these diseases should be widened on both ends. Close interaction between pediatric and adult nephrology is key to appropriate care of patients suffering from genetic kidney disease to profit from each other’s experience.

BackgroundAcute kidney injury is a frequent complication after cardiac surgery and is associated with adverse outcomes. Although short‐term calorie restriction (CR) has proven protective in rodent models of acute kidney injury, similar effects have not yet been demonstrated in humans. Methods and ResultsCR_KCH (Effect of a Preoperative Calorie Restriction on Renal Function After Cardiac Surgery) is a randomized controlled trial in patients scheduled for cardiac surgery. Patients were randomly assigned to receive either a formula diet containing 60% of the daily energy requirement (CR group) or ad libitum food (control group) for 7 days before surgery. In total, 82 patients were enrolled between April 16, 2012, and February 5, 2015. There was no between‐group difference in the primary end point of median serum creatinine increment after 24 hours (control group: 0.0 mg/dL [−0.1 – (+0.2) mg/dL]; CR group: 0.0 mg/dL [−0.2 – (+0.2) mg/dL]; P=0.39). CR prevented a rise in median creatinine at 48 hours (control group: +0.1 mg/dL [0.0 – 0.3 mg/dL]; CR group: −0.1 mg/dL [−0.2 – (+0.1) mg/dL]; P=0.03), with most pronounced effects observed in male patients and patients with a body mass index >25. This benefit persisted until discharge: Median creatinine decreased by 0.1 mg/dL (−0.2 – 0.0 mg/dL) in the CR group, whereas it increased by 0.1 mg/dL (0.0 – 0.3 mg/dL; P=0.0006) in the control group. Incidence of acute kidney injury was reduced by 5.8% (41.7% in the CR group compared with 47.5% in the control group). Safety‐related events did not differ between groups. ConclusionsDespite disappointing results with respect to creatinine rise within the first 24 hours, the benefits observed at later time points and the subgroup analyses suggest the protective potential of short‐term CR in patients at risk for acute kidney injury, warranting further investigation. Clinical Trial RegistrationURL: http://www.clinicaltrials.gov. Unique identifier: NCT01534364.

Abstract Mitochondrial dysfunction and alterations in energy metabolism have been implicated in a variety of human diseases. Mitochondrial fusion is essential for maintenance of mitochondrial function and requires the prohibitin ring complex subunit prohibitin‐2 (PHB2) at the mitochondrial inner membrane. Here, we provide a link between PHB2 deficiency and hyperactive insulin/IGF‐1 signaling. Deletion of PHB2 in podocytes of mice, terminally differentiated cells at the kidney filtration barrier, caused progressive proteinuria, kidney failure, and death of the animals and resulted in hyperphosphorylation of S6 ribosomal protein (S6RP), a known mediator of the mTOR signaling pathway. Inhibition of the insulin/IGF‐1 signaling system through genetic deletion of the insulin receptor alone or in combination with the IGF‐1 receptor or treatment with rapamycin prevented hyperphosphorylation of S6RP without affecting the mitochondrial structural defect, alleviated renal disease, and delayed the onset of kidney failure in PHB2‐deficient animals. Evidently, perturbation of insulin/IGF‐1 receptor signaling contributes to tissue damage in mitochondrial disease, which may allow therapeutic intervention against a wide spectrum of diseases.

BACKGROUND:Recent data emphasize that thin basement membrane nephropathy (TBMN) should not be viewed as a form of benign familial hematuria since chronic renal failure (CRF) and even end-stage renal disease (ESRD), is a possible development for a subset of patients on long-term follow-up, through the onset of focal and segmental glomerulosclerosis (FSGS). We hypothesize that genetic modifiers may explain this variability of symptoms. METHODS:We looked in silico for potentially deleterious functional SNPs, using very strict criteria, in all the genes significantly expressed in the slit diaphragm (SD). Two variants were genotyped in a cohort of well-studied adult TBMN patients from 19 Greek-Cypriot families, with a homogeneous genetic background. Patients were categorized as "Severe" or "Mild", based on the presence or not of proteinuria, CRF and ESRD. A larger pooled cohort (HEMATURIA) of 524 patients, including IgA nephropathy patients, was used for verification. Additionally, three large general population cohorts [Framingham Heart Study (FHS), KORAF4 and SAPHIR] were used to investigate if the NEPH3-V353M variant has any renal effect in the general population. RESULTS AND CONCLUSIONS:Genotyping for two high-scored variants in 103 TBMN adult patients with founder mutations who were classified as mildly or severely affected, pointed to an association with variant NEPH3-V353M (filtrin). This promising result prompted testing in the larger pooled cohort (HEMATURIA), indicating an association of the 353M variant with disease severity under the dominant model (p = 3.0x10-3, OR = 6.64 adjusting for gender/age; allelic association: p = 4.2x10-3 adjusting for patients' kinships). Subsequently, genotyping 6,531 subjects of the Framingham Heart Study (FHS) revealed an association of the homozygous 353M/M genotype with microalbuminuria (p = 1.0x10-3). Two further general population cohorts, KORAF4 and SAPHIR confirmed the association, and a meta-analysis of all three cohorts (11,258 individuals) was highly significant (p = 1.3x10-5, OR = 7.46). Functional studies showed that Neph3 homodimerization and Neph3-Nephrin heterodimerization are disturbed by variant 353M. Additionally, 353M was associated with differential activation of the unfolded protein response pathway, when overexpressed in stressed cultured undifferentiated podocyte cells, thus attesting to its functional significance. Genetics and functional studies support a "rare variant-strong effect" role for NEPH3-V353M, by exerting a negative modifier effect on primary glomerular hematuria. Additionally, genetics studies provide evidence for a role in predisposing homozygous subjects of the general population to micro-albuminuria.

Acute kidney injury is a leading contributor to morbidity and mortality in the ageing population. Proteotoxic stress response pathways have been suggested to contribute to the development of acute renal injury. Recent evidence suggests that increased synthesis of N-glycan precursors in the hexosamine pathway as well as feeding of animals with aminosugars produced in the hexosamine pathway may increase stress resistance through reducing proteotoxic stress and alleviate pathology in model organisms. As feeding of the hexosamine pathway metabolite glucosamine to aged mice increased their life expectancy we tested whether supplementation of this aminosugar may also protect mice from acute kidney injury after renal ischemia and reperfusion. Animals were fed for 4 weeks ad libitum with standard chow or standard chow supplemented with 0.5% N-acetylglucosamine. Preconditioning with caloric restriction for four weeks prior to surgery served as a positive control for protective dietary effects. Whereas caloric restriction demonstrated the known protective effect both on renal function as well as survival in the treated animals, glucosamine supplementation failed to promote any protection from ischemia-reperfusion injury. These data show that although hexosamine pathway metabolites have a proven role in enhancing protein quality control and survival in model organisms oral glucosamine supplementation at moderate doses that would be amenable to humans does not promote protection from ischemia-reperfusion injury of the kidney.

RATIONALE: Preeclampsia is a devastating medical complication of pregnancy which leads to maternal and fetal morbidity and mortality. While the etiology of preeclampsia is unclear, human and animal studies suggest that excessive circulating levels of soluble fms-like tyrosine-kinase-1 (sFlt-1), an alternatively spliced variant of VEGF-receptor1, contribute to the signs and symptoms of preeclampsia. Since sFlt-1 binds to heparin and heparan sulfate proteoglycans, we hypothesized that the anticoagulant heparin, which is often used in pregnancy, may interfere with the levels, distribution and elimination of sFlt-1 in vivo. OBJECTIVE: We systematically determined serum and urine levels of angiogenic factors in preeclamptic women before and after administration of low molecular weight heparin and further characterized the interaction with heparin in biochemical studies. METHODS AND RESULTS: Serum and urine samples were used to measure sFlt-1 levels before and after heparin administration. Serum levels of sFlt-1 increased by 25% after heparin administration in pregnant women. The magnitude of the increase in circulating sFlt-1 correlated with initial sFlt-1 serum levels. Urinary sFlt-1 levels were also elevated following heparin administration and levels of elimination were dependent on the underlying integrity of the glomerular filtration barrier. Biochemical binding studies employing cation exchange chromatography revealed that heparin bound sFlt-1 had decreased affinity to negatively charged surfaces when compared to sFlt-1 alone. CONCLUSION: Low molecular weight heparin administration increased circulating sFlt1 levels and enhanced renal elimination. We provide evidence that both effects may be due to heparin binding to sFlt1 and masking the positive charges on sFlt1 protein.

Fabry's disease results from an inborn error of glycosphingolipid metabolism that is due to deficiency of the lysosomal hydrolase α-galactosidase A. This X-linked defect results in the accumulation of enzyme substrates with terminally α-glycosidically bound galactose, mainly the neutral glycosphingolipid Globotriaosylceramide (Gb3) in various tissues, including the kidneys. Although end-stage renal disease is one of the most common causes of death in hemizygous males with Fabry's disease, the pathophysiology leading to proteinuria, hematuria, hypertension, and kidney failure is not well understood. Histological studies suggest that the accumulation of Gb3 in podocytes plays an important role in the pathogenesis of glomerular damage. However, due to the lack of appropriate animal or cellular models, podocyte damage in Fabry's disease could not be directly studied yet. As murine models are insufficient, a human model is needed. Here, we developed a human podocyte model of Fabry's disease by combining RNA interference technology with lentiviral transduction of human podocytes. Knockdown of α-galactosidase A expression resulted in diminished enzymatic activity and slowly progressive accumulation of intracellular Gb3. Interestingly, these changes were accompanied by an increase in autophagosomes as indicated by an increased abundance of LC3-II and a loss of mTOR kinase activity, a negative regulator of the autophagic machinery. These data suggest that dysregulated autophagy in α-galactosidase A-deficient podocytes may be the result of deficient mTOR kinase activity. This finding links the lysosomal enzymatic defect in Fabry's disease to deregulated autophagy pathways and provides a promising new direction for further studies on the pathomechanism of glomerular injury in Fabry patients.

Polo-like kinase (Plk1) plays a central role in regulating the cell cycle. Plk1-mediated phosphorylation is essential for centrosome maturation, and for numerous mitotic events. Although Plk1 localizes to multiple subcellular sites, a major site of action is the centrosomes, which supports mitotic functions in control of bipolar spindle formation. In G0 or G1 untransformed cells, the centriolar core of the centrosome differentiates into the basal body of the primary cilium. Primary cilia are antenna-like sensory organelles dynamically regulated during the cell cycle. Whether Plk1 has a role in ciliary biology has never been studied. Nephrocystin-1 (NPHP1) is a ciliary protein; loss of NPHP1 in humans causes nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. We here demonstrate that Plk1 colocalizes with nephrocystin-1 to the transition zone of primary cilia in epithelial cells. Plk1 co-immunoprecipitates with NPHP1, suggesting it is part of the nephrocystin protein complex. We identified a candidate Plk1 phosphorylation motif (D/E-X-S/T-φ-X-D/E) in nephrocystin-1, and demonstrated in vitro that Plk1 phosphorylates the nephrocystin N-terminus, which includes the specific PLK1 phosphorylation motif. Further, induced disassembly of primary cilia rapidly evoked Plk1 kinase activity, while small molecule inhibition of Plk1 activity or RNAi-mediated downregulation of Plk1 limited the first and second phase of ciliary disassembly. These data identify Plk1 as a novel transition zone signaling protein, suggest a function of Plk1 in cilia dynamics, and link Plk1 to the pathogenesis of NPH and potentially other cystic kidney diseases.

BACKGROUND: Thrombotic microangiopathies (TMA) in adults such as thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are life-threatening disorders if untreated. Clinical presentation is highly variable and prognostic factors for clinical course and outcome are not well established. METHODS: We performed a retrospective observational study of 62 patients with TMA, 22 males and 40 females aged 16 to 76 years, treated with plasma exchange at one center to identify clinical risk factors for the development of renal insufficiency. RESULTS: On admission, 39 of 62 patients (63%) had acute renal failure (ARF) with 32 patients (52%) requiring dialysis treatment. High systolic arterial pressure (SAP, p = 0.009) or mean arterial pressure (MAP, p = 0.027) on admission was associated with acute renal failure. Patients with SAP>140 mmHg on admission had a sevenfold increased risk of severe kidney disease (OR 7.464, CI 2.097-26.565). MAP>100 mmHg indicated a fourfold increased risk for acute renal failure (OR 4.261, CI 1.400-12.972). High SAP, diastolic arterial pressure (DAP), and MAP on admission were also independent risk factors for persistent renal insufficiency with the strongest correlation for high MAP. Moreover, a high C-reactive protein (CRP) level on admission correlated with renal failure in the course of the disease (p = 0.003). At discharge, renal function in 11 of 39 patients (28%) had fully recovered, 14 patients (23%) remained on dialysis, and 14 patients (23%) had non-dialysis-dependent chronic kidney disease. Seven patients (11%) died. We identified an older age as risk factor for death. CONCLUSIONS: High blood pressure as well as high CRP serum levels on admission are associated with renal insufficiency in TMA. High blood pressure on admission is also a strong predictor of sustained renal insufficiency. Thus, adult TMA patients with high blood pressure may require special attention to prevent persistent renal failure.

In contrast to effort-induced symptoms in obstructive coronary disease, spasm in normal coronary arteries is characterized by angina at rest. We describe a 44-year-old patient with minor coronary plaques and pure exercised-induced coronary spasm. The case questions the differential pathogenic considerations of variant of the variant as opposed to Prinzmetal's variant angina.