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Background/aim Vimentin3 (Vim3) was recently described as a tumour marker for the direct discrimination between benign and malignant kidney tumours. Here, we examined its expression in prostate cancer (PCa) cell lines and the regulation of its expression by endothelin receptors. Materials and methods Prostate cancer cell lines (PC3, DU145, LNCap) were incubated with endothelin 1 (ET-1), BQ123 [endothelin A receptor (ETAR) antagonist], BQ788 [endothelin B receptor (ETBR) antagonist], BQ123+ET-1, BQ788+ET-1 for 24 h and a scratch assay was performed. Cell extracts were analysed by western blotting and qRT-PCR. Results ET-1 induced Vim3 overexpression. Blocking the ETBR in the different prostate cancer cell lines yielded a higher migration rate, whereby Vim3 expression was significantly increased. Conclusion Vim3 concentration increases in cell lines without a functional ETBR and may be used as a marker for PCas where ETBR is frequently methylated.

Autosomal recessive polycystic kidney disease (ARPKD) is mainly caused by variants in the PKHD1 gene, encoding fibrocystin (FC), a large transmembrane protein of incompletely understood cellular function. Here, we show that a C‐terminal fragment of human FC can suppress a signalling module of the kinase SRC and signal transducer and activator of transcription 3 (STAT3). Consistently, we identified truncating genetic variants specifically affecting the cytoplasmic tail in ARPKD patients, found SRC and the cytoplasmic tail of fibrocystin in a joint dynamic protein complex and observed increased activation of both SRC and STAT3 in cyst‐lining renal epithelial cells of ARPKD patients.

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.

Prematurity is linked to incomplete nephrogenesis and risk of chronic kidney diseases (CKDs). Oxygen is life-saving in that context but induces injury in numerous organs. Here, we studied the structural and functional impact of hyperoxia on renal injury and its IL-6 dependency. Newborn wild-type (WT) and IL-6 knockout (IL-6-/-) mice were exposed to 85% O2 for 28 d, followed by room air until postnatal d (P) 70. Controls were in room air throughout life. At P28, hyperoxia reduced estimated kidney cortex area (KCA) in WT; at P70, KCA was greater, number of glomeruli was fewer, fractional potassium excretion was higher, and glomerular filtration rate was slightly lower than in controls. IL-6-/- mice were protected from these changes after hyperoxia. Mechanistically, the acute renal injury phase (P28) showed in WT but not in IL-6-/- mice an activation of IL-6 (signal transducer and activator of transcription 3) and TGF-β [mothers against decapentaplegic homolog (Smad)2] signaling, increased inflammatory markers, disrupted mitochondrial biogenesis, and reduced tubular proliferation. Regenerative phase at P70 was characterized by tubular proliferation in WT but not in IL-6-/- mice. These data demonstrate that hyperoxia increases the risk of CKD through a novel IL-6-Smad2 axis. The amenability of these pathways to pharmacological approaches may offer new avenues to protect premature infants from CKD.-Mohr, J., Voggel, J., Vohlen, C., Dinger, K., Dafinger, C., Fink, G., Gobel, H., Liebau, M. C., Dotsch, J., Alejandre Alcazar, M. A. IL-6/Smad2 signaling mediates acute kidney injury and regeneration in a murine model of neonatal hyperoxia.

Acute kidney injury (AKI) is a frequent and critical complication in the clinical setting. In rodents AKI can be prevented effectively 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, in particular 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.

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.

Kidney transplantation is the preferred renal replacement therapy available. Yet, long‐term transplant survival is unsatisfactory, partially due to insufficient possibilities of longitudinal monitoring and understanding of the biological processes after transplantation. Small urinary extracellular vesicles (suEVs) – as a non‐invasive source of information – were collected from 22 living donors and recipients. Unbiased proteomic analysis revealed temporal patterns of suEV protein signature and cellular processes involved in both early response and longer‐term graft adaptation. Complement activation was among the most dynamically regulated components. This unique atlas of the suEV proteome is provided through an online repository allowing dynamic interrogation by the user. Additionally, a correlative analysis identified putative prognostic markers of future allograft function. One of these markers – phosphoenol pyruvate carboxykinase (PCK2) – could be confirmed using targeted MS in an independent validation cohort of 22 additional patients. This study sheds light on the impact of kidney transplantation on urinary extracellular vesicle content and allows the first deduction of early molecular processes in transplant biology. Beyond that our data highlight the potential of suEVs as a source of biomarkers in this setting.

Background/aim Endothelin-1 (ET-1) is overexpressed in many types of cancer, inhibiting the release of the microRNA 15a (miR-15a) and inducing the production of Mxi-2. Our aim was to identify a molecular complex regulating p53 activity in prostate cancer (PCa). Materials and methods DU145 cells were treated with ET-1, MAPK p38 inhibitor, Endothelin A receptor inhibitor (ETAR inhibitor) and Endothelin B receptor inhibitor (ETBR inhibitor). Extracts were analysed using Western Blot, immunoprecipitation and qRT-PCR. Furthermore, prostate cancer patient samples were analysed using qRT-PCR and ELISA. Results The hypothesised molecular complex was identified, with miR-15a, microRNA 1285 (miR-1285) and Mxi-2 levels up-regulated in patients in relation to increasing aggressiveness of PCa. Conclusion A complex composed of Argonaut 2 (Ago2)/Mxi-2/miR-1285 is involved in PCa. The expression of Mxi-2 correlates with increasing PCa aggressiveness and might be used as a non-invasive marker for the diagnosis and progression of PCa.

Background: Lipopolysaccharide-responsive and beige-like anchor protein (LRBA) deficiency is characterized by autoimmunity, chronic diarrhea, and immunodeficiency. Minor renal manifestations have been found in a few patients, but kidney disease has not been systematically studied and may remain underdiagnosed in this highly variable entity. Results: Our patient initially presented with pancytopenia, enteropathy, hypogammaglobulinemia, and failure to thrive at the age of 15 months. Chronic kidney disease was diagnosed at 6 years. A renal biopsy taken at 11 years of age showed interstitial nephritis. The patient progressed rapidly to end-stage renal disease (ESRD) and underwent kidney transplantation at the age of 12 years. Bronchiolitis obliterans, post-transplant lymphoproliferative disease (PTLD), and chronic rejection complicated the post-transplant management. Graft loss required reinstitution of hemodialysis within 3 years. After negative results of different targeted sequencing strategies, exome sequencing identified a homozygous nonsense mutation (p.Q1010*) in the LRBA gene more than 21 years after the patient's initial presentation. Conclusions: We report here the development of ESRD and long-term follow-up in a patient with LRBA deficiency. A molecular diagnosis in rare (kidney) disease like LRBA deficiency bears many advantages over a descriptive diagnosis. A precise diagnosis may result in improved (symptomatic) treatment and allows differentiating treatment- and procedure-related complications from manifestations of the primary 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.

Acute kidney injury (AKI) leads to significant morbidity and mortality; unfortunately, strategies to prevent or treat AKI are lacking. In recent years, several preconditioning protocols have been shown to be effective in inducing organ protection in rodent models. Here, we characterized two of these interventions—caloric restriction and hypoxic preconditioning—in a mouse model of cisplatin-induced AKI and investigated the underlying mechanisms by acquisition of multi-layered omic data (transcriptome, proteome, N-degradome) and functional parameters in the same animals. Both preconditioning protocols markedly ameliorated cisplatin-induced loss of kidney function, and caloric restriction also induced lipid synthesis. Bioinformatic analysis revealed mRNA-independent proteome alterations affecting the extracellular space, mitochondria, and transporters. Interestingly, our analyses revealed a strong dissociation of protein and RNA expression after cisplatin treatment that showed a strong correlation with the degree of damage. N-degradomic analysis revealed that most posttranscriptional changes were determined by arginine-specific proteolytic processing. This included a characteristic cisplatin-activated complement signature that was prevented by preconditioning. Amyloid and acute-phase proteins within the cortical parenchyma showed a similar response. Extensive analysis of disease-associated molecular patterns suggested that transcription-independent deposition of amyloid P-component serum protein may be a key component in the microenvironmental contribution to kidney damage. This proof-of-principle study provides new insights into the pathogenesis of cisplatin-induced AKI and the molecular mechanisms underlying organ protection by correlating phenotypic and multi-layered omics data.

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.

Ciliopathies comprise a large number of hereditary human diseases and syndromes caused by mutations resulting in dysfunction of either primary or motile cilia. Both types of cilia share a similar architecture. While primary cilia are present on most cell types, expression of motile cilia is limited to specialized tissues utilizing ciliary motility. We characterized protein complexes of ciliopathy proteins and identified the conserved AAA-ATPase Ruvbl1 as a common novel component. Here, we demonstrate that Ruvbl1 is crucial for the development and maintenance of renal tubular epithelium in mice: both constitutive and inducible deletion in tubular epithelial cells result in renal failure with tubular dilatations and fewer ciliated cells. Moreover, inducible deletion of Ruvbl1 in cells carrying motile cilia results in hydrocephalus, suggesting functional relevance in both primary and motile cilia. Cilia of Ruvbl1-negative cells lack crucial proteins, consistent with the concept of Ruvbl1-dependent cytoplasmic pre-assembly of ciliary protein complexes., 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.

Background Adenocarcinomas or combined adeno-neuroendocrine carcinomas (MANEC) of small bowel usually have a dismal prognosis with limited systemic therapy options. This is the first description of a patient showing a germline-related BRCA1 mutated MANEC of his ileum. The tumor presented a susceptibility to a combined chemotherapy and the PARP1-inhibitor olaparib. Case presentation A 74-year old male patient presented with a metastasized MANEC of his ileum. Due to clinical symptoms his ileum-tumor and the single brain metastasis were removed. We verified the same pathogenic (class 5) BRCA1 mutation in different tumor locations. There was no known personal history of a previous malignant tumor. Nevertheless we identified his BRCA1 mutation as germline-related. A systemic treatment was started including Gemcitabine followed by selective internal radiotherapy (SIRT) to treat liver metastases and in the further course Capecitabine but this treatment finally failed after 9 months and all liver metastases showed progression. The treatment failure was the reason to induce an individualized therapeutic approach using combined chemotherapy of carboplatin, paclitaxel and the Poly (ADP-ribose) polymerase- (PARP)-inhibitor olaparib analogous to the treatment protocol of Oza et al. All liver metastases demonstrated with significant tumor regression after 3 months and could be removed. In his most current follow up from December 2017 (25 months after his primary diagnosis) the patient is in a very good general condition without evidence for further metastases. Conclusion We present first evidence of a therapy susceptible germline-related BRCA1 mutation in small bowel adeno-neuroendocrine carcinoma (MANEC). Our findings offer a personalized treatment option. The germline background was unexpected in a 74-year old man with no previously known tumor burden. We should be aware of the familiar background in tumors of older patients as well.

Background Immune infiltration is implicated in the development of acquired resistance to anti-angiogenic cancer therapy. We therefore investigated the correlation between neutrophil infiltration in metastasis of colorectal cancer (CRC) patients and survival after treatment with bevacizumab. Our study identifies CD177+ tumour neutrophil infiltration as an adverse prognostic factor for bevacizumab treatment. We further demonstrate that a novel anti-VEGF/anti-Ang2 compound (BI-880) can overcome resistance to VEGF inhibition in experimental tumour models. Methods A total of 85 metastatic CRC patients were stratified into cohorts that had either received chemotherapy alone (n = 39) or combined with bevacizumab (n = 46). Tumour CD177+ neutrophil infiltration was correlated to clinical outcome. The impact of neutrophil infiltration on anti-VEGF or anti-VEGF/anti-Ang2 therapy was studied in both xenograft and syngeneic tumour models by immunohistochemistry. Results The survival of bevacizumab-treated CRC patients in the presence of CD177+ infiltrates was significantly reduced compared to patients harbouring CD177− metastases. BI-880 treatment reduced the development of hypoxia associated with bevacizumab treatment and improved vascular normalisation in xenografts. Furthermore, neutrophil depletion or BI-880 treatment restored treatment sensitivity in a syngeneic tumour model of anti-VEGF resistance. Conclusions Our findings implicate CD177 as a biomarker for bevacizumab and suggest VEGF/Ang2 inhibition as a strategy to overcome neutrophil associated resistance to anti-angiogenic treatment.

Recent human genetic studies have suggested an intriguing link between ciliary signaling defects and altered DNA damage responses in nephronophthisis (NPH) and related ciliopathies. However, the molecular mechanism and the role of altered DNA damage response in kidney degeneration and fibrosis have remained elusive. We recently identified the kinase-regulated DNA damage response target Apoptosis Antagonizing Transcription Factor (AATF) as a master regulator of the p53 response. Here, we characterized the phenotype of mice with genetic deletion of Aatf in tubular epithelial cells. Mice were born without an overt phenotype, but gradually developed progressive kidney disease. Histology was notable for severe tubular atrophy and interstitial fibrosis as well as cysts at the corticomedullary junction, hallmarks of human nephronophthisis. Aatf deficiency caused ciliary defects as well as an accumulation of DNA double strand breaks. In addition to its role as a p53 effector, we found that AATF suppressed RNA:DNA hybrid (R loop) formation, a known cause of DNA double strand breaks, and enabled DNA double strand break repair in vitro. Genome-wide transcriptomic analysis of Aatf deficient tubular epithelial cells revealed several deregulated pathways that could contribute to the nephronophthisis phenotype, including alterations in the inflammatory response and anion transport. These results suggest that AATF is a regulator of primary cilia and a modulator of the DNA damage response, connecting two pathogenetic mechanisms in nephronophthisis and related ciliopathies.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12881-017-0416-5) contains supplementary material, which is available to authorized users.

Polycystic kidney disease (PKD) is among the leading causes of end-stage renal disease. Increasing evidence exists that molecular therapeutic strategies targeted to cyst formation and growth might be more efficacious in early disease stages, highlighting the growing need for sensitive biomarkers. Here we apply quantitative magnetic resonance imaging techniques of T2 mapping and diffusion-weighted imaging in the jck mouse model for PKD using a clinical 3.0 T scanner. We tested whether kidney T2 values and the apparent diffusion coefficient (ADC) are superior to anatomical imaging parameters in the detection of early cystogenesis, as shown on macro- and histopathology. We also tested whether kidney T2 values and ADC have the potential to monitor early treatment effects of therapy with the V2 receptor antagonist Mozavaptane. Kidney T2 values and to a lesser degree ADC were found to be highly sensitive markers of early cystogenesis and superior to anatomical-based imaging parameters. Furthermore, kidney T2 values exhibited a nearly perfect correlation to the histological cystic index, allowing a clear separation of the two mouse genotypes. Additionally, kidney T2 values and ADC were able to monitor early treatment effects in the jck mouse model in a proof-of-principle experiment. Thus, given the superiority of kidney T2 values and ADC over anatomical-based imaging in mice, further studies are needed to evaluate the translational impact of these techniques in patients with PKD.

Podocytes are terminally differentiated cells of the kidney filtration barrier. They are subjected to physiological filtration pressure and considerable mechanical strain, which can be further increased in various kidney diseases. When injury causes cytoskeletal reorganization and morphological alterations of these cells, the filtration barrier may become compromised and allow proteins to leak into the urine (a condition called proteinuria). Using time-resolved proteomics, we showed that podocyte injury stimulated the activity of the transcriptional coactivator YAP and the expression of YAP target genes in a rat model of glomerular disease before the development of proteinuria. Although the activities of YAP and its ortholog TAZ are activated by mechanical stress in most cell types, injury reduced YAP and TAZ activity in cultured human and mouse podocyte cell lines grown on stiff substrates. Culturing these cells on soft matrix or inhibiting stress fiber formation recapitulated the damage-induced YAP up-regulation observed in vivo, indicating a mechanotransduction-dependent mechanism of YAP activation in podocytes. YAP overexpression in cultured podocytes increased the abundance of extracellular matrix-related proteins that can contribute to fibrosis. YAP activity was increased in mouse models of diabetic nephropathy, and the YAP target CTGF was highly expressed in renal biopsies from glomerular disease patients. Although overexpression of human YAP in mice induced mild proteinuria, pharmacological inhibition of the interaction between YAP and its partner TEAD in rats ameliorated glomerular disease and reduced damage-induced mechanosignaling in the glomeruli. Thus, perturbation of YAP-dependent mechanosignaling is a potential therapeutic target for treating some glomerular diseases. 2017

We describe a consanguineous Iraqi family with Leber congenital amaurosis (LCA), Joubert syndrome (JBTS), and polycystic kidney disease (PKD). Targeted next-generation sequencing for excluding mutations in known LCA and JBTS genes, homozygosity mapping, and whole-exome sequencing identified a homozygous missense variant, c.317G>C (p.Arg106Pro), in POC1B, a gene essential for ciliogenesis, basal body, and centrosome integrity. In silico modeling suggested a requirement of p.Arg106 for the formation of the third WD40 repeat and a protein interaction interface. In human and mouse retina, POC1B localized to the basal body and centriole adjacent to the connecting cilium of photoreceptors and in synapses of the outer plexiform layer. Knockdown of Poc1b in zebrafish caused cystic kidneys and retinal degeneration with shortened and reduced photoreceptor connecting cilia, compatible with the human syndromic ciliopathy. A recent study describes homozygosity for p.Arg106ProPOC1B in a family with nonsyndromic cone-rod dystrophy. The phenotype associated with homozygous p.Arg106ProPOC1B may thus be highly variable, analogous to homozygous p.Leu710Ser in WDR19 causing either isolated retinitis pigmentosa or Jeune syndrome. Our study indicates that POC1B is required for retinal integrity, and we propose POC1B mutations as a probable cause for JBTS with severe PKD.

Mutations affecting the integrity and function of cilia have been identified in various genes over the last decade accounting for a group of diseases called ciliopathies. Ciliopathies display a broad spectrum of phenotypes ranging from mild manifestations to lethal combinations of multiple severe symptoms and most of them share cystic kidneys as a common feature. Our starting point was a consanguineous pedigree with three affected fetuses showing an early embryonic phenotype with enlarged cystic kidneys, liver and pancreas and developmental heart disease. By genome-wide linkage analysis, we mapped the disease locus to chromosome 17q11 and identified a homozygous nonsense mutation in NEK8/NPHP9 that encodes a kinase involved in ciliary dynamics and cell cycle progression. Missense mutations in NEK8/NPHP9 have been identified in juvenile cystic kidney jck mice and in patients suffering from nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. This work confirmed a complete loss of NEK8 expression in the affected fetuses due to nonsense-mediated decay. In cultured fibroblasts derived from these fetuses, the expression of prominent polycystic kidney disease genes (PKD1 and PKD2) was decreased, whereas the oncogene c-MYC was upregulated, providing potential explanations for the observed renal phenotype. We furthermore linked NEK8 with NPHP3, another NPH protein known to cause a very similar phenotype in case of null mutations. Both proteins interact and activate the Hippo effector TAZ. Taken together, our study demonstrates that NEK8 is essential for organ development and that the complete loss of NEK8 perturbs multiple signalling pathways resulting in a severe early embryonic phenotype.

MicroRNAs have emerged as essential regulators of gene expression and may play important roles in a variety of human disorders. To understand the role of microRNA-mediated gene regulation in the kidney, we deleted the microRNA-processing enzyme Dicer in developing renal tubules and parts of the ureteric bud in mice. Genetic deletion of Dicer resulted in renal failure and death of the animals at 4-6 weeks of age. Interestingly, the kidneys of microRNA-deficient animals were small due to a reduced number of nephrons and showed massive hydronephrosis due to ureteropelvic junction obstruction. This phenotype is reminiscent of congenital anomalies of the kidney and urinary tract (CAKUT), an important group of human disorders characterized by a combination of renal hypoplasia with congenital abnormalities of the urinary tract. We used metanephric kidney cultures to examine the developmental defects underlying these pathologies. Dicer knockout kidneys showed a significant reduction of tubular branching explaining renal hypoplasia. Moreover, the ureters of these kidneys showed an altered morphology and impaired motility. These functional changes went along with altered expression of smooth muscle actin implying a defect in the differentiation of ureteric smooth muscle cells. In addition, we show the polycystic kidney disease gene Pkd1 to be a target of miR-20 implying that this interaction may contribute to the molecular basis for the cystogenesis in our model. In conclusion, these data demonstrate an essential role for microRNA-dependent gene regulation in mammalian kidney development and suggest that deregulation of microRNAs may underlie CAKUT, the most important group of renal disorders in humans.

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.

Background Three pregnancies with male offspring in one family were complicated by severe polyhydramnios and prematurity. One fetus died; the other two had transient massive salt-wasting and polyuria reminiscent of antenatal Bartter's syndrome. Methods To uncover the molecular cause of this possibly X-linked disease, we performed whole-exome sequencing of DNA from two members of the index family and targeted gene analysis of other members of this family and of six additional families with affected male fetuses. We also evaluated a series of women with idiopathic polyhydramnios who were pregnant with male fetuses. We performed immunohistochemical analysis, knockdown and overexpression experiments, and protein-protein interaction studies. Results We identified a mutation in MAGED2 in each of the 13 infants in our analysis who had transient antenatal Bartter's syndrome. MAGED2 encodes melanoma-associated antigen D2 (MAGE-D2) and maps to the X chromosome. We also identified two different MAGED2 mutations in two families with idiopathic polyhydramnios. Four patients died perinatally, and 11 survived. The initial presentation was more severe than in known types of antenatal Bartter's syndrome, as reflected by an earlier onset of polyhydramnios and labor. All symptoms disappeared spontaneously during follow-up in the infants who survived. We showed that MAGE-D2 affects the expression and function of the sodium chloride cotransporters NKCC2 and NCC (key components of salt reabsorption in the distal renal tubule), possibly through adenylate cyclase and cyclic AMP signaling and a cytoplasmic heat-shock protein. Conclusions We found that MAGED2 mutations caused X-linked polyhydramnios with prematurity and a severe but transient form of antenatal Bartter's syndrome. MAGE-D2 is essential for fetal renal salt reabsorption, amniotic fluid homeostasis, and the maintenance of pregnancy. (Funded by the University of Groningen and others.).

The primary hyperoxalurias (PH) are rare, but underdiagnosed disorders where the loss of enzymatic activity in key compounds of glyoxylate metabolism results in excessive endogenous oxalate generation. Clinically, they are characterized by recurrent urolithiasis and/or nephrocalcinosis. PH type I is the most frequent and most devastating subtype often leading to early end-stage renal failure.Profound overview of clinical, diagnostic, and currently available treatment options with a focus on PH I at different stages of the disease. Discussion of future therapeutic avenues including pharmacological chaperones (small molecules rescuing protein function), gene therapy with safer adenoviral vectors, and potential application of cell-based transplantation strategies is provided.Due to lack of familiarity with PH and its heterogeneous clinical expression, diagnosis is often delayed until advanced disease is present, a condition, requiring intensive hemodialysis and timely transplantation. Achieving the most beneficial outcome largely depends on the knowledge of the clinical spectrum, early diagnosis, and initiation of treatment before renal failure ensues. A number of preconditions required for substantial improvement in the care of orphan disease like PH have now been achieved or soon will come within reach, so new treatment options can be expected in the near future.

The case of a 47-year-old patient is described with a carcinoid of the middle ear. Initial symptoms were hearing impairment, feeling of pressure and dizziness. Mastoidectomy was carried out for mastoid shadowing with space encroachment in the auditory canal. The histological examination initially revealed an atypical cholesteatoma and the subsequent immunohistochemical investigation revealed a carcinoid. A radical excavation with complete excision of the tumor and tympanoplasty was carried out. A carcinoid of the middle ear is definitely a rare finding and the primary treatment is complete surgical removal. If metastases are suspected octreotide scintigraphy has proved to be the best option in analogy to intestinal carcinoids. Radiation therapy has not proved successful but the use of the somatostatin analog octreotide, interferon-alpha or palliative chemotherapy (e.g. streptozotosine, 5-fluorouracil) for metastases are further therapy options.

Objective—To assess the possible role of proinflammatory CD28−T cells in abdominal aortic aneurysms (AAAs). Animal studies and human tissue studies suggest a role for interferon (IFN)-γ–producing T cells in the development and progression of AAAs.Methods and Results—Fluorescence-activated cells sorter analysis of peripheral blood samples and measurement of AAA size using sonography were performed in 101 AAA patients and 38 healthy controls. Peripheral percentages of CD28−T cells of the CD3+CD4+and the CD3+CD8+were enriched in AAA patients with 7.8±8.8% and 41.9±15.7% compared with healthy controls with 2.2±6.1% and 24.9±15.5%, respectively (P=0.002 andP+CD28−and CD8+CD28−T cells produced large amounts of IFN-γ and perforin. Patients with small AAAs (+CD28−T cells than those with larger AAAs (P=0.025). Immunohistological examinations revealed 39.1±17.2% CD4+CD28−and 44.0±13.8% CD8+CD28−in AAA tissue specimens with inflammatory infiltrates.Conclusions—IFN-γ– and perforin-producing CD28−T cells are present in the periphery and the vessel wall of a majority of AAAs. This observation in humans favors the concept of a T cell–mediated pathophysiology of AAAs, especially during the early development of AAAs.

Extracapillary-proliferative glomerulonephritis is a rare complication of multiple myeloma. Partial remission of kidney involvement with cyclophosphamide therapy has previously been described. We report the case of a 60-year-old male patient diagnosed with rapidly progressive glomerulonephritis associated with IgG kappa monoclonal gammopathy. His kidney biopsy revealed pauci-immune extracapillary-proliferative glomerulonephritis without cryoglobulinaemia. Treatment with the proteasome inhibitor bortezomib induced rapid clinical and histological remission of his kidney disease. The patient's renal function remained stable on bortezomib maintenance therapy. Our findings suggest that bortezomib is a promising therapeutic approach to ameliorate severe kidney damage in monoclonal gammopathy- and myeloma-associated pauci-immune extracapillary-proliferative glomerulonephritis.

Objective: Peri-operative ischemic episodes following coronary artery bypass grafting with the internal mammary artery (IMA) are thought to be due to a vasospasm of this conduit. Endothelin-1 (ET-1) is a potent vasoconstrictor by itself and increases the response to other vasoconstrictor stimuli. This study focused on the possible role of an enhanced tissue ET-1-like immunoreactivity in the perioperative reaction of the IMA in patients with diabetes or hypercholesterolemia. Methods: Specimens of the distal part of the IMA from 46 patients (mean age 58.5 years, four women, 42 men) were studied prospectively. Nine of those patients were diabetic and 26 had evidence of hypercholesterolemia. Another cohort of 20 IMA specimens was stained retrospectively; 10 of those biopsies were from patients that had experienced transient ischemic events peri-operatively in the myocardial area supplied by the IMA. The biopsies were examined histologically and immunohistochemically (rabbit polyclonal ET-1 antiserum, three-step avidin-biotin complex) with regard to their immunoreactivity to tissue ET-1. Results: An immunoreactivity to ET-1 (graded 0‐3) was present in 89% of the biopsies. The reactivity was significantly higher in patients with hypercholesterolemia (1.92 ∠ 0.74) when compared to controls (1.0 ∠ 0.63) (P = 0.04). The reactivity was also increased in patients with non-insulin-dependent diabetes mellitus (2.1 ∠ 0.79), when compared to controls (P = 0.02). Mostly transient ischemic events in the area supplied by the IMA seemed to occur more frequently when the biopsies revealed a higher immunoreactivity to ET-1. They showed an increased reactivity to ET-1 (2.27 ∠ 0.76) compared to 10 patients with an uneventful peri-operative course (1.66 ∠ 0.71) (P = 0.04). Conclusions: This study provides evidence that the internal mammary artery is not a passive conduit. Vasospasm or vasoconstriction, in particular at its distal end, may occur more frequently in patients with hypercholesterolemia or diabetes, and may lead to post-operative ischemic events. © 1998 Elsevier Science B.V. All rights reserved

Bei einem 63-jahrigen Mann mit progredienter Dyspnoe soll eine Globalherzinsuffizienz abgeklart werden. Im EKG wird ein kompletter Rechtsschenkelblock nachgewiesen. In der Herzkatheteruntersuchung kann eine koronare Makroangiopathie ausgeschlossen werden. Echokardiografisch fallen ein dilatierter rechter Ventrikel sowie eine eingeschrankte links- und rechtsventrikulare Pumpfunktion auf. Das Herz-MRT und die von rechtsventrikular durchgefuhrte Endomyokardbiopsie ergeben drei pathologische Befunde ( ▸ Abb. 1 ).

Several standard protocols for ABO-incompatible kidney transplantation use scheduled preemptive antigen-specific immunoadsorption during the postoperative period. Our center has developed a different approach. Our patients undergo antigen-specific immunoadsorption postoperatively only if their isoagglutinine titers (immunoglobulin G anti-A/B) exceed 1:8 in the first postoperative week and 1:16 in the second postoperative week. Using this strategy, 22 ABO-incompatible kidney transplantations have been performed at our center since 2004. Only 32% of these patients (7 of 22) needed to undergo postoperative immunoadsorption (mean 4.1 immunoadsorption sessions per patient). The renal outcome in patients receiving postoperative immunoadsorption treatment versus the outcome in patients without postoperative immunoadsorption remained equal at a mean follow-up of 17 months. We identified a shorter pretransplant time on dialysis, a blood type constellation of donor A1/recipient O, and high initial starting titers as predictors for the need for postoperative immunoadsorption treatment. A more detailed version of this study, with modified tables and figures, has been accepted for publication in Nephrology Dialysis Transplantation.

In mice,Anaplasma phagocytophilumcontrol is independent of phagocyte oxidase (phox), inducible NO synthase (NOS2), tumor necrosis factor (TNF), and MyD88 Toll-like receptor signaling. We show that despite evasion of these host responses, phox, NOS2, TNF, and MyD88 are activated and contribute to inflammation and hepatic injury more thanA. phagocytophilumitself.

Background We sought to determine the impact of kidney transplantation with simultaneous unilateral nephrectomy on perioperative morbidity and patient and graft survival. Methods From January 1990 to May 2004, 75 kidney transplantations with simultaneous unilateral nephrectomy (group NE+) were performed at the University of Freiburg. Of these, 49 had polycystic kidney disease. Patients of group NE+ were matched with 75 kidney transplants without nephrectomy (group NE-). Immunosuppressive maintenance therapy in both groups was based on cyclosporine A, mycophenolate mofetil or azathioprine, and prednisone. Mean follow up was 4.1 yrs (range 0.3-11.7 yrs). Results Patient survival rate at 1 and 5 yrs was 95% and 84% versus 95% and 93% in group NE+ and NE-, resp. (P=0.56). Accordingly, kidney function rate was 85% and 74% in group NE+ versus 89% and 79% in group NE- (P=0.89). Perioperative (90 days) mortality rate in group NE+ was 1.3% and 2.7% in group NE- (P=0.56). Perioperative surgical complications were similar in both groups. Conclusions Kidney transplantation with concomitant unilateral nephrectomy has no negative impact on patient or graft survival and is associated with a reasonable morbidity rate.

Claudia Heilmann · Friedhelm Beyersdorf Abteilung Herzund Gefaschirurgie Herz-Kreislauf Zentrum Freiburg Hugstetterstr. 55 79106 Freiburg, Germany Sirs: Congenital cardiac ventricular diverticula or aneurysms are very uncommon. They were reported in only 0.076% of over 13000 congenital heart operations and usually occur in the left ventricle [1–7]. Right ventricular diverticula, even more seldom, are frequently associated with other congenital anomalies, including intracardiac, midline thoracic, diaphragmatic, and abdominal wall defects [8–10], which was first described in detail by Cantrell et al. in 1958 [11]. Only rarely is no associated cardiac anomaly present [10]. Most congenital ventricular diverticula have been observed in newborns and children, but they affect all age groups [2, 4].

Introduction Growth factor therapy provides a therapeutic alternative for “no option” patients with coronary disease. Fibroblast Growth Factor-2 (FGF-2) predominantly stimulates angiogenesis, the growth of new capillaries, whereas Monocyte Chemoattractant Protein-1 (MCP-1) is considered an arteriogenic agent. We hypothesised a synergetic effect of FGF-2 and MCP-1 in ischemic myocardium. Methods A severe coronary stenosis was created in pigs. After one week, chronic ischemia was confirmed by angiography, echocardiography, reduced ejection fraction, and increase of marker enzymes. FGF-2, MCP-1, both, or vector only were then injected intramyocardially as plasmid DNA in the impaired area. Regional contractility and number of capillaries and arterial vessels were evaluated after three months. Results FGF-2, FGF-2 + MCP-1, and vector, but not MCP-1 alone improved regional contractility at rest, whereas only FGF-2 alone ameliorated function under stress conditions. Angiogenesis in the ischemic area was stimulated by FGF-2 compared to MCP-1. In contrast, MCP-1 induced arteriogenesis relative to FGF-2. Conclusion Differences for vessel growth and regional function were apparent between FGF-2 and MCP-1. This contrast could allow the speculation that development of a flow reserve in chronically ischemic myocardium is linked to angiogenesis rather than to arteriogenesis. No additional benefits were seen following combined therapy.

Objective: Low-flow ischemia results in glucose transporter translocation and in increased glucose uptake. After total ischemia in rat heart, we found no increase in glucose uptake. Here we test the hypothesis that total ischemia is associated with decreased activation of GLUT4 despite translocation. Methods: Isolated working hearts (n=70, Sprague–Dawley rats) were perfused for 70 min at physiological workload with Krebs–Henseleit buffer containing [2-3H]glucose (5 mmol/l, 0.05 μCi/ml) with either oleate (0.4 mmol/l, 1%BSA) or pyruvate (5 mmol/l, 1%BSA). After 20 min, hearts were subjected to 15 min of total ischemia followed by 35 min of reperfusion. We measured glucose uptake and intracellular free glucose (IFG) using [2-3H]glucose and [14C]sucrose, and determined the distribution of GLUT4 by colocalization immunofluorescence with Na–K ATP-ase. Results: Cardiac power was 10.1 ± 0.90 mW before ischemia and did not differ between groups. Recovery was the same in both groups (55.7 ± 24.8$%). Glucose uptake did not differ between groups before ischemia, and did not increase during reperfusion. Despite evidence of GLUT4 translocation after reperfusion in both groups, IFG did not increase compared with before ischemia. Conclusion: We conclude that there is a discrepancy between glucose transporter availability and glucose uptake after ischemia, which may be due to inhibition of GLUT4 in the plasma membrane. (Mol Cell Biochem 278: 129–137, 2005)

Background Although AKI lacks effective therapeutic approaches, preventive strategies using preconditioning protocols, including caloric restriction and hypoxic preconditioning, have been shown to prevent injury in animal models. A better understanding of the molecular mechanisms that underlie the enhanced resistance to AKI conferred by such approaches is needed to facilitate clinical use. We hypothesized that these preconditioning strategies use similar pathways to augment cellular stress resistance. Methods To identify genes and pathways shared by caloric restriction and hypoxic preconditioning, we used RNA-sequencing transcriptome profiling to compare the transcriptional response with both modes of preconditioning in mice before and after renal ischemia-reperfusion injury. Results The gene expression signatures induced by both preconditioning strategies involve distinct common genes and pathways that overlap significantly with the transcriptional changes observed after ischemia-reperfusion injury. These changes primarily affect oxidation-reduction processes and have a major effect on mitochondrial processes. We found that 16 of the genes differentially regulated by both modes of preconditioning were strongly correlated with clinical outcome; most of these genes had not previously been directly linked to AKI. Conclusions This comparative analysis of the gene expression signatures in preconditioning strategies shows overlapping patterns in caloric restriction and hypoxic preconditioning, pointing toward common molecular mechanisms. Our analysis identified a limited set of target genes not previously known to be associated with AKI; further study of their potential to provide the basis for novel preventive strategies is warranted. To allow for optimal interactive usability of the data by the kidney research community, we provide an online interface for user-defined interrogation of the gene expression datasets (http://shiny.cecad.uni-koeln.de:3838/IRaP/).

Meeting abstract Genetic cystic kidney diseases, including autosomal recessive polycystic kidney disease (ARPKD), are among the most common causes of end stage renal disease in children and in adolescents. While the detailed biological events resulting in cyst formation remain incompletely understood, nowadays there is wide agreement that dysfunction of a specialized cellular organelle, the primary cilium, underlies genetic cystic kidney disease. In addition to cystic kidney disease, phenotypes resulting from impaired ciliary function can affect nearly every organ of the human body and are nowadays termed ciliopathies. We recently identified the AAA ATPase RUVBL1 as part of various ciliopathy-associated renal protein complexes and could show that loss of Ruvbl1 in the renal tubule leads to a severe ARPKD-like cystic kidney phenotype in mice. RUVBL1 has previously been linked to regulation of cilia-associated signaling pathways. To obtain novel insights into the cellular function of RUVBL1 and its partner protein RUVBL2, we generated stable murine cell lines with single genomic integration of coding sequences for fluorescence-tagged Ruvbl1 and Ruvbl2. Out of these cell lines we performed protein interaction screens by repetitive and independent immunoprecipitations followed by quantitative mass spectrometry. Along with multiple known interaction partners we identified novel candidates including proteins that had already been linked to ciliary function. In addition to the important role of RUVBL1 in the renal tubule our data point to a more general function of the RUVBL proteins and the RUVBLs-containing chaperone-like R2TP protein complex for ciliary function. Novel murine in vivo data strongly supports this concept. In summary, we obtained novel functional insights into the cell biological link of the AAA ATPases RUVBL1 and RUVBL2 to primary cilia. Our data suggest a role of the RUVBL proteins in the cytosolic pre-assembly of ciliary protein complexes.