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In light of the organ shortage, there is a great responsibility to assess postmortal organs for which procurement has been consented and to increase the life span of transplanted organs. The former responsibility has moved many centers to accept extended criteria organs. The latter responsibility requires an exact diagnosis and, if possible, omission of the harmful influence on the transplant. We report the course of a kidney transplant that showed a steady decline of function over a decade, displaying numerous cysts of different sizes. Clinical workup excluded the most frequent causes of chronic transplant failure. The filed allocation documents mentioned the donor’s disease of oral‐facial‐digital syndrome, a rare ciliopathy, which can also affect the kidney. Molecular diagnosis was performed by culturing donor tubular cells from the recipient´s urine more than 10 years after transplantation. Next‐generation panel sequencing with DNA from tubular urinary cells revealed a novel truncating mutation in OFD1, which sufficiently explains the features of the kidney transplants, also found in the second kidney allograft. Despite this severe donor disease, lifesaving transplantation with good long‐term outcome was enabled for 5 recipients.

PurposeKidney transplantation (KT) is the treatment of choice for end-stage chronic kidney disease (CKD) and is well known to improve the clinical outcome of patients. However, the impact of KT on comorbid psychological symptoms, particularly depression and anxiety, is less clear, and recipients of living-donor (LD) organs may have a different psychological outcome from recipients of dead-donor (DD) organs.Design/methodology/approachIn total, 152 patients were included and analyzed using a cross-sectional design. Of these patients, 25 were pre-KT, 13 were post-KT with a LD transplant and 114 were post-KT with a DD transplant. The patients were tested for a variety of psychometric outcomes using the Hospital Anxiety and Depression Scale, the 12-Item Short Form Health Survey (assessing physical and mental health-related quality of life), the Resilience Scale, the Coping Self-Questionnaire and the Social Support Questionnaire.FindingsThe mean age of the patients was 51.25 years and 40 per cent of the patients were female. As expected, the post-KT patients had significantly better scores on the physical component of the Short Form Health Survey than the pre-KT patients, and there were no significant differences between the two post-KT groups. There were no significant differences among the groups in any of the other psychometric outcome parameters tested, including anxiety, depression and the mental component of health-related quality of life.Research limitations/implicationsKT and the origin of the donor organ do not appear to have a significant impact on the psychological well-being of transplant patients with CKD. Although the diagnosis and early treatment of psychological symptoms, such as depression and anxiety, remain important for these patients, decisions regarding KT, including the mode of transplantation, should not be fundamentally influenced by concerns about psychological impairments at the population level.Originality/valueCKD is a serious condition involving profound impairment of the physical and psychological well-being of patients. KT is considered the treatment of choice for most of these patients. KT has notable advantages over dialysis with regard to the long-term physical functioning of the renal and cardiovascular system and increases the life expectancy of patients. However, the data on the improvement of psychological impairments after KT are less conclusive.

PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2. At the cellular level, PIK3C2A is critical for the formation of cilia and for receptor mediated endocytosis, among other biological functions. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. In particular, the considerable phenotypic overlap, yet distinct features, between this syndrome and Lowe’s syndrome, which is caused by mutations in the PI-5-phosphatase OCRL, highlight the key role of PI metabolizing enzymes in specific developmental processes and demonstrate the unique non-redundant functions of each enzyme. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease., Author summary Identifying the genetic basis of rare disorders can provide insight into gene function, susceptibility to disease, guide the development of new therapeutics, improve opportunities for genetic counseling, and help clinicians evaluate and potentially treat complicated clinical presentations. However, it is estimated that the genetic basis of approximately one-half of all rare genetic disorders remains unknown. We describe one such rare disorder based on genetic and clinical evaluations of individuals from 3 unrelated consanguineous families with a similar constellation of features including short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations including stroke, among other findings. We discovered that these features were due to deficiency of the PIK3C2A enzyme. PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of the lipids phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2 that are essential for a variety of cellular processes including cilia formation and vesicle trafficking. This syndrome is the first monogenic disorder caused by mutations in a class II PI3K family member and thus sheds new light on their role in human development.

An ill-defined number of patients with chronic kidney disease (CKD) receive an incorrect diagnosis. Numerous diseases are rather unspecific in terms of clinical appearance and histological characteristics, particularly hereditary kidney diseases. Autosomal dominant tubulointerstitial kidney diseases (ADTKD) can be seen as a paradigm of the diagnostic challenge, where only molecular genetics can assure the diagnosis. We report a young lady with CKD who historically received a diagnosis of “biopsy-proven” IgA nephropathy (IgAN). She received 8 months of oral steroids, with side effects but further renal deterioration. Despite a positive family history with CKD over at least three generations, this clue was not considered. We established a doubtless diagnosis of MUC1-associated ADTKD by a heterozygous frameshift-causing mutation by SNaPshot minisequencing in our index patient and her affected father. Furthermore, the mucin 1 frameshift protein was readily detectable in the 10 year old kidney biopsy. We hypothesize that similar diagnostic failures are frequent in the current routine. Interestingly, the misdiagnosis of IgAN may be an underestimated problem since a substantial fraction of the healthy population may show mesangial proliferation and IgA deposition. The correct diagnosis of hereditary diseases has several clinical implications and should be the ultimate aim of precision nephrology.

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

Basierend auf neuen pathophysiologischen Erkenntnissen der vorausgehenden Jahre, erfolgte im Jahr 2010 die Einfuhrung einer neuen Klassifikation einer Glomerulonephritis mit dominanten oder kodominanten C3-Ablagerungen, deren wesentliche Untergruppe die C3-Glomerulopathie (C3G) darstellt. Wahrend sekundare Ursachen zu einer immunkomplexvermittelten membranoproliferativen Glomerulonephritis (MPGN) fuhren, liegen die Hauptursachen der C3-Glomerulopathie primar in Storungen des Komplementsystems, die dann haufig in Form einer MPGN, aber auch anderer lichtmikroskopischer Erscheinungsbilder apparent werden. Die Kenntnis der zugrunde liegenden Pathophysiologie ist fur die weiterfuhrende Diagnostik zur Abklarung sekundarer Ursachen wegweisend. Eine umfassende Komplementanalytik, begleitet von Antikorperscreening und humangenetischer Analyse, sollte konsequent erfolgen. Wenn auch bis heute durch Studien nicht systematisch validiert, bieten die vorliegenden Erkenntnisse doch eine robuste Grundlage fur die Anwendung verfugbarer Therapieansatze fur diese haufig rasch progredienten und nach Nierentransplantation oftmals wiederkehrenden Krankheitsbilder. In der vorliegenden Ubersicht werden die derzeitigen Erkenntnisse systematisch dargestellt und in Form eines (nationalen) Expertenkonsensus zur Diagnostik der C3-dominanten Glomerulonephritis und der MPGN zusammengefasst.

In der Nachsorge nierentransplantierter Patienten kommt der nephropathologischen Begutachtung der Transplantatnierenbiopsie eine wesentliche Bedeutung zu. Die kontinuierliche Uberarbeitung der diagnostischen Kriterien der Transplantatabstosung durch die sog. BANFF-Konferenzen seit 1997 hat einen wesentlichen Anteil zur Systematisierung und damit zur Diagnosesicherheit beigetragen. Die letzte Uberarbeitung 2013 mit dem Einschluss der C4d-negativen antikorpervermittelten Abstosung schliest hier eine diagnostische Lucke, hat aber auch dazu beigetragen, dass mehr antikorpervermittelte Abstosungen diagnostiziert werden. Die therapeutischen Optionen bei zellularen und antikorpervermittelten Abstosungen sind multimodal, basieren jedoch auf geringer Evidenz. Zellulare Rejektionen und akute/aktive humorale Rejektionen sind in der Regel dennoch gut behandelbar. Vor allem die chronisch, aktive humorale Rejektion stellt den Kliniker weiterhin vor grose Herausforderungen, da viele der Therapieoptionen eingreifend und teuer sind und potenziell zu schweren Infektkomplikationen fuhren konnen. Der Artikel gibt eine Ubersicht uber die pathologischen Veranderungen entsprechend der BANFF-Klassifikation und erortert spezifisch zu den unterschiedlichen Rejektionsformen die therapeutischen Optionen.

Background/Aims: Hypercalcemia can result in nephrocalcinosis/nephrolithiasis and may lead to renal failure. Idiopathic infantile hypercalcemia is caused by mutations of the CYP24A1 gene, which regulates vitamin D activity. Classically infants present with hypercalcemia. Recently, a number of individuals have been reported with late onset clinical manifestation or late diagnosis in adulthood. All these patients are believed to show hypercalciuria. Methods: We report a 24 year old patient of healthy consanguine parents. Genetic analysis was performed by Sanger sequencing of the CYP24A1 gene in the index patient and targeted exon 2 analysis of all other family members. Results: The patient was hospitalized with severe malaise during an acute EBV-infection. He showed hypercalcemia > 3mmol/l and acute, hypovolemic renal failure with profound nephrocalcinosis, but no hypercalciuria. Genetic workup revealed a homozygous loss-of-function mutation p.E143del in the CYP24A1 gene. His clinically asymptomatic brother showed nephrocalcinosis of lesser degree. Repeatedly, low parathyroid hormone levels were detected in both brothers. Conclusion: This family displays the highly variable phenotype of CYP24A1 biallelic mutation carriers. CYP24A1 associated disease is an important differential diagnosis for the workup and counseling of infants as well as adults with hypercalcemia since a proper genetic diagnosis may result in therapeutic consequences.

A health economic analysis was undertaken based on the one-year database from a randomized study of rabbit anti-human thymocyte immunoglobulin (rATG) versus basiliximab, in kidney transplantation using resource utilization data and cost estimates from three German hospitals. A three-state Markov model was applied to estimate cost effectiveness to 10 years post-transplant. Total mean treatment cost per patient to year 1 post-transplant was €62,075 versus €59,767 for rATG versus basiliximab (p

Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor. Here we demonstrate a critical role for the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored oxygen-dependent instability. pVHL and HIF alpha-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF alpha-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumours. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing.

Hypoxia-inducible transcription factors (HIF) protect cells against oxygen deprivation, and HIF stabilization before ischemia mitigates tissue injury. Because ischemic acute kidney injury (AKI) often involves the thick ascending limb (TAL), modulation of HIF in this segment may be protective. Here, we generated mice with targeted TAL deletion of the von Hippel-Lindau protein (Vhl), which mediates HIF degradation under normoxia, using Tamm-Horsfall protein (Thp)-driven Cre expression. These mice showed strong expression of HIF-1α in TALs but no changes in kidney morphology or function under control conditions. Deficiency of Vhl in the TAL markedly attenuated proximal tubular injury and preserved TAL function following ischemia-reperfusion, which may be partially a result of enhanced expression of glycolytic enzymes and lactate metabolism. These results highlight the importance of the thick ascending limb in the pathogenesis of AKI and suggest that pharmacologically targeting the HIF system may have potential to prevent and mitigate AKI.

The reasons for inadequate production of erythropoietin (EPO) in patients with ESRD are poorly understood. A better understanding of EPO regulation, namely oxygen-dependent hydroxylation of the hypoxia-inducible transcription factor (HIF), may enable targeted pharmacological intervention. Here, we tested the ability of fibrotic kidneys and extrarenal tissues to produce EPO. In this phase 1 study, we used an orally active prolyl-hydroxylase inhibitor, FG-2216, to stabilize HIF independent of oxygen availability in 12 hemodialysis (HD) patients, six of whom were anephric, and in six healthy volunteers. FG-2216 increased plasma EPO levels 30.8-fold in HD patients with kidneys, 14.5-fold in anephric HD patients, and 12.7-fold in healthy volunteers. These data demonstrate that pharmacologic manipulation of the HIF system can stimulate endogenous EPO production. Furthermore, the data indicate that deranged oxygen sensing--not a loss of EPO production capacity--causes renal anemia.

The growth and differentiation of renal tubular epithelial cells is normally tightly controlled. Disturbances can lead to the development of renal cysts or renal cell carcinomas, clinically relevant disease entities, which have so far been considered as being caused by entirely distinct mechanisms. Clear cell renal carcinoma, the most frequent type of renal cancer is associated with inactivation of the von Hippel Lindau (VHL) protein. Genetic defects leading to cystic kidney disease usually affect proteins that play a role in structure or function of primary cilia of renal epithelial cells. Accumulating evidence suggests that the VHL protein also controls cilia function and that its inactivation may result in both malignant or nonmalignant growth of epithelial cells and that this effect is in part mediated through the accumulation of hypoxia-inducible factors. Unraveling the complex role of VHL in renal epithelial cells is likely to shed further insight into mechanism of epithelial growth control, epithelial-mesenchymal transformation, and tumor development.

Background. Hypoxia plays an important role in kidney injury. By the stabilization of the transcription factor HIF-1, hypoxia affects gene expression also in tubular epithelial cells. Increased expression of connective tissue growth factor (CTGF) is observed in different kidney diseases and is associated with deteriorating renal function. Therefore, we hypothesized that the expression of CTGF might be modulated under hypoxic conditions. Methods. The human proximal tubular epithelial cell lines HK-2 and HKC-8 were treated with reduced oxygen tension (1% O2) or the hypoxia mimetic dimethyloxalyl glycine (DMOG). CTGF was analysed by Western blotting, real-time RT-PCR and luciferase gene expression assays. Results. Exposure of HK-2 or HKC-8 cells to hypoxia or treatment with DMOG for up to 24 h reduced cellular as well as secreted CTGF protein synthesis. Downregulation was also detectable at the mRNA level and was confirmed by reporter gene assays. Hypoxic repression of CTGF synthesis was dependent on HIF-1, as shown by HIF-1α knockdown by siRNA. Furthermore, exposure to hypoxia reduced CTGF synthesis in response to TGF-β. A negative correlation between HIF-1α accumulation and CTGF synthesis was also observed in renal cell carcinoma cells (RCC4 and RCC10). Reexpression of von HippelLindau protein reduced HIF-1α and increased CTGF synthesis. Conclusions. We provide evidence that hypoxia inhibits CTGF synthesis in human proximal tubular epithelial cells, involving HIF-1α. Under hypoxic conditions, induction of CTGF by TGF-β was repressed. The reduced synthesis of the profibrotic factor CTGF may contribute to a potential protective effect of hypoxic preconditioning in acute renal injury.

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

In kidney transplant recipients with chronic graft dysfunction, long-term immunosuppression with calcineurin inhibitors (CNIs) or mTOR inhibitors (mTORi) can be challenging due to adverse effects, such as nephrotoxicity and proteinuria. Seventy-nine kidney transplant recipients treated with CNI-based or mTORi-based maintenance immunosuppression who had CNI-induced nephrotoxicity or severe adverse events were switched to belatacept. Mean time from transplantation to belatacept conversion was 69.0 months. Mean estimated glomerular filtration rate (eGFR) ± standard deviation at baseline was 26.1 ± 15.0 ml/min/1.73 m2 , increasing to 34.0 ± 15.2 ml/min/1.73 m2 at 12 months postconversion (P 500 mg/l) at conversion. The Kaplan-Meier estimates for patient and graft survival at 12 months were 95.0% and 85.6%, respectively. The discontinuation rate due to adverse events was 7.9%. One case of post-transplant lymphoproliferative disorder occurred at 17 months postconversion. For comparison, a historical control group of 41 patients converted to mTORi-based immunosuppression because of biopsy-confirmed CNI-induced toxicity was examined; eGFR increased from 27.6 ± 7.2 ml/min/1.73 m2 at baseline to 31.1 ± 11.9 ml/min/1.73 m2 at 12 months (P = 0.018). Belatacept-based immunosuppression may be an alternative regimen for kidney transplant recipients with CNI- or mTORi-induced toxicity.

Activation of hypoxia-inducible transcription factor (HIF) has been identified as an important mechanism of cellular adaptation to low oxygen. Normoxic degradation of HIF is mediated by oxygen-dependent hydroxylation of specific prolyl residues of the regulative alpha-subunits by HIF prolyl hydroxylases (PHD). It was hypothesized that inhibition of HIF degradation by either hypoxia or pharmacologic inhibition of PHD would confer protection against subsequent ischemic injury. For testing this hypothesis ischemic acute renal failure was induced in rats by 40 min of clamping of the left renal artery after right-sided nephrectomy. Before surgery, pretreatment with either carbon monoxide, leading to tissue hypoxia, or the novel PHD inhibitor FG-4487 was applied. No toxic effects of FG-4487 were observed. Both pretreatments strongly induced the accumulation of HIF-1alpha and HIF-2alpha in tubular and peritubular cells, respectively, as well as HIF target gene expression. The course of subsequent ischemic injury was significantly ameliorated by both strategies of preconditioning, as evident from a significant improvement of serum creatinine and serum urea after 24 and 72 h. Furthermore, tissue injury and apoptosis were less severe, which were quantified by application of a standardized histologic scoring system in a blinded manner. In conclusion, the data provide proof of principle that preconditional activation of the HIF system protects against ischemic injury. Inhibiting the activity of HIF hydroxylases therefore seems to have considerable clinical perspectives.

Despite a high overall oxygen supply, the tissue oxygen tensions in the kidney are comparatively low and render the kidneys prone to hypoxic injury. However, the role of hypoxia in the pathogenesis of different types of renal disease remains incompletely understood. The importance of hypoxic cell injury is most obvious in renal vascular disease, in which occlusion of the renal artery or one of its branches can induce tissue necrosis. In acute renal failure, circumstantial evidence suggests that hypoxic injury to the renal medulla plays a significant role. In addition, chronically impaired oxygenation may also be an important factor in the progression of chronic renal disease. Destruction of the glomerular capillaries leads to hypoperfusion of the peritubular interstitium. Moreover, in focal disease, a compensatory increase in perfusion of other glomeruli may increase flow and pressure in peritubular capillaries derived from their vasa efferentia which could be a cause of microvascular injury. The interstitial capillary density is reduced in chronic renal disease, and results of animal experiments suggest that this is due to an imbalance in the expression of pro- and antiangiogenic factors. Besides its essential role in energy generation, oxygen is increasingly recognized as an important regulator of cellular functions. Hypoxia induces specific genes through increased expression of hypoxia-inducible transcription factors (HIF). Different HIF isoforms have recently been shown to be inducible in glomerular, tubular, and interstitial cells of the kidney. While the majority of HIF-dependent genes confer protection against hypoxia, hypoxia-inducible gene expression has been suggested to contribute also to increased interstitial matrix deposition.

Oxygen tensions in the kidney are heterogeneous, and their changes presumably play an important role in renal physiologic and pathophysiologic processes. A family of hypoxia-inducible transcription factors (HIF) have been identified as mediators of transcriptional responses to hypoxia, which include the regulation of erythropoietin, metabolic adaptation, vascular tone, and neoangiogenesis. In vitro, the oxygen-regulated subunits HIF-1alpha and -2alpha are expressed in inverse relationship to oxygen tensions in every cell line investigated to date. The characteristics and functional significance of the HIF response in vivo are largely unknown. High-amplification immunohistochemical analyses were used to study the expression of HIF-1alpha and -2alpha in kidneys of rats exposed to systemic hypoxia bleeding anemia, functional anemia (0.1% carbon monoxide), renal ischemia, or cobaltous chloride (which is known to mimic hypoxia). These treatments led to marked nuclear accumulation of HIF-1alpha and -2alpha in different renal cell populations. HIF-1alpha was mainly induced in tubular cells, including proximal segments with exposure to anemia/carbon monoxide, in distal segments with cobaltous chloride treatment, and in connecting tubules and collecting ducts with all stimuli. Staining for HIF-1alpha colocalized with inducible expression of the target genes heme oxygenase-1 and glucose transporter-1. HIF-2alpha was not expressed in tubular cells but was expressed in endothelial cells of a small subset of glomeruli and in peritubular endothelial cells and fibroblasts. The kidney demonstrates a marked potential for upregulation of HIF, but accumulation of HIF-1alpha and HIF-2alpha is selective with respect to cell type, kidney zone, and experimental conditions, with the expression patterns partly matching known oxygen profiles. The expression of HIF-2alpha in peritubular fibroblasts suggests a role in erythropoietin regulation.

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

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

Background/Aims: Crucial steps in the initiation of lupus nephritis are the deposition of (auto-)antibodies and consequent complement activation. In spite of aggressive treatment patients may develop terminal renal failure. Therefore, new treatment strategies are needed. In extension to our previously published data we here analyzed the potential renoprotective mechanisms of bortezomib (BZ) in experimental lupus nephritis by focusing on morphological changes. Methods: Female NZB×NZW F1 mice develop lupus-like disease with extensive nephritis that finally leads to lethal renal failure. Treatment with 0.75 mg/kg BZ i.v. or placebo (PBS) twice per week started at 18 or 24 weeks of age. Antibody production was measured with ELISA and kidney damage was determined by quantitative morphological and immunohistochemical methods. Results: BZ treatment completely inhibited antibody production in both BZ-treated groups and prevented the development of nephritis in comparison to PBS-treated animals. Glomerular and tubulointerstitial damage scores, collagen IV expression, mean glomerular volume as well as tubulointerstitial proliferation and apoptosis were significantly lower after BZ treatment. Glomerular ultrastructure and in particular podocyte damage and loss were prevented by BZ treatment. Conclusions: BZ effectively prevents the development of nephritis in the NZB/W F1 mouse model. Specific protection of podocyte ultrastructure may critically contribute to renoprotection by BZ, which may also represent a potential new treatment option in human lupus nephritis.

Cellular responses to oxygen are increasingly recognized as critical in normal development and physiology, and are implicated in pathological processes. Many of these responses are mediated by the transcription factors HIF-1 and HIF-2. Their regulation occurs through oxygen-dependent proteolysis of the alpha subunits HIF-1alpha and HIF-2alpha, respectively. Both are stabilized in cell lines exposed to hypoxia, and recently HIF-1alpha was reported to be widely expressed in vivo. In contrast, regulation and sites of HIF-2alpha expression in vivo are unknown, although a specific role in endothelium was suggested. We therefore analyzed HIF-2alpha expression in control and hypoxic rats. Although HIF-2alpha was not detectable under baseline conditions, marked hypoxic induction occurred in all organs investigated, including brain, heart, lung, kidney, liver, pancreas, and intestine. Time course and amplitude of induction varied between organs. Immunohistochemistry revealed nuclear accumulation in distinct cell populations of each tissue, which were exclusively non-parenchymal in some organs (kidney, pancreas, and brain), predominantly parenchymal in others (liver and intestine) or equally distributed (myocardium). These data indicate that HIF-2 plays an important role in the transcriptional response to hypoxia in vivo, which is not confined to the vasculature and is complementary to rather than redundant with HIF-1.

The contribution of hypoxia to cisplatin-induced renal tubular injury is controversial. Because the hypoxia-inducible factor (HIF) pathway is a master regulator of adaptation to hypoxia, we measured the effects of cisplatin on HIF accumulation in vitro and in vivo, and tested whether hypoxic preconditioning is protective against cisplatin-induced injury. We found that cisplatin did not stabilize HIF-1alpha protein in vitro or in vivo under normoxic conditions. However, hypoxic preconditioning of cisplatin-treated proximal tubular cells in culture reduced apoptosis in an HIF-1alpha-dependent fashion and increased cell proliferation as measured by BrdU incorporation. In vivo, rats preconditioned with carbon monoxide before cisplatin administration had significantly better renal function than rats kept in normoxic conditions throughout. Moreover, the histomorphological extent of renal damage and tubular apoptosis was reduced by the preconditional treatment. Therefore, development of pharmacologic agents to induce renal HIF might provide a new approach to ameliorate cisplatin-induced nephrotoxicity.

In polycystic kidney disease (PKD), erythropoietin (EPO) production and interstitial vascularization are increased compared with other kidney diseases. EPO and several angiogenic factors are controlled by hypoxia-inducible transcription factors (HIFs), which are composed of a constitutive beta-subunit and two alternative alpha-subunits (HIF-1alpha, HIF-2alpha). We hypothesized that cyst expansion may result in pericystic hypoxia and consecutive up-regulation of HIF and thus examined the expression of HIF-alpha and HIF target genes in human PKD and in a rodent PKD model. HIF-1alpha and HIF-2alpha were found to be up-regulated in cyst epithelium and cells of cyst walls, respectively. The distinct expression pattern of the HIF-alpha isoforms closely resembles the respective pattern in normal kidneys under systemic hypoxia. Pimonidazole staining, a marker for tissue hypoxia, confirmed the existence of regional hypoxia in polycystic kidneys. Immunohistochemistry for selected target genes implicated a role for HIF-1alpha in vascular endothelial growth factor and Glut-1 activation and HIF-2alpha in endoglin and EPO stimulation. Polycystin-deficient cells showed physiological, oxygen-dependent HIF-alpha modulation, excluding a direct influence of polycystin deficiency on HIF-alpha regulation. In conclusion, HIF accumulation in human and rat PKD seems to be responsible for increased EPO production and pericystic hypervascularity and may have an impact on progression of PKD.

Hypoxia-inducible factor (HIF) regulates expression of genes involved in adaptation to hypoxia and ischemia. Three prolyl hydroxylases (PHD1-3) underlie oxygen-regulated destruction of HIFalpha chains. We have investigated the organ distribution of the PHDs in the rat, their regulation by hypoxia and changes in local expression after experimental myocardial infarction using RNase protection assays, in situ hybridization and immunohistochemistry. mRNAs of all isoforms were detectable in heart, liver, kidney, brain, testis and lung. In normal animals, highest levels for PHD2 mRNA and PHD3 mRNA were found in myocardium, whereas PHD1 mRNA was detected predominantly in the testis. PHD1 mRNA was constitutively expressed. PHD2 mRNA was induced by hypoxia in the liver and PHD3 mRNA in liver, testis and heart. Overall our results show that PHD2 mRNA is ubiquitously expressed in normal animals, in keeping with a general role in oxygen sensing. PHD1 and 3 mRNA distributions suggest particular roles in testis and heart, respectively. In a model of myocardial infarction, in situ hybridization showed periischemic enhancement for PHD2 mRNA and PHD3 mRNA, but not PHD1 mRNA. Immunostaining of PHD2 and 3 in infarcted hearts showed enhanced protein expression, maximal 7 days after infarction. Levels were strongest in regions neighboring areas of HIF staining but also partially overlapped with these zones. Inducibility of PHD2 and 3 by hypoxia and ischemia in vivo has important implications both for the pathophysiology of conditions where oxygen supply is deranged and for attempts to manipulate the HIF system therapeutically.

Chronic hypoxia induces pulmonary vascular remodeling, leading to pulmonary hypertension, right ventricular hypertrophy, and heart failure. Heterozygous deficiency of hypoxia-inducible factor-1alpha (HIF-1alpha), which mediates the cellular response to hypoxia by increasing expression of genes involved in erythropoiesis and angiogenesis, has been previously shown to delay hypoxia-induced pulmonary hypertension. HIF-2alpha is a homologue of HIF-1alpha and is abundantly expressed in the lung, but its role in pulmonary hypertension remains unknown. Therefore, we analyzed the pulmonary response of WT and viable heterozygous HIF-2alpha-deficient (Hif2alpha(+/-)) mice after exposure to 10% O(2) for 4 weeks. In contrast to WT mice, Hif2alpha(+/-) mice were fully protected against pulmonary hypertension and right ventricular hypertrophy, unveiling a critical role of HIF-2alpha in hypoxia-induced pulmonary vascular remodeling. Pulmonary expression levels of endothelin-1 and plasma catecholamine levels were increased threefold and 12-fold respectively in WT but not in Hif2alpha(+/-) mice after hypoxia, suggesting that HIF-2alpha-mediated upregulation of these vasoconstrictors contributes to the development of hypoxic pulmonary vascular remodeling.

itself is the direct regulator of the transcriptional rate of Epo. A DNA element was identified adjacent to the coding region of the Epo gene, which is essential and sufficient to convey increased gene expression during hypoxia (hypoxia responsive element, HRE). Activation of the HRE requires binding of a specific, hypoxia-inducible transcription factor (HIF) w2x. The role of this transcriptional regulator was soon found not to be limited to the control of Epo production. In vitro, virtually all cells respond to hypoxia with an upregulation of HIF, and besides Epo many other genes have meanwhile been identified as downstream targets of HIF. Amongst others, these include genes involved in the regulation of vascular tone (NO synthase, endothelin-1, adrenomedullin), new vessel formation (vascular endothelial growth factor, VEGF; platelet derived growth factor, PDGF), catecholamine synthesis (tyrosine hydroxylase) and anaerobic glycolysis and cellular glucose uptake w3–6x. HIF, therefore, appears to be a ubiquitously expressed key regulator of adaptation to reduced oxygen supply.

Introduction Chondrocytes have to withstand considerable hypoxic conditions within the avascular articular cartilage. The present study investigated the effects of inhibiting or stabilizing hypoxia-inducible factor (HIF)-1α by 2-methoxyestradiol or dimethyloxaloylglycine on the progression of osteoarthritis in murine knee joints. Methods 2-Methoxyestradiol was injected six times over a period of 2 weeks into the left knee joint of Balb/C mice. Joints were assessed by histochemical and immunohistochemical methods, 3 weeks and 12 weeks following the first injection. Dimethyloxaloylglycine, an inhibitor of HIF-degrading prolyl-hydroxylases, was injected into the left knee joints of STR/ORT mice once a week over the entire period of 12 weeks. Right knee joints that received a saline solution served as controls. In addition, the effects of dimethyloxaloylglycine on HIF-1 target gene expression and on collagen metabolism were analyzed in vitro. Results Injection of 2-methoxyestradiol led to osteoarthritic changes in the treated knee joints of Balb/C mice. The first signs of osteophyte formation were observed in the knee joints after 3 weeks, followed by progressive destruction of the articular cartilage at 12 weeks that was not, however, accompanied by inflammatory reactions. Injection of dimethyloxaloylglycine could not prevent severe osteoarthritis that spontaneously developed in the knee joints of STR/ORT mice. In chondrocyte cultures, administration of dimethyloxaloylglycine resulted in an upregulation of Sox9 expression. Such a stimulatory effect was not observed, however, for the expression of type II collagen, which might be the indirect consequence of intracellular collagen retention observed by immunofluorescence or of increased expression of IL-1β and IL-6. Conclusions Induction of osteoarthritis by 2-methoxyestradiol demonstrates the importance of HIF-1 in maintaining the integrity of hypoxic articular cartilage. Stabilization of HIF-1 by dimethyloxaloylglycine, however, was not of therapeutic value, since this nonselective prolyl-hydroxylase inhibitor also interferes with proper collagen metabolism and induces the expression of catabolic cytokines

Cellular responses to hypoxia after renal segmental infarction. Background Hypoxia is believed to play an important role in the pathogenesis of acute and chronic kidney disease. However, the impact of low oxygen tensions on cellular functions in the kidney and potential adaptive responses are poorly understood. Methods In order to assess the effects of regional hypoxia, we induced large segmental renal infarcts in rats by renal artery branch ligation to create an oxygen gradient vertical to the corticomedullary axis and studied the effects on cell morphology, the induction of hypoxia-inducible transcription factors (HIF), the expression of HIF target genes, and cell proliferation. Results Pimonidazol protein adduct immunohistochemistry, a marker for severe tissue hypoxia, verified a continuous area of hypoxic renal tissue extending from the cortex to the papilla, in which tubular necrosis developed subsequently. Within this area local sparing of pimonidazol staining and tissue preservation was found around arcuate veins, indicating regional oxygen supply via diffusion from venous blood. HIF-1α was up-regulated within 1 hour and for up to 7 days predominantly in the border zone of the infarct in tubular cells, glomerular cells, resident interstitial cells, capillary endothelial cells, and infiltrating macrophages. HIF-2α expression was less prominent and confined to resident and infiltrating peritubular cells in the cortex. HIF expression was colocalized with regional up-regulation of the hypoxia-inducible genes heme oxygenase-1 and vascular endothelial growth factor (VEGF), and was followed by capillary and tubular proliferation. Conclusion Our findings illustrate a marked potential of renal tissue to respond to regional ischemia and initiate adaptive reactions, including angiogenesis.

Early kidney development is associated with the coordinated branching of the renal tubular and vascular system and hypoxia has been proposed to be a major regulatory factor in this process. Under low oxygen levels, the hypoxia-inducible transcription factor (HIF) regulates the expression of genes involved in angiogenesis, erythropoiesis and glycolysis. To investigate the role of HIF in kidney development, we analyzed the temporal and spatial expression of the oxygen regulated HIF-1alpha and -2alpha subunits at different stages of rat and human kidney development. Using double-staining procedures, localization of the HIF target geneproducts vascular endothelial growth factor (VEGF) and endoglin was studied in relation to HIFalpha. In both species, we found marked nuclear expression of HIF-1alpha in medullary and cortical collecting ducts and in glomerular cells. In contrast, HIF-2alpha was expressed in interstitial and peritubular cells podocytes of the more mature glomeruli. After completion of glomerulogenesis and nephrogenesis, HIF-1alpha and -2alpha were no longer detectable. The HIF-target gene VEGF colocalized with HIF-1alpha protein in glomeruli and medullary collecting ducts. HIF-2alpha colocalized with the endothelium-associated angiogenic factor, endoglin. Both HIFalpha isoforms are activated in the developing kidney in a cell-specific and temporally controlled manner, indicating a regulatory role of oxygen tension in nephrogenesis. HIF-1alpha seems to be primarily involved in tubulogenesis and HIF-2alpha in renal vasculogenesis. Both isoforms are found in glomerulogenesis, potentially having synergistic effects.

Adaptation to hypoxic environment is conferred through hypoxia-inducible transcription factors (HIFs). We have previously shown that the HIF system is transiently activated in vivo in radiocontrast-induced acute renal failure, associated with profound hypoxia in the renal medulla. Medullary thick ascending limbs (mTALs), the most affected nephron segments in this model, were virtually unable to mount an adaptive HIF response. Here, we study correlations between oxygenation, HIF activation, and cell viability in a related ex vivo model, the isolated perfused rat kidney (IPK). In IPKs perfused with cell-free oxygenated medium, severe medullary hypoxic damage developed, affecting 42+/-9% of mTALs in the mid-inner stripe. HIF-1alpha tubular immunostaining was noted with a zonal and tubular pattern largely similar to our findings in vivo: in 34+/-3% of collecting ducts (CDs) within the mid-inner stripe and extensively in the papillary tip, whereas mTALs were all HIF-negative. In IPKs supplemented with RBCs (improved oxygen supply), mTAL damage was totally prevented and CDs' HIF expression was attenuated (22+/-4%). By contrast, although measures designed to reduce medullary hypoxia by decreasing tubular reabsorptive activity (furosemide, ouabain, or high-albumin-non-filtering system) reduced mTAL damage, all paradoxically resulted in increased HIF expression in CDs (51+/-4%), and 17+/-3% of mTALs became immunostained as well. Our data confirm that CDs and mTALs have markedly different HIF responses, which correlate with their viability under hypoxic stress. mTALs transcriptional adaptation occurs within a narrow hypoxic range, and it appears that workload reduction can shift mTALs into this window of opportunity for HIF activation and survival.