Daprodustat prevents cyclosporine-A-mediated anemia and peritubular capillary loss.

Chronic Cyclosporine-A treatment is associated with serious side effects, including kidney toxicity and anemia. Although pathophysiology of Cyclosporine-A-induced kidney injury remains incompletely understood, hypoxia is likely involved. Here, we investigated the effect of the hypoxia inducible factor activator daprodustat on Cyclosporine-A -induced kidney toxicity. As Cyclosporine-A profoundly alters protein phosphorylation by inhibiting the phosphatase calcineurin, special attention was directed towards the kidney phospho-proteome. Mice received Cyclosporine-A with or without daprodustat for up to eight weeks. In kidney homogenates, 1360 selected proteins were analyzed at expression and phosphorylation levels. Of these, Cyclosporine-A changed the expression of 79 and the phosphorylation of 86 proteins. However, when Cyclosporine-A treatment was combined with daprodustat, the expression of 95 proteins and phosphorylation of only six proteins was altered suggesting that daprodustat prevented most protein phosphorylation brought about by Cyclosporine-A. Although daprodustat showed only marginal effect on its own, angiogenesis-related pathways were among the most profoundly impacted by daprodustat when given on top of Cyclosporine-A. Additionally, Cyclosporine-A lowered the blood hemoglobin concentration and caused kidney capillary rarefaction, which daprodustat prevented. Thus, combined daprodustat/Cyclosporine-A treatment prevented deleterious Cyclosporine-A effects on microcirculation and hemoglobin, and the protective action of daprodustat involves suppression of broad protein phosphorylation changes caused by Cyclosporine-A.

Pathophysiology of unilateral ischemia-reperfusion injury: importance of renal counterbalance and implications for the AKI-CKD transition.

Unilateral ischemia-reperfusion (UIR) injury leads to progressive renal atrophy and tubulointerstitial fibrosis (TIF) and is commonly used to investigate the pathogenesis of the acute kidney injury-chronic kidney disease transition. Although it is well known that contralateral nephrectomy (CNX), even 2 wk post-UIR injury, can improve recovery, the physiological mechanisms and tubular signaling pathways mediating such improved recovery remain poorly defined. Here, we examined the renal hemodynamic and tubular signaling pathways associated with UIR injury and its reversal by CNX. Male Sprague-Dawley rats underwent left UIR or sham UIR and 2 wk later CNX or sham CNX. Blood pressure, left renal blood flow (RBF), and total glomerular filtration rate were assessed in conscious rats for 3 days before and over 2 wk after CNX or sham CNX. In the presence of a contralateral uninjured kidney, left RBF was lower (P < 0.05) from 2 to 4 wk following UIR (3.6 ± 0.3 mL/min) versus sham UIR (9.6 ± 0.3 mL/min). Without CNX, extensive renal atrophy, TIF, and tubule dedifferentiation, but minimal pimonidazole and hypoxia-inducible factor-1α positivity in tubules, were present at 4 wk post-UIR injury. Conversely, CNX led (P < 0.05) to sustained increases in left RBF (6.2 ± 0.6 mL/min) that preceded the increases in glomerular filtration rate. The CNX-induced improvement in renal function was associated with renal hypertrophy, more redifferentiated tubules, less TIF, and robust pimonidazole and hypoxia-inducible factor-1α staining in UIR injured kidneys. Thus, contrary to expectations, indexes of hypoxia are not observed with the extensive TIF at 4 wk post-UIR injury in the absence of CNX but are rather associated with the improved recovery of renal function and structure following CNX.

Episodic Hypoxia Promotes Defence Against Cellular Stress.

BACKGROUND/AIMS: Recently, we have demonstrated that episodic hypoxia occurs in kidneys of mice challenged repetitively with the immunosuppressant cyclosporine A (CsA), in analogy to humans on CsA treatment. However, the molecular consequences of episodic hypoxia remain poorly defined, as is its impact on cell survival. Here, we systematically study cell response to episodic, as compared to single course hypoxia. METHODS: In vivo, kidneys of mice challenged daily with CsA for one week were analyzed by microarray analysis, gene ontology analysis, and qPCR. In vitro, renal cells were subjected to hypoxia (1 % O2) which was either episodic (4 h for 6 consecutive days), short-term (4 h), or sustained (24 h). Western blot analysis quantified hypoxia-inducible factor-1α (HIF-1α). 2',7'-dichlorofluorescein diacetate detected intracellular ROS. After re-oxygenation, staurosporine served to induce apoptosis, quantified by active caspase-3. RESULTS: In vivo, HIF target gene expression was suppressed by daily CsA treatment. Yet, we found up-regulation of genes involved in defence against cellular stress, notably against ROS. Renal cells in vitro behaved largely different under episodic and sustained hypoxia, while their response to short-term hypoxia oscillated between the previous two. Episodic hypoxia exhibited the highest total HIF-1α protein level, lowest nucleus-to-cytoplasm ratio, and lowest HIF target gene expression. When compared with normoxia, re-oxygenation after sustained hypoxia increased ROS by 3.04 ± 1.04 fold (p<0.001), and re-oxygenation after episodic hypoxia by 1.26 ± 0.16 fold (p<0.01). Staurosporine-induced active caspase-3 was highest after sustained, and lowest after episodic hypoxia. CONCLUSION: In vitro episodic hypoxia mimics the largely HIF-independent transcriptome observed after repetitive CsA treatment in vivo. In vitro preconditioning with episodic hypoxia protects against stress-induced apoptosis. Despite of its long-term adverse effects, CsA derived episodic hypoxia induces a unique renal hypoxia response that provides adaptation to re-oxygenation mediated ROS damage.

Urinary Calprotectin loses specificity as tumour marker due to sterile leukocyturia associated with bladder cancer.

BACKGROUND: Urinary Calprotectin, a mediator of the innate immune system, has been identified as a biomarker in bladder cancer. Our aim was to investigate the association between sterile leukocyturia and urinary Calprotectin in low-grade and high-grade bladder cancer. MATERIALS AND METHODS: We performed a prospective cross-sectional study including 52 patients with bladder cancer and 40 healthy controls. Definition of sterile leukocyturia was > 5.0 leukocytes per visual field in absence of bacteriuria. RESULTS: The rate of sterile leukocyturia in low-grade (60.0%) and high-grade (62.0%) bladder cancer was comparable (p = 0.87). However, the median absolute urinary leukocyte count in patients with sterile leukocyturia was significantly higher in high-grade than in low-grade bladder cancer (p < 0.01). Spearman correlation revealed a significant correlation between urinary Calprotectin and leucocyte concentration (R = 0.4, p < 0.001). Median urinary Calprotectin concentration was 4.5 times higher in bladder cancer patients with than in patients without sterile leukocyturia (p = 0.03). Subgroup analysis revealed a significant difference in urinary Calprotectin regarding the presence of sterile leukocyturia in high-grade patients (596.8 [91.8-1655.5] vs. 90.4 [28.0-202.3] ng ml-1, p = 0.02). Multivariate analysis identified the leukocyte concentration to be the only significant impact factor for urinary Calprotectin (OR 3.2, 95% CI 2.5-3.8, p = 0.001). Immunohistochemistry showed Calprotectin positive neutrophils and tumour cells in high-grade bladder cancer with sterile leukocyturia. CONCLUSIONS: Urinary Calprotectin cannot be regarded as a specific tumour marker for bladder cancer, but rather as a surrogate parameter for tumour inflammation.

Loss of the Hematopoietic Stem Cell Factor GATA2 in the Osteogenic Lineage Impairs Trabecularization and Mechanical Strength of Bone.

The transcription factor GATA2 is required for expansion and differentiation of hematopoietic stem cells (HSCs). In mesenchymal stem cells (MSCs), GATA2 blocks adipogenesis, but its biological relevance and underlying genomic events are unknown. We report a dual function of GATA2 in bone homeostasis. GATA2 in MSCs binds near genes involved in skeletal system development and colocalizes with motifs for FOX and HOX transcription factors, known regulators of skeletal development. Ectopic GATA2 blocks osteoblastogenesis by interfering with SMAD1/5/8 activation. MSC-specific deletion of GATA2 in mice increases the numbers and differentiation capacity of bone-derived precursors, resulting in elevated bone formation. Surprisingly, MSC-specific GATA2 deficiency impairs the trabecularization and mechanical strength of bone, involving reduced MSC expression of the osteoclast inhibitor osteoprotegerin and increased osteoclast numbers. Thus, GATA2 affects bone turnover via MSC-autonomous and indirect effects. By regulating bone trabecularization, GATA2 expression in the osteogenic lineage may contribute to the anatomical and cellular microenvironment of the HSC niche required for hematopoiesis.

Urinary Calprotectin Differentiates Between Prerenal and Intrinsic Acute Renal Allograft Failure.

BACKGROUND: Urinary calprotectin has recently been identified as a promising biomarker for the differentiation between prerenal and intrinsic acute kidney injury (AKI) in the nontransplant population. The present study investigates whether calprotectin is able to differentiate between these 2 entities in transplant recipients as well. METHODS: Urinary calprotectin was assessed by enzyme-linked immunosorbent assay in 328 subjects including 125 cases of intrinsic acute allograft failure, 27 prerenal graft failures, 118 patients with stable graft function, and 58 healthy controls. Acute graft failure was defined as AKI stages 1 to 3 (Acute Kidney Injury Network criteria), exclusion criteria were obstructive uropathy, urothelial carcinoma, and metastatic cancer. The clinical differentiation of prerenal and intrinsic graft failure was performed either by biopsy or by a clinical algorithm including response to fluid repletion, history, physical examination, and urine dipstick examination. RESULTS: Reasons for intrinsic graft failure comprised rejection, acute tubular necrosis, urinary tract infection/pyelonephritis, viral nephritis, and interstitial nephritis. Calprotectin concentrations of patients with stable graft function (50.4 ng/mL) were comparable to healthy controls (54.8 ng/mL, P = 0.70) and prerenal graft failure (53.8 ng/mL, P = 0.62). Median urinary calprotectin was 36 times higher in intrinsic AKI (1955 ng/mL) than in prerenal AKI (P < 0.001). Receiver-operating characteristic curve analysis revealed a high accuracy of calprotectin (area under the curve, 0.94) in the differentiation of intrinsic versus prerenal AKI. A cutoff level of 134.5 ng/mL provided a sensitivity of 90.4% and a specificity of 74.1%. Immunohistochemical stainings for calprotectin in renal allograft biopsy specimens confirmed the serological results. CONCLUSIONS: Urinary calprotectin is a promising biomarker for the differentiation of prerenal and intrinsic acute renal allograft failure.

Urinary calprotectin: a new diagnostic marker in urothelial carcinoma of the bladder.

PURPOSE: Recently, a proteomic study of sera from patients with bladder cancer identified S100A8 and S100A9 as tumor-associated proteins. The present cross-sectional study investigates whether calprotectin, the heterodimer of S100A8/S100A9 may serve as a urinary biomarker for the detection of urothelial bladder cancer. METHODS: Urinary calprotectin concentrations were assessed in a population of 181 subjects including 46 cases of bladder cancer. 41 cases of renal cell cancer, 54 cases of prostate cancer, and 40 healthy subjects served as control. Acute kidney injury, urinary tract infection, previous BCG-treatment and secondary transurethral resection of the bladder tumor were defined as exclusion criteria. Assessment was performed by enzyme-linked immunosorbent assay and immunohistochemistry detecting calprotectin. RESULTS: Median calprotectin concentrations (ng/ml) were significantly higher in patients with bladder cancer than in healthy controls (522.3 vs. 51.0, p < 0.001), renal cell cancer (90.4, p < 0.001), and prostate cancer (71.8, p < 0.001). In urothelial carcinoma prominent immunostaining occurred in a subset of tumor cells and in infiltrating myeloid cells. Receiver operating characteristic analysis provided an area under the curve of 0.88 for the differentiation of bladder cancer and healthy control. A cut-off value of 140 ng/ml (determined by Youden's index) resulted in sensitivity and specificity values of 80.4 and 92.5 %. Low grade tumors were associated with significantly lower calprotectin concentrations than high grade tumors (351.9 vs. 1635.2 ng/ml, p = 0.004). CONCLUSIONS: Urothelial malignancies are associated with highly increased concentrations of calprotecin in the urine. In absence of renal failure and pyuria, calprotectin constitutes a promising biomarker for the detection of bladder cancer.

Stabilization of hypoxia inducible factor-1α ameliorates acute renal neurogenic hypertension.

OBJECTIVES: Renal neurogenic hypertension (RNH) contributes to cardiovascular morbidity. Renal hypoxia may cause RNH and vice versa, leading to a vicious circle. Hypoxia adaptation is conferred through hypoxia-inducible factors (HIFs). We hypothesized that acute RNH is accompanied by increased renal vascular resistance (RVR) and that hypertension and increased RVR are countered by increasing HIF-1α by cobalt chloride (CoCl2) preconditioning. METHODS: First, we studied mean arterial pressure (MAP) and RVR in innervated or denervated contralateral kidneys in anesthetized rats before and after unilateral intrarenal injection of phenol, a manoeuvre known to elicit acute RNH. Then HIFα was induced by CoCl2 in drinking water (2 mM, 10 days) after which we compared intrarenal isotonic saline or phenol injection on MAP and RVR in CoCl2 preconditioned and control rats. HIF-1α was determined by immunohistochemistry. RESULTS: Unilateral intrarenal phenol induced immediate rise in MAP and contralateral RVR, and comparable HIF-1α upregulation in both kidneys, consistent with bi-renal hypoxia. Removing the phenol-injected kidney immediately normalized MAP. Contralateral renal denervation had no effect on the rise in MAP, but abrogated the contralateral increase in RVR, suggesting mediation by increased efferent nerve activity. Strong renal staining for HIF-1α confirmed efficacy of CoCl2 preconditioning, and time-dependent increase in heme oxygenase-1 gene expression stabilization of HIFα. CoCl2 preconditioning prior to phenol reduced both ΔMAP (+10 ± 2 vs. +20 ± 3%, P=0.015) and ΔRVR (+21 ± 11 vs. +90 ± 26%, P=0.003). CONCLUSION: Acute RNH leads to renal vasoconstriction and increased renal HIF-1α. Increasing HIF-1α by CoCl2 preconditioning ameliorates intrarenal phenol-induced RNH and renal vasoconstriction.

Tubular von Hippel-Lindau knockout protects against rhabdomyolysis-induced AKI.

Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia-inducible factor (HIF), is incomplete in these conditions. Preconditional HIF activation protects against renal ischemia-reperfusion injury, yet the mechanisms involved are largely unknown, and HIF-mediated renoprotection has not been examined in other causes of AKI. Here, we show that selective activation of HIF in renal tubules, through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO), protects from rhabdomyolysis-induced AKI. In this model, HIF activation correlated inversely with tubular injury. Specifically, VHL deletion attenuated the increased levels of serum creatinine/urea, caspase-3 protein, and tubular necrosis induced by rhabdomyolysis in wild-type mice. Moreover, HIF activation in nephron segments at risk for injury occurred only in VHL-KO animals. At day 1 after rhabdomyolysis, when tubular injury may be reversible, the HIF-mediated renoprotection in VHL-KO mice was associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal, as demonstrated by quantitative PCR, pathway enrichment analysis, and immunohistochemistry. In conclusion, a HIF-mediated shift toward improved energy supply may protect against acute tubular injury in various forms of AKI.

Odd-skipped related genes regulate differentiation of embryonic limb mesenchyme and bone marrow mesenchymal stromal cells.

The regulation of progenitor cell differentiation to a specific tissue type is one of the fundamental questions of biology. Here, we identify Osr1 and Osr2, 2 closely related genes encoding zinc finger transcription factors, as being strongly expressed in irregular connective tissue (ICT) fibroblasts in the chicken embryo, suitable as a developmental marker. We provide evidence that both Osr1 and Osr2 regulate mesenchymal cell-type differentiation. Both Osr1 and Osr2 can promote the formation of ICT, a cell type of so far unknown molecular specification, while suppressing differentiation of other tissues such as cartilage and tendon from uncommitted progenitors. Conversely, knockdown of either Osr gene alone or in combination reverses this effect, thereby leading to decreased differentiation of ICT fibroblasts and increased chondrogenesis in vitro. This indicates that Osr genes play a pivotal role in ICT fibroblast differentiation. Undifferentiated mesenchymal cells reside in the adult body in the form of mesenchymal stem cells in the bone marrow cavity. Using bone marrow stromal cells (BMSCs) isolated from chicken fetal long bones, we show that Osr1 and Osr2 have an intrinsic role in BMSC differentiation similar to their role in early embryonic development, that is, the enforcement of CT fibroblast differentiation and the repression of other cell types as exemplified here by osteoblast differentiation.

Deeply conserved chordate noncoding sequences preserve genome synteny but do not drive gene duplicate retention.

Animal genomes possess highly conserved cis-regulatory sequences that are often found near genes that regulate transcription and development. Researchers have proposed that the strong conservation of these sequences may affect the evolution of the surrounding genome, both by repressing rearrangement, and possibly by promoting duplicate gene retention. Conflicting data, however, have made the validity of these propositions unclear. Here, we use a new computational method to identify phylogenetically conserved noncoding elements (PCNEs) in a manner that is not biased by rearrangement and duplication. This method is powerful enough to identify more than a thousand PCNEs that have been conserved between vertebrates and the basal chordate amphioxus. We test 42 of our PCNEs in transgenic zebrafish assays--including examples from vertebrates and amphioxus--and find that the majority are functional enhancers. We find that PCNEs are enriched around genes with ancient synteny conservation, and that this association is strongest for extragenic PCNEs, suggesting that cis-regulatory interdigitation plays a key role in repressing genome rearrangement. Next, we classify mouse and zebrafish genes according to association with PCNEs, synteny conservation, duplication history, and presence in bidirectional promoter pairs, and use these data to cluster gene functions into a series of distinct evolutionary patterns. These results demonstrate that subfunctionalization of conserved cis-regulation has not been the primary determinate of gene duplicate retention in vertebrates. Instead, the data support the gene balance hypothesis, which proposes that duplicate retention has been driven by selection against dosage imbalances in genes with many protein connections.