Tubular deficiency of von Hippel-Lindau attenuates renal disease progression in anti-GBM glomerulonephritis.
ABSTRACT: In many kidney diseases, the original insult primarily involves the glomerulus and may then pass onto the tubulointerstitium. Several hypotheses link glomerular disease to tubular injury; perhaps the foremost hypothesis involves chronic tubular hypoxia. The reported effects of hypoxia and consecutive stabilization of hypoxia-inducible factors (HIFs), however, are controversial. Hypoxia induces interstitial fibrosis but also has beneficial effects on renal disease progression when HIF is activated pharmacologically. To analyze the impact of HIF on tubulointerstitial disease development in primary glomerular disease, transgenic von Hippel Lindau (VHL)-knockout mice were generated and null expression was induced before the onset of autoimmune IgG-mediated anti-glomerular basement membrane glomerulonephritis (GN). Tubular VHL knockout and, thus, local HIF-? stabilization increased renal production of vascular endothelial growth factor, tumor growth factor-?(1), and platelet-derived growth factor-B, resulting in augmented formation of capillaries and interstitial matrix, and conversion of fibroblasts to myofibroblasts. Within the glomerular disease, VHL knockout reduced the glomerular damage and attenuated tubulointerstitial injury. Likewise, proteinuria, plasma urea concentration, and tubulointerstitial matrix were decreased in VHL knockout with GN. These findings shown that tubular HIF-? stabilization in glomerular disease is beneficial for disease outcome. In comparison with VHL knockout alone, GN is a much stronger activator of fibrosis such that stimuli other than hypoxia may be considered important for renal disease progression.
Project description: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.
Project description:The Hypoxia-inducible transcription Factor (HIF) represents an important adaptive mechanism under hypoxia, whereas sustained activation may also have deleterious effects. HIF activity is determined by the oxygen regulated ?-subunits HIF-1? or HIF-2?. Both are regulated by oxygen dependent degradation, which is controlled by the tumor suppressor "von Hippel-Lindau" (VHL), the gatekeeper of renal tubular growth control. HIF appears to play a particular role for the kidney, where renal EPO production, organ preservation from ischemia-reperfusion injury and renal tumorigenesis are prominent examples. Whereas HIF-1? is inducible in physiological renal mouse, rat and human tubular epithelia, HIF-2? is never detected in these cells, in any species. In contrast, distinct early lesions of biallelic VHL inactivation in kidneys of the hereditary VHL syndrome show strong HIF-2? expression. Furthermore, knockout of VHL in the mouse tubular apparatus enables HIF-2? expression. Continuous transgenic expression of HIF-2? by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to multiple renal cysts. In conclusion, VHL appears to specifically repress HIF-2? in renal epithelia. Unphysiological expression of HIF-2? in tubular epithelia has deleterious effects. Our data are compatible with dedifferentiation of renal epithelial cells by sustained HIF-2? expression. However, HIF-2? overexpression alone is insufficient to induce tumors. Thus, our data bear implications for renal tumorigenesis, epithelial differentiation and renal repair mechanisms.
Project description:Hyperglycemia results in increased oxygen consumption and decreased oxygen tension in the kidney. We tested the hypothesis that activation of hypoxia-inducible factors (HIFs) protects against diabetes-induced alterations in oxygen metabolism and kidney function. Experimental groups consisted of control and streptozotocin-induced diabetic rats treated with or without chronic cobalt chloride to activate HIFs. We elucidated the involvement of oxidative stress by studying the effects of acute administration of the superoxide dismutase mimetic tempol. Compared with controls, diabetic rats displayed tissue hypoxia throughout the kidney, glomerular hyperfiltration, increased oxygen consumption, increased total mitochondrial leak respiration, and decreased tubular sodium transport efficiency. Diabetic kidneys showed proteinuria and tubulointerstitial damage. Cobalt chloride activated HIFs, prevented the diabetes-induced alterations in oxygen metabolism, mitochondrial leak respiration, and kidney function, and reduced proteinuria and tubulointerstitial damage. The beneficial effects of tempol were less pronounced after activation of HIFs, indicating improved oxidative stress status. In conclusion, activation of HIFs prevents diabetes-induced alteration in kidney oxygen metabolism by normalizing glomerular filtration, which reduces tubular electrolyte load, preventing mitochondrial leak respiration and improving tubular transport efficiency. These improvements could be related to reduced oxidative stress and account for the reduced proteinuria and tubulointerstitial damage. Thus, pharmacologic activation of the HIF system may prevent development of diabetic nephropathy.
Project description:Ketohexokinase (KHK) is the first and rate-limiting enzyme of fructose metabolism. Expression of the two alternatively spliced KHK isoforms, KHK-A and KHK-C, is tissue-specific and KHK-C is predominantly expressed in liver, kidney and intestine and responsible for the fructose-catabolizing function. While KHK isoform choice has been linked to the development of disorders such as obesity, diabetes, cardiovascular disease and cancer, little is known about the regulation of total KHK expression. In the present study, we investigated how hypoxic signaling influences fructose metabolism in the liver. Hypoxia or von Hippel-Lindau (VHL) tumor suppressor loss leads to the stabilization of hypoxia-inducible factors alpha (HIF-1? and HIF-2?) and the activation of their signaling to mediate adaptive responses. By studying liver-specific Vhl, Vhl/Hif1a, and Vhl/Epas1 knockout mice, we found that KHK expression is suppressed by HIF-2? (encoded by Epas1) but not by HIF-1? signaling on mRNA and protein levels. Reduced KHK levels were accompanied by downregulation of aldolase B (ALDOB) in the livers of Vhl and Vhl/Hif1a knockout mice, further indicating inhibited fructose metabolism. HIF-1? and HIF-2? have both overlapping and distinct target genes but are differentially regulated depending on the cell type and physiologic or pathologic conditions. HIF-2? activation augments peroxisome degradation in mammalian cells by pexophagy and thereby changes lipid composition reminiscent of peroxisomal disorders. We further demonstrated that fructose metabolism is negatively regulated by peroxisome-deficiency in a Pex2 knockout Zellweger mouse model, which lacks functional peroxisomes and is characterized by widespread metabolic dysfunction. Repression of fructolytic genes in Pex2 knockout mice appeared to be independent of PPAR? signaling and nutritional status. Interestingly, our results demonstrate that both HIF-2? and peroxisome-deficiency result in downregulation of Khk independent of splicing as both isoforms, Khka as well as Khkc, are significantly downregulated. Hence, our study offers new and unexpected insights into the general regulation of KHK, and therefore fructolysis. We revealed a novel regulatory function of HIF-2?, suggesting that HIF-1? and HIF-2? have tissue-specific opposing roles in the regulation of Khk expression, isoform choice and fructolysis. In addition, we discovered a previously unknown function of peroxisomes in the regulation of fructose metabolism.
Project description:Renal hypoxia is widespread in acute kidney injury (AKI) of various aetiologies. Hypoxia adaptation, conferred through the hypoxia-inducible factor (HIF), appears to be insufficient. Here we show that HIF activation in renal tubules through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO) protects from rhabdomyolysis-induced AKI. In this model, histological observations indicate that injury mainly affects proximal convoluted tubules, with 5% necrosis at d1 and 40% necrosis at d2. HIF-1alpha up-regulation in distal tubules reflects renal hypoxia. However, lack of HIF in proximal tubules suggests insufficient adaptation by HIF. AKI in VHL-KO mice leads to prominent HIF activation in all nephron segments, as well as to reduced serum creatinine, serum urea, tubular necrosis, and apoptosis marker caspase-3 protein. At d1 after rhabdomyolysis, when tubular injury is potentially reversible, HIF mediated protection in AKI is associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal as demonstrated by qPCR, pathway enrichment analysis and immunohistochemistry. Together, our data provide evidence for a HIF-orchestrated multi-level shift towards glycolysis as a major mechanism for protection against acute tubular injury. All experiments were carried out in transgenic mice in which selective renal tubular VHL knockout (VHL-KO) was inducible by doxycycline (Reference: Mathia S, Paliege A, Koesters R, Peters H, Neumayer HH, Bachmann S, Rosenberger C. Action of hypoxia-inducible factor in liver and kidney from mice with Pax8-rtTA-based deletion of von Hippel-Lindau protein. Acta Physiol (Oxf). 2013; 207(3):565-76.). Four groups of animals were used: 1) controls: untreated mice; 2) VHL-KO: injected with doxycycline (0.1 mg per 10 g body weight SC), 4 days prior to sacrifice; 3) AKI: rhabdomyolysis; 4) VHL-KO/AKI: doxycycline plus rhabdomyolysis. To induce AKI, 50% glycerol (0.05 ml per 10 g body weight) was injected IM into the left hind limb under isoflurane narcosis. Drinking water was withdrawn between 20 h prior and 24 h after glycerol injection.
Project description:Metastatic renal cell carcinoma (mRCC) is nearly incurable and accounts for most of the mortality associated with RCC. Von Hippel Lindau (VHL) is a tumour suppressor that is lost in the majority of clear cell RCC (ccRCC) cases. Its role in regulating hypoxia-inducible factors-1? (HIF-1?) and -2? (HIF-2?) is well-studied. Recent work has demonstrated that VHL knock down induces an epithelial-mesenchymal transition (EMT) phenotype. In this study we showed that a CRISPR/Cas9-mediated knock out of VHL in the RENCA model leads to morphologic and molecular changes indicative of EMT, which in turn drives increased metastasis to the lungs. RENCA cells deficient in HIF-1? failed to undergo EMT changes upon VHL knockout. RNA-seq revealed several HIF-1?-regulated genes that are upregulated in our VHL knockout cells and whose overexpression signifies an aggressive form of ccRCC in the cancer genome atlas (TCGA) database. Independent validation in a new clinical dataset confirms the upregulation of these genes in ccRCC samples compared to adjacent normal tissue. Our findings indicate that loss of VHL could be driving tumour cell dissemination through stabilization of HIF-1? in RCC. A better understanding of the mechanisms involved in this phenomenon can guide the search for more effective treatments to combat mRCC.
Project description: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-1? 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.
Project description:Mitochondrial dysfunction leads to loss of renal function and structure; however, the precise mechanisms by which mitochondrial function can regulate renal fibrosis remain unclear. Proximal tubular cells (PTCs) prefer fatty acid oxidation as their energy source and dysregulation of lipid metabolism has been linked to tubulointerstitial fibrosis (TIF). Here, we demonstrated that mitochondrial uncoupling protein 2 (UCP2) regulates TIF through the stimulation of lipid deposition and extracellular matrix (ECM) accumulation. We show that UCP2 expression was increased in human biopsy sample and mouse kidney tissues with TIF. Moreover, UCP2-deficient mice displayed mitigated renal fibrosis in I/R-induced mouse model of TIF. Consistent with these results, UCP2 deficiency displayed reduced lipid deposition and ECM accumulation in vivo and in vitro. In UCP2-deficient PTCs, inhibition of TIF resulted from downregulation of hypoxia-inducible factor-1? (HIF-1?), a key regulator of lipid metabolism and ECM accumulation. Furthermore, we describe a molecular mechanism by which UCP2 regulates HIF-1? stabilization through regulation of mitochondrial respiration and tissue hypoxia during TIF. HIF-1? inhibition by siRNA suppressed lipid and ECM accumulation by restoration of PPAR? and CPT1?, as well as suppression of fibronectin and collagen I expression in PTCs. In conclusion, our results suggest that UCP2 regulates TIF by inducing the HIF-1? stabilization pathway in tubular cells. These results identify UCP2 as a potential therapeutic target in treating chronic renal fibrosis.
Project description:Progressive cyst growth leads to decline of renal function in polycystic kidney disease. Macrophage migration inhibitory factor (MIF) was found to be upregulated in cyst-lining cells in a mouse model of polycystic kidney disease and to promote cyst growth. In addition, MIF can be secreted by tubular cells and may contribute to cyst growth in an autocrine manner. However, the underlying mechanisms leading to induction of MIF in cyst-lining cells remained elusive. Here, we demonstrate that hypoxia-inducible transcription factor (HIF) 1? upregulates MIF in cyst-lining cells in a tubule-specific PKD1 knockout mouse. Pharmacological stabilization of HIF-1? resulted in significant increase of MIF in cyst epithelial cells whereas tubule-specific knockout of HIF-1? prevented MIF upregulation. Identical regulation could be found for ABCA1, which has been shown to act as a transport protein for MIF. Furthermore, we show that MIF and ABCA1 are direct target genes of HIF-1? in human primary tubular cells. Next to HIF-1? and hypoxia, we found MIF being additionally regulated by cAMP which is a strong promotor of cyst growth. In line with these findings, HIF-1?- and cAMP-dependent in vitro cyst growth could be decreased by the MIF-inhibitor ISO-1 which resulted in reduced cyst cell proliferation. In conclusion, HIF-1? and cAMP regulate MIF in primary tubular cells and cyst-lining epithelial cells, and MIF promotes cyst growth in the absence of macrophages. In line with these findings, the MIF inhibitor ISO-1 attenuates HIF-1?- and cAMP-dependent in vitro cyst enlargement. KEY MESSAGES: • MIF is upregulated in cyst-lining cells in a polycystic kidney disease mouse model. • MIF upregulation is mediated by hypoxia-inducible transcription factor (HIF) 1?. • ABCA1, transport protein for MIF, is also regulated by HIF-1? in vitro and in vivo. • MIF is additionally regulated by cAMP, a strong promotor of cyst growth. • MIF-inhibitor ISO-1 reduces HIF-1?- and cAMP-dependent cyst growth.
Project description:Inactivation of the von Hippel-Lindau (VHL) gene is associated with the development of highly vascularized tumors. pVHL targets the alpha subunits of hypoxia inducible factor (HIF) for ubiquitin-mediated degradation in an oxygen-dependent manner. Although pVHL-deficient tumor cell lines demonstrate constitutive stabilization and activation of HIF, it has yet to be shown that loss of murine Vhl alone is sufficient to dysregulate HIF. We utilized a genetic approach to demonstrate that loss of Vhl is sufficient not only to stabilize HIF-alpha subunits under normoxia, but also fully activate HIF-mediated responses. These studies have implications for the hierarchy of signaling events leading to HIF stabilization, nuclear translocation, and target gene expression. We further demonstrate that loss of murine Vhl does not promote teratocarcinoma growth, indicating that other genetic changes must occur to facilitate Vhl-mediated tumorigenesis.