Inhibition of hypoxia-inducible factor-prolyl hydroxylation protects from cyclophosphamide-induced bladder injury and urinary dysfunction.

Disruption of the blood-urine barrier can result in acute or chronic inflammatory bladder injury. Activation of the oxygen-regulated hypoxia-inducible factor (HIF) pathway has been shown to protect mucosal membranes by increasing the expression of cytoprotective genes and by suppressing inflammation. The activity of HIF is controlled by prolyl hydroxylase domain (PHD) dioxygenases, which have been exploited as therapeutic targets for the treatment of anemia of chronic kidney disease. Here, we established a mouse model of acute cyclophosphamide (CYP)-induced blood-urine barrier disruption associated with inflammation and severe urinary dysfunction to investigate the HIF-PHD axis in inflammatory bladder injury. We found that systemic administration of dimethyloxalylglycine or molidustat, two small-molecule inhibitors of HIF-prolyl hydroxylases, profoundly mitigated CYP-induced bladder injury and inflammation as assessed by morphological analysis of transmural edema and urothelial integrity and by measuring tissue cytokine expression. Void spot analysis to examine bladder function quantitatively demonstrated that HIF-prolyl hydroxylase inhibitor administration normalized micturition patterns and protected against CYP-induced alteration of urinary frequency and micturition patterns. Our study highlights the therapeutic potential of HIF-activating small-molecule compounds for the prevention or therapy of bladder injury and urinary dysfunction due to blood-urine barrier disruption.NEW & NOTEWORTHY Disruption of the blood-urine barrier can result in acute or chronic inflammatory bladder injury. Here, we demonstrate that pharmacological inhibition of hypoxia-inducible factor (HIF)-prolyl hydroxylation prevented bladder injury and protected from urinary dysfunction in a mouse model of cyclophosphamide-induced disruption of the blood-urine barrier. Our study highlights a potential role for HIF-activating small-molecule compounds in the prevention or therapy of bladder injury and urinary dysfunction and provides a rationale for future clinical studies.

EPO synthesis induced by HIF-PHD inhibition is dependent on myofibroblast transdifferentiation and colocalizes with non-injured nephron segments in murine kidney fibrosis.

AIM: Erythropoietin (EPO) is regulated by hypoxia-inducible factor (HIF)-2. In the kidney, it is produced by cortico-medullary perivascular interstitial cells, which transdifferentiate into collagen-producing myofibroblasts in response to injury. Inhibitors of prolyl hydroxylase domain (PHD) dioxygenases (HIF-PHIs) activate HIF-2 and stimulate kidney and liver EPO synthesis in patients with anemia of chronic kidney disease (CKD). We examined whether HIF-PHIs can reactivate EPO synthesis in interstitial cells that have undergone myofibroblast transdifferentiation in established kidney fibrosis. METHODS: We investigated Epo transcription in myofibroblasts and characterized the histological distribution of kidney Epo transcripts by RNA in situ hybridization combined with immunofluorescence in mice with adenine nephropathy (AN) treated with HIF-PHI molidustat.  Lectin absorption chromatography was used to assess liver-derived EPO.  In addition, we examined kidney Epo transcription in Phd2 knockout mice with obstructive nephropathy. RESULTS: In AN, molidustat-induced Epo transcripts were not found in areas of fibrosis and did not colocalize with interstitial cells that expressed α-smooth muscle actin, a marker of myofibroblast transdifferentiation. Epo transcription was associated with megalin-expressing, kidney injury molecule 1-negative nephron segments and contingent on residual renal function. Liver-derived EPO did not contribute to serum EPO in molidustat-treated mice. Epo transcription was not associated with myofibroblasts in Phd2 knockout mice with obstructive nephropathy. CONCLUSIONS: Our studies suggest that HIF-PHIs do not reactivate Epo transcription in interstitial myofibroblasts and that their efficacy in inducing kidney EPO in CKD is dependent on the degree of myofibroblast formation, the preservation of renal parenchyma and the level of residual renal function.

Disruption of mitochondrial complex III in cap mesenchyme but not in ureteric progenitors results in defective nephrogenesis associated with amino acid deficiency.

Oxidative metabolism in mitochondria regulates cellular differentiation and gene expression through intermediary metabolites and reactive oxygen species. Its role in kidney development and pathogenesis is not completely understood. Here we inactivated ubiquinone-binding protein QPC, a subunit of mitochondrial complex III, in two types of kidney progenitor cells to investigate the role of mitochondrial electron transport in kidney homeostasis. Inactivation of QPC in sine oculis-related homeobox 2 (SIX2)-expressing cap mesenchyme progenitors, which give rise to podocytes and all nephron segments except collecting ducts, resulted in perinatal death from severe kidney dysplasia. This was characterized by decreased proliferation of SIX2 progenitors and their failure to differentiate into kidney epithelium. QPC inactivation in cap mesenchyme progenitors induced activating transcription factor 4-mediated nutritional stress responses and was associated with a reduction in kidney tricarboxylic acid cycle metabolites and amino acid levels, which negatively impacted purine and pyrimidine synthesis. In contrast, QPC inactivation in ureteric tree epithelial cells, which give rise to the kidney collecting system, did not inhibit ureteric differentiation, and resulted in the development of functional kidneys that were smaller in size. Thus, our data demonstrate that mitochondrial oxidative metabolism is critical for the formation of cap mesenchyme-derived nephron segments but dispensable for formation of the kidney collecting system. Hence, our studies reveal compartment-specific needs for metabolic reprogramming during kidney development.

Inactivation of HIF-prolyl 4-hydroxylases 1, 2 and 3 in NG2-expressing cells induces HIF2-mediated neurovascular expansion independent of erythropoietin.

AIM: NG2 cells in the brain are comprised of pericytes and NG2 glia and play an important role in the execution of cerebral hypoxia responses, including the induction of erythropoietin (EPO) in pericytes. Oxygen-dependent angiogenic responses are regulated by hypoxia-inducible factor (HIF), the activity of which is controlled by prolyl 4-hydroxylase domain (PHD) dioxygenases and the von Hippel-Lindau (VHL) tumour suppressor. However, the role of NG2 cells in HIF-regulated cerebral vascular homeostasis is incompletely understood. METHODS: To examine the HIF/PHD/VHL axis in neurovascular homeostasis, we used a Cre-loxP-based genetic approach in mice and targeted Vhl, Epo, Phd1, Phd2, Phd3 and Hif2a in NG2 cells. Cerebral vasculature was assessed by immunofluorescence, RNA in situ hybridization, gene and protein expression analysis, gel zymography and in situ zymography. RESULTS: Vhl inactivation led to a significant increase in angiogenic gene and Epo expression. This was associated with EPO-independent expansion of capillary networks in cortex, striatum and hypothalamus, as well as pericyte proliferation. A comparable phenotype resulted from the combined inactivation of Phd2 and Phd3, but not from Phd2 inactivation alone. Concomitant PHD1 function loss led to further expansion of the neurovasculature. Genetic inactivation of Hif2a in Phd1/Phd2/Phd3 triple mutant mice resulted in normal cerebral vasculature. CONCLUSION: Our studies establish (a) that HIF2 activation in NG2 cells promotes neurovascular expansion and remodelling independently of EPO, (b) that HIF2 activity in NG2 cells is co-controlled by PHD2 and PHD3 and (c) that PHD1 modulates HIF2 transcriptional responses when PHD2 and PHD3 are inactive.

Kidney epithelial targeted mitochondrial transcription factor A deficiency results in progressive mitochondrial depletion associated with severe cystic disease.

Abnormal mitochondrial function is a well-recognized feature of acute and chronic kidney diseases. To gain insight into the role of mitochondria in kidney homeostasis and pathogenesis, we targeted mitochondrial transcription factor A (TFAM), a protein required for mitochondrial DNA replication and transcription that plays a critical part in the maintenance of mitochondrial mass and function. To examine the consequences of disrupted mitochondrial function in kidney epithelial cells, we inactivated TFAM in sine oculis-related homeobox 2-expressing kidney progenitor cells. TFAM deficiency resulted in significantly decreased mitochondrial gene expression, mitochondrial depletion, inhibition of nephron maturation and the development of severe postnatal cystic disease, which resulted in premature death. This was associated with abnormal mitochondrial morphology, a reduction in oxygen consumption and increased glycolytic flux. Furthermore, we found that TFAM expression was reduced in murine and human polycystic kidneys, which was accompanied by mitochondrial depletion. Thus, our data suggest that dysregulation of TFAM expression and mitochondrial depletion are molecular features of kidney cystic disease that may contribute to its pathogenesis.

Hypoxia-inducible factor prolyl-4-hydroxylation in FOXD1 lineage cells is essential for normal kidney development.

Hypoxia in the embryo is a frequent cause of intra-uterine growth retardation, low birth weight, and multiple organ defects. In the kidney, this can lead to low nephron endowment, predisposing to chronic kidney disease and arterial hypertension. A key component in cellular adaptation to hypoxia is the hypoxia-inducible factor pathway, which is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3. In the adult kidney, PHD oxygen sensors are differentially expressed in a cell type-dependent manner and control the production of erythropoietin in interstitial cells. However, the role of interstitial cell PHDs in renal development has not been examined. Here we used a genetic approach in mice to interrogate PHD function in FOXD1-expressing stroma during nephrogenesis. We demonstrate that PHD2 and PHD3 are essential for normal kidney development as the combined inactivation of stromal PHD2 and PHD3 resulted in renal failure that was associated with reduced kidney size, decreased numbers of glomeruli, and abnormal postnatal nephron formation. In contrast, nephrogenesis was normal in animals with individual PHD inactivation. We furthermore demonstrate that the defect in nephron formation in PHD2/PHD3 double mutants required intact hypoxia-inducible factor-2 signaling and was dependent on the extent of stromal hypoxia-inducible factor activation. Thus, hypoxia-inducible factor prolyl-4-hydroxylation in renal interstitial cells is critical for normal nephron formation.

Prolyl-4-hydroxylase 2 and 3 coregulate murine erythropoietin in brain pericytes.

A classic response to systemic hypoxia is the increased production of red blood cells due to hypoxia-inducible factor (HIF)-mediated induction of erythropoietin (EPO). EPO is a glycoprotein hormone that is essential for normal erythropoiesis and is predominantly synthesized by peritubular renal interstitial fibroblast-like cells, which express cellular markers characteristic of neuronal cells and pericytes. To investigate whether the ability to synthesize EPO is a general functional feature of pericytes, we used conditional gene targeting to examine the von Hippel-Lindau/prolyl-4-hydroxylase domain (PHD)/HIF axis in cell-expressing neural glial antigen 2, a known molecular marker of pericytes in multiple organs. We found that pericytes in the brain synthesized EPO in mice with genetic HIF activation and were capable of responding to systemic hypoxia with the induction of Epo. Using high-resolution multiplex in situ hybridization, we determined that brain pericytes represent an important cellular source of Epo in the hypoxic brain (up to 70% of all Epo-expressing cells). We furthermore determined that Epo transcription in brain pericytes was HIF-2 dependent and cocontrolled by PHD2 and PHD3, oxygen- and 2-oxoglutarate-dependent prolyl-4-hydroxylases that regulate HIF activity. In summary, our studies provide experimental evidence that pericytes in the brain have the ability to function as oxygen sensors and respond to hypoxia with EPO synthesis. Our findings furthermore suggest that the ability to synthesize EPO may represent a functional feature of pericytes in the brain and kidney.

Distinct subpopulations of FOXD1 stroma-derived cells regulate renal erythropoietin.

Renal peritubular interstitial fibroblast-like cells are critical for adult erythropoiesis, as they are the main source of erythropoietin (EPO). Hypoxia-inducible factor 2 (HIF-2) controls EPO synthesis in the kidney and liver and is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3, which function as cellular oxygen sensors. Renal interstitial cells with EPO-producing capacity are poorly characterized, and the role of the PHD/HIF-2 axis in renal EPO-producing cell (REPC) plasticity is unclear. Here we targeted the PHD/HIF-2/EPO axis in FOXD1 stroma-derived renal interstitial cells and examined the role of individual PHDs in REPC pool size regulation and renal EPO output. Renal interstitial cells with EPO-producing capacity were entirely derived from FOXD1-expressing stroma, and Phd2 inactivation alone induced renal Epo in a limited number of renal interstitial cells. EPO induction was submaximal, as hypoxia or pharmacologic PHD inhibition further increased the REPC fraction among Phd2-/- renal interstitial cells. Moreover, Phd1 and Phd3 were differentially expressed in renal interstitium, and heterozygous deficiency for Phd1 and Phd3 increased REPC numbers in Phd2-/- mice. We propose that FOXD1 lineage renal interstitial cells consist of distinct subpopulations that differ in their responsiveness to Phd2 inactivation and thus regulation of HIF-2 activity and EPO production under hypoxia or conditions of pharmacologic or genetic PHD inactivation.

Renal epithelium regulates erythropoiesis via HIF-dependent suppression of erythropoietin.

The adult kidney plays a central role in erythropoiesis and is the main source of erythropoietin (EPO), an oxygen-sensitive glycoprotein that is essential for red blood cell production. Decreases of renal pO2 promote hypoxia-inducible factor 2-mediated (HIF-2-mediated) induction of EPO in peritubular interstitial fibroblast-like cells, which serve as the cellular site of EPO synthesis in the kidney. It is not clear whether HIF signaling in other renal cell types also contributes to the regulation of EPO production. Here, we used a genetic approach in mice to investigate the role of renal epithelial HIF in erythropoiesis. Specifically, we found that HIF activation in the proximal nephron via induced inactivation of the von Hippel-Lindau tumor suppressor, which targets the HIF-α subunit for proteasomal degradation, led to rapid development of hypoproliferative anemia that was associated with a reduction in the number of EPO-producing renal interstitial cells. Moreover, suppression of renal EPO production was associated with increased glucose uptake, enhanced glycolysis, reduced mitochondrial mass, diminished O2 consumption, and elevated renal tissue pO2. Our genetic analysis suggests that tubulointerstitial cellular crosstalk modulates renal EPO production under conditions of epithelial HIF activation in the kidney.

Endothelial HIF-2 mediates protection and recovery from ischemic kidney injury.

The hypoxia-inducible transcription factors HIF-1 and HIF-2 mediate key cellular adaptions to hypoxia and contribute to renal homeostasis and pathophysiology; however, little is known about the cell type-specific functions of HIF-1 and HIF-2 in response to ischemic kidney injury. Here, we used a genetic approach to specifically dissect the roles of endothelial HIF-1 and HIF-2 in murine models of hypoxic kidney injury induced by ischemia reperfusion or ureteral obstruction. In both models, inactivation of endothelial HIF increased injury-associated renal inflammation and fibrosis. Specifically, inactivation of endothelial HIF-2α, but not endothelial HIF-1α, resulted in increased expression of renal injury markers and inflammatory cell infiltration in the postischemic kidney, which was reversed by blockade of vascular cell adhesion molecule-1 (VCAM1) and very late antigen-4 (VLA4) using monoclonal antibodies. In contrast, pharmacologic or genetic activation of HIF via HIF prolyl-hydroxylase inhibition protected wild-type animals from ischemic kidney injury and inflammation; however, these same protective effects were not observed in HIF prolyl-hydroxylase inhibitor-treated animals lacking endothelial HIF-2. Taken together, our data indicate that endothelial HIF-2 protects from hypoxia-induced renal damage and represents a potential therapeutic target for renoprotection and prevention of fibrosis following acute ischemic injury.

Hypoxia-inducible factor regulates hepcidin via erythropoietin-induced erythropoiesis.

Iron demand in bone marrow increases when erythropoiesis is stimulated by hypoxia via increased erythropoietin (EPO) synthesis in kidney and liver. Hepcidin, a small polypeptide produced by hepatocytes, plays a central role in regulating iron uptake by promoting internalization and degradation of ferroportin, the only known cellular iron exporter. Hypoxia suppresses hepcidin, thereby enhancing intestinal iron uptake and release from internal stores. While HIF, a central mediator of cellular adaptation to hypoxia, directly regulates renal and hepatic EPO synthesis under hypoxia, the molecular basis of hypoxia/HIF-mediated hepcidin suppression in the liver remains unclear. Here, we used a genetic approach to disengage HIF activation from EPO synthesis and found that HIF-mediated suppression of the hepcidin gene (Hamp1) required EPO induction. EPO induction was associated with increased erythropoietic activity and elevated serum levels of growth differentiation factor 15. When erythropoiesis was inhibited pharmacologically, Hamp1 was no longer suppressed despite profound elevations in serum EPO, indicating that EPO by itself is not directly involved in Hamp1 regulation. Taken together, we provide in vivo evidence that Hamp1 suppression by the HIF pathway occurs indirectly through stimulation of EPO-induced erythropoiesis.

Hepatic HIF-2 regulates erythropoietic responses to hypoxia in renal anemia.

The kidney is the main physiologic source of erythropoietin (EPO) in the adult and responds to decreases in tissue oxygenation with increased EPO production. Although studies in mice with liver-specific or global gene inactivation have shown that hypoxia-inducible factor 2 (Hif-2) plays a major role in the regulation of Epo during infancy and in the adult, respectively, the contribution of renal HIF-2 signaling to systemic EPO homeostasis and the role of extrarenal HIF-2 in erythropoiesis, in the absence of kidney EPO, have not been examined directly. Here, we used Cre-loxP recombination to ablate Hif-2α in the kidney, whereas Hif-2-mediated hypoxia responses in the liver and other Epo-producing tissues remained intact. We found that the hypoxic induction of renal Epo is completely Hif-2 dependent and that, in the absence of renal Hif-2, hepatic Hif-2 takes over as the main regulator of serum Epo levels. Furthermore, we provide evidence that hepatocyte-derived Hif-2 is involved in the regulation of iron metabolism genes, supporting a role for HIF-2 in the coordination of EPO synthesis with iron homeostasis.