The present study was designed to test the hypothesis that hypoxia inducible factor (HIF)-1α mediates profibrotic effects of angiotensin (ANG) II and to determine whether HIF prolyl-hydroxylase, the enzyme that promotes the degradation of HIF-1α, is involved in the profibrotic action of ANG II. In cultured renal medullary interstitial cells, ANG II (10−6 M) treatment for 20 hours remarkably increased HIF-1α levels, which was accompanied by the significant upregulation of collagen I/III and tissue inhibitor of metalloproteinases (TIMP)-1. HIF-1α siRNA decreased HIF-1α levels and completely blocked the effects of ANG II on collagen I/III and TIMP-1. HIF-1α siRNA also abolished ANG II-induced elevation of proliferating cell nuclear antigen, a marker of cell proliferation, and vimentin, a marker of cell transdifferentiation. HIF-2α siRNA did not affect the action of ANG II on collagen I/III and TIMP-1. Overexpression of PHD2 transgene, the predominant renal HIF prolyl-hydroxylase, attenuated ANG II-induced profibrotic action and silencing of PHD2 gene enhanced ANG II-induced profibrotic action. Removal of H2O2 eliminated ANG II-induced profibrotic effects. Two week ANG II infusion (150 ng/Kg/min) increased the expression of HIF-1α and α-smooth muscle actin in the renal medullary interstitial cells in vivo. Our data suggest that HIF-1α mediates ANG II-induced profibrotic effects through activation of cell transdifferentiation and that redox regulation of PHD plays a critical role in ANG II-induced activation of HIF-1α and consequent cell proliferation, transdifferentiation and abnormal extracellular matrix metabolism in renal cells.

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Abstract-High salt induces the expression of transcription factor hypoxia-inducible factor (HIF) 1␣ and its target genes in the renal medulla, which is an important renal adaptive mechanism to high-salt intake. HIF prolyl-hydroxylase domain-containing proteins (PHDs) have been identified as major enzymes to promote the degradation of HIF-1␣. PHD2 is the predominant isoform of PHDs in the kidney and is primarily expressed in the renal medulla. The present study tested the hypothesis that PHD2 responds to high salt and mediates high-salt-induced increase in HIF-1␣ levels in the renal medulla. In normotensive rats, high-salt intake (4% NaCl, 10 days) significantly inhibited PHD2 expressions and enzyme activities in the renal medulla. Renal medullary overexpression of the PHD2 transgene significantly decreased HIF-1␣ levels. PHD2 transgene also blocked high-salt-induced activation of HIF-1␣ target genes heme oxygenase 1 and NO synthase 2 in the renal medulla. In Dahl salt-sensitive hypertensive rats, however, high-salt intake did not inhibit the expression and activities of PHD2 in the renal medulla. Correspondingly, renal medullary HIF-1␣ levels were not upregulated by high-salt intake in these rats. After transfection of PHD2 small hairpin RNA, HIF-1␣ and its target genes were significantly upregulated by high-salt intake in Dahl salt-sensitive rats. Overexpression of PHD2 transgene in the renal medulla impaired renal sodium excretion after salt loading. These data suggest that high-salt intake inhibits PHD2 in the renal medulla, thereby upregulating the HIF-1␣ expression. The lack of PHD-mediated response to high salt may represent a pathogenic mechanism producing salt-sensitive hypertension. (Hypertension. 2010;55:1129-1136.)

Although it has been shown that up-regulation of hypoxia-inducible factor (HIF)-1α is protective in acute ischemic renal injury, long-term over-activation of HIF-1α is implicated to be injurious in chronic kidney diseases. Angiotensin II (ANG II) is a well-known pathogenic factor producing chronic renal injury and has also been shown to increase HIF-1α. However, the contribution of HIF-1α to ANG II-induced renal injury has not been evidenced. The present study tested the hypothesis that HIF-1α mediates ANG II-induced renal injury in Sprague-Dawley rats. Chronic renal injury was induced by ANG II infusion (200ng/kg/min) for 2 weeks in uninephrectomized rats. Transfection of vectors expressing HIF-1α shRNA into the kidneys knocked down HIF-1α gene expression by 70%, blocked ANG II-induced HIF-1α activation and significantly attenuated ANG II-induced albuminuria, which was accompanied by inhibition of ANG II-induced vascular endothelial growth factor, a known glomerular permeability factor, in glomeruli. HIF-1α shRNA also significantly improved the glomerular morphological damage induced by ANG II. Furthermore, HIF-1α shRNA blocked ANG II-induced upregulation of collagen and α-smooth muscle actin in tubulointerstitial region. There was no difference in creatinine clearance and ANG II-induced increase in blood pressure. HIF-1α shRNA had no effect on ANG II-induced reduction in renal blood flow and hypoxia in the kidneys. These data suggested that over-activation of HIF-1α-mediated gene regulation in the kidney is a pathogenic pathway mediating ANG II-induced chronic renal injuries and normalization of over-activated HIF-1α may be used as a treatment strategy for chronic kidney damages associated with excessive ANG II.

Vascular endothelial growth factor (VEGF)-dependent angiogenesis is crucial for corpus leteum formation and their functional maintenance in mammalian ovaries. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1α-mediated transcriptional activation contributes to the increased expression of VEGF gene in response to hypoxia in the bovine developing luteal cells (LCs). By real-time RT-PCR analysis, VEGF messenger RNA (mRNA) expression was found to significantly increase under hypoxia or treatment with desferrioxamine (DFX), cobalt chloride (CoCl(2)) or even N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal (MG-132), while these increased VEGF mRNA expressions could also be blocked by ferrous ammonium sulfate (FAS) or cis-element oligodeoxynucleotide (dsODN) transfection under hypoxia. Further analysis also found that these changes of VEGF mRNA were consistent with HIF-1α expression or HIF-1 activity. Taken together, our results indicate that VEGF is transcriptionally activated by hypoxia through HIF-1α-mediated mechanisms in LCs. This hypoxia-induced transcriptional activation may be one of the important mechanisms mediating the increase of VEGF expression in developing LCs during mammalian corpus leteum formation.

Silencing of hypoxia-inducible factor-1␣ gene attenuates chronic ischemic renal injury in two-kidney, one-clip rats. Am J Physiol Renal Physiol 306: F1236 -F1242, 2014. First published March 12, 2014 doi:10.1152/ajprenal.00673.2013.-Overactivation of hypoxia-inducible factor (HIF)-1␣ is implicated as a pathogenic factor in chronic kidney diseases (CKD). However, controversy exists regarding the roles of HIF-1␣ in CKD. Additionally, although hypoxia and HIF-1␣ activation are observed in various CKD and HIF-1␣ has been shown to stimulate fibrogenic factors, there is no direct evidence whether HIF-1␣ is an injurious or protective factor in chronic renal hypoxic injury. The present study determined whether knocking down the HIF-1␣ gene can attenuate or exaggerate kidney damage using a chronic renal ischemic model. Chronic renal ischemia was induced by unilaterally clamping the left renal artery for 3 wk in Sprague-Dawley rats. HIF-1␣ short hairpin (sh) RNA or control vectors were transfected into the left kidneys. Experimental groups were shamϩcontrol vector, clipϩcontrol vector, and clipϩHIF-1␣ shRNA. Enalapril was used to normalize blood pressure 1 wk after clamping the renal artery. HIF-1␣ protein levels were remarkably increased in clipped kidneys, and this increase was blocked by shRNA. Morphological examination showed that HIF-1␣ shRNA significantly attenuated injury in clipped kidneys: glomerular injury indices were 0.71 Ϯ 0.04, 2.50 Ϯ 0.12, and 1.34 Ϯ 0.11, and the percentage of globally damaged glomeruli was 0.02, 34.3 Ϯ 5.0, and 6.3 Ϯ 1.6 in sham, clip, and clipϩshRNA groups, respectively. The protein levels of collagen and ␣-smooth muscle actin also dramatically increased in clipped kidneys, but this effect was blocked by HIF-1␣ shRNA. In conclusion, long-term overactivation of HIF-1␣ is a pathogenic factor in chronic renal injury associated with ischemia/hypoxia. collagen; ␣-smooth muscle actin; renal fibrosis; chronic kidney diseases REDUCED RENAL TISSUE OXYGEN levels have been demonstrated in a large variety of chronic kidney diseases (CKD) in both human patients and in experimental animal models. Hypoxia in CKD results from a combination of structural and functional changes (12, 36). As a result, hypoxia-inducible factor (HIF)-1␣ has been reported to be consistently upregulated in almost all types of CKD (7, 16, 17, 36 -38). However, it is unclear whether upregulation of HIF-1␣ is beneficial or deleterious in progressive CKD. HIF-1␣ is a transcription factor and has been shown to stimulate collagen accumulation (7,15,43,44) and promote the epithelial-to-mesenchymal transition (EMT) (11,35), an important mechanism involved in the progression of CKD (3,33,57,67). Therefore, although upregulation of HIF-1␣ is protective in acute kidney injury (9, 18, 37, 53), ample evidence indicates that long-term overactivation of HIF-1␣ may be a pathogenic factor in CKD (10,16,21,24,36,45).Previous studies have shown that genetic ablation of renal epithelial HIF-1␣ inhibits the development of renal tubulointerstitia...

This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O 2 ·À production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs. Antioxid. Redox Signal. 11, 703-712.

Zhu Q, Xia M, Wang Z, Li P, Li N. A novel lipid natriuretic factor in the renal medulla: sphingosine-1-phosphate. Am J Physiol Renal Physiol 301: F35-F41, 2011. First published April 6, 2011 doi:10.1152/ajprenal.00014.2011.-Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite formed by phosphorylation of sphingosine. S1P has been indicated to play a significant role in the cardiovascular system. It has been shown that the enzymes for S1P metabolism are expressed in the kidneys. The present study characterized the expression of S1P receptors in the kidneys and determined the role of S1P in the control of renal hemodynamics and sodium excretion. Real-time RT-PCR analyses showed that S1P receptors S1P1, S1P2, and S1P3 were most abundantly expressed in the renal medulla. Immunohistochemistry revealed that all three types of S1P receptors were mainly located in collecting ducts. Intramedullary infusion of FTY720, an S1P agonist, produced a dramatic increase in sodium excretion by twofold and a small but significant increase in medullary blood flow (16%). Administration of W146, an S1P1 antagonist, into the renal medulla blocked the effect of FTY720 and decreased the sodium excretion by 37% when infused alone. The antagonists of S1P2 and S1P3 had no effect. FTY720 produced additive natriuretic effects in combination with different sodium transporter inhibitors except amiloride, an epithelial sodium channel blocker. In the presence of nitric oxide synthase inhibitor L-NAME, FTY720 still increased sodium excretion. These data suggest that S1P produces natriuretic effects via activation of S1P1 in the renal medulla and this natriuretic effect may be through inhibition of epithelial sodium channel, which is nitric oxide independent. It is concluded that S1P is a novel diuretic factor in the renal medulla and may be an important regulator of sodium homeostasis. renal blood flow; sodium transporter; nitric oxide; collecting duct SPHINGOLIPIDS WERE ORIGINALLY thought to serve only as structural components of mammalian cell membranes. In recent years, sphingolipid metabolites are emerging as important lipid signaling molecules. Among them, sphingosine-1-phosphate (S1P) is known to play important roles in cellular processes in various organ systems including cardiovascular system and kidney (15,29). S1P is formed by phosphorylation of sphingosine catalyzed by sphingosine kinase and acts via its G protein-coupled receptors (17,29). Five members of S1P receptor family have been identified and termed S1P1-S1P5 (17, 29). The S1P1, S1P2, and S1P3 are ubiquitously expressed, while S1P4 is predominantly expressed in the lung and lymphoid system, and S1P5 is mainly in brain tissue (17,29). The expression and activity of sphingosine kinase have been detected in the kidneys (1, 10) and the mRNAs of S1P receptors are also present in the kidneys (17). It has been shown that S1P protects the kidneys from ischemic injury (2,27). It has also been demonstrated that S1P regulates vascular functions (14, 23). Therefore, it is p...