Palmitate deranges erythropoietin production via transcription factor ATF4 activation of unfolded protein response.

Lipotoxicity plays an important role in the progression of chronic kidney damage via various mechanisms, such as endoplasmic reticulum stress. Several studies proposed renal lipotoxicity in glomerular and tubular cells but the effect of lipid on renal erythropoietin (EPO)-producing (REP) cells in the interstitium has not been elucidated. Since renal anemia is caused by derangement of EPO production in REP cells, we evaluated the effect of palmitate, a representative long-chain saturated fatty acid, on EPO production and the endoplasmic reticulum stress pathway. EPO production was suppressed by palmitate (palmitate-conjugated bovine serum albumin [BSA]) or a high palmitate diet, but not oleic acid-conjugated BSA or a high oleic acid diet, especially under cobalt-induced pseudo-hypoxia both in vitro and in vivo. Importantly, suppression of EPO production was not induced by a decrease in transcription factor HIF activity, while it was significantly associated with endoplasmic reticulum stress, particularly transcription factor ATF4 activation, which suppresses 3'-enhancer activity of the EPO gene. ATF4 knockdown by siRNA significantly attenuated the suppressive effect of palmitate on EPO production. Studies utilizing inherited super-anemic mice (ISAM) mated with EPO-Cre mice (ISAM-REC mice) for lineage-labeling of REP cells showed that ATF4 activation by palmitate suppressed EPO production in REP cells. Laser capture microdissection confirmed ATF4 activation in the interstitial area of ISAM-REC mice treated with palmitate-conjugated BSA. Thus, endoplasmic reticulum stress induced by palmitate suppressed EPO expression by REP cells in a manner independent of HIF activation. The link between endoplasmic reticulum stress, dyslipidemia, and hypoxia may contribute to development and progression of anemia in CKD.

Albumin suppresses vascular endothelial growth factor via alteration of hypoxia-inducible factor/hypoxia-responsive element pathway.

Reduction of vascular endothelial growth factor (VEGF) expression plays a crucial role in chronic kidney disease (CKD). In order to clarify a cause of VEGF suppression in CKD, we examined an interaction between proteinuria and VEGF. Rat proximal tubular cells were subjected to hypoxia with or without albumin to mimic proteinuric conditions, and VEGF expression was assessed by real-time quantitative PCR and enzyme-linked immunosorbent assays. Albumin significantly reduced VEGF expression under hypoxia. Luciferase activity controlled by hypoxia-responsive element (HRE) was suppressed by albumin, demonstrating suppression of the hypoxia-inducible factor (HIF)/HRE pathway. Studies utilizing a proteasome inhibitor and a prolyl hydroxylase inhibitor showed that mechanisms of HIF/HRE pathway suppression by albumin load did not involve degradation of HIF protein levels. Further, albumin did not change HIF mRNA levels. Our data, for the first time, suggest a clear 'link' between proteinuria and hypoxia, the two principal pathogenic factors for CKD progression.

Erythropoietin induces heme oxygenase-1 expression and attenuates oxidative stress.

Recent studies have established that erythropoietin (EPO) is a pleiotropic cytokine. In this study we investigated whether pleiotropic effects of EPO may involve regulation of heme oxygenase (HO)-1, an anti-oxidative stress protein. A stimulatory effect of EPO on HO-1 expression was demonstrated in cultured renal endothelial cells, in which EPO decreased intracellular oxidative stress and provided cytoprotection against H(2)O(2). These beneficial effects were partially reversed by a HO-1 inhibitor. We then evaluated whether EPO induces HO-1 and ameliorates renal injury in vivo. Administration of EPO to Dahl salt-sensitive (DS) rats with low salt diet, a model of chronic tubulointerstitial injury, reduced proteinuria, and renal injury including peritubular capillaries rarefaction as compared to vehicle-treated DS rats. This renoprotection was associated with up-regulation of HO-1 in the kidney. In conclusion, EPO-induced HO-1 expression is likely to provide cytoprotection against oxidative stress.

High glucose blunts vascular endothelial growth factor response to hypoxia via the oxidative stress-regulated hypoxia-inducible factor/hypoxia-responsible element pathway.

Vascular endothelial growth factor (VEGF) is an important survival factor for endothelial cells in hypoxic environments. High glucose regulates certain aspects of VEGF expression in various cell types, including proximal tubular cells. Thus, ambient glucose levels may modulate the progression of chronic kidney disease, especially diabetic nephropathy. Immortalized rat proximal tubular cells (IRPTC) were cultured for 24 h under hypoxic conditions (1% O(2)), with or without high d-glucose (25 mM), or with or without high l-glucose (25 mM). Controls included culture in normoxic conditions and normal d-glucose (5.5 mM). VEGF mRNA expression was assessed by real-time quantitative PCR, and VEGF protein in the supernatant was assessed by ELISA. Hypoxia increased VEGF expression. This response was significantly blunted by high d-glucose (1.98 +/- 0.11- versus 2.65 +/- 0.27-fold increase for VEGF mRNA expression, 252.8 +/- 14.7 versus 324.0 +/- 11.5 pg/10(5) cells for VEGF protein; P < 0.05 both) but not by high l-glucose. It is interesting that hydrogen peroxide also blunted this response, whereas alpha-tocopherol restored the VEGF response to hypoxia in the presence of high d-glucose. For determination of involvement of the hypoxia-inducible factor (HIF)/hypoxia-responsible element (HRE) pathway, IRPTC that were stably transfected with HRE-luciferase were cultured under the previous conditions. High d-glucose also reduced luciferase activity under hypoxia, whereas alpha-tocopherol restored activity. In vivo experiments using streptozotocin-induced diabetic rats confirmed that hyperglycemia blunted HIF-HRE pathway activation. Insulin treatment restored activation of the HIF-HRE pathway in streptozotocin-induced diabetic rats. In conclusion, high glucose blunts VEGF response to hypoxia in IRPTC. This effect is mediated by the oxidative stress-regulated HIF-HRE pathway.

A biologic role of HIF-1 in the renal medulla.

BACKGROUND: Activation of hypoxia-inducible factor-1 (HIF-1) is the primary defensive mechanism against hypoxia. HIF-1 activation generally occurs in pathologic disruption of tissue oxygenation. However, a biologic role of HIF-1 in the medulla of the kidney, which is considered perpetually hypoxic under physiologic conditions due to its unique circulation, remains to be elucidated. METHODS: The expression of HIF-1alpha was detected by immunohistochemical analysis. Functional studies of HIF in medulla were carried out by gene transfer of various plasmids by retrograde injection via ureter. RESULTS: Our immunohistochemical analysis detected HIF-1alpha in the inner stripe and the inner medulla of normal rats. Water deprivation increased the number of HIF-1alpha-positive cells, which may be mediated by an increase in medullar workload and a decrease in local blood flow. To perform functional studies, we performed gene transfer. Efficient expression of the transgene was confirmed using an enhanced green fluorescent protein (E-GFP) expressing vector. Our histologic and immunoblotting analysis detected the transgene product at the inner medulla and the inner stripe 48 hours after injection. Administration of negative-dominant HIF induced severe damage in the medulla of normal rats. In contrast, gene transfer of constitutively active HIF (HIF/VP16) induced expression of various HIF-regulated genes and protected the medulla against ischemic insults. CONCLUSION: Our studies demonstrated a crucial role of HIF in the renal medulla under normal and hypoxic circumstances.