PPAR-α Insufficiency Enhances Doxorubicin-Induced Nephropathy in PPAR-α Knockout Mice and a Murine Podocyte Cell Line.

Peroxisome proliferator-activated receptor-alpha (PPAR-α) and its exogenous activators (fibrates) promote autophagy. However, whether the deleterious effects of PPAR-α deficiency on doxorubicin (DOX)-induced podocytopathy are associated with reduced autophagy remains to be clarified. We investigated the mechanisms of PPAR-α in DOX-induced podocytopathy and tubular injury in PPAR-α knockout (PAKO) mice and in a murine podocyte cell line. DOX-treated PAKO mice showed higher serum levels of triglycerides and non-esterified fatty acids and more severe podocytopathy than DOX-treated wild-type mice, as evidenced by higher urinary levels of proteins and podocalyxin at 3 days to 2 weeks and higher blood urea nitrogen and serum creatinine levels at 4 weeks. Additionally, there was an increased accumulation of p62, a negative autophagy marker, in the glomerular and tubular regions in DOX-treated PAKO mice at Day 9. Moreover, DOX-treated PAKO mice showed more severe glomerulosclerosis and tubular damage and lower podocalyxin expression in the kidneys than DOX-treated control mice at 4 weeks. Furthermore, DOX treatment increased p-p53, an apoptosis marker, and cleaved the caspase-3 levels and induced apoptosis, which was ameliorated by fenofibrate, a PPAR-α activator. Fenofibrate further enhanced AMPK activation and autophagy under fed and fasting conditions. Conclusively, PPAR-α deficiency enhances DOX-induced podocytopathy, glomerulosclerosis, and tubular injury, possibly by reducing autophagic activity in mouse kidneys.

Fenofibrate reduces cisplatin-induced apoptosis by inhibiting the p53/Puma/Caspase-9 pathway and the MAPK/Caspase-8 pathway rather than by promoting autophagy in murine renal proximal tubular cells.

The main lesion of cisplatin nephrotoxicity is damage to proximal tubular cells due to increased apoptosis via the mitochondrial and death receptor pathways, which may be alleviated by appropriate promotion of autophagy. Fenofibrate, a peroxisome proliferator-activated receptor-alpha (PPAR-α) activator, is recently reported to promote autophagy as well as protect against cisplatin nephrotoxicity, although the mechanisms were only partially analyzed. Here, the detailed mechanisms of these putative protective effects were investigated in a murine renal proximal tubular (mProx) cell line. Fenofibrate attenuated cisplatin-induced apoptosis of mProx cells based on flow cytometry. As for the mitochondrial apoptotic pathway, the reagent reduced cisplatin-stimulated caspase-3 activation by decreasing the phosphorylation of p53, JNK, and 14-3-3, cytosolic and mitochondrial Puma accumulation, cytochrome C release to the cytosol, and resulting cytosolic caspase-9 activation. Fenofibrate also decreased cisplatin-stimulated activation of caspases-8 by suppressing MAPK and NFkB pathways and reducing the gene expression of TNF-α, TL1A, and Fas, main mediators of the death receptor apoptotic pathway. Autophagy defined by p62 reduction and an increase in LC3 II/I was promoted by fenofibrate in mProx cells under starvation. Autophagy inhibition using 3-MA further increased basal and cisplatin-induced caspase-3 and -8 activation, but had no influence on the inhibitory effects of fenofibrate on caspase activation. In conclusion, our study suggests fenofibrate to be a candidate agent to mitigate cisplatin nephrotoxicity by inhibiting the mitochondrial and death apoptotic pathways rather than by promoting autophagy.

PPAR-δ activation reduces cisplatin-induced apoptosis via inhibiting p53/Bax/caspase-3 pathway without modulating autophagy in murine renal proximal tubular cells.

BACKGROUND: Cisplatin-induced injury of renal proximal tubular cells results basically from increased apoptosis via mitochondrial damage, and is mitigated by appropriate enhancement of autophagy. Peroxisome proliferator-activated receptor-delta (PPAR-δ) reportedly protects against not only mitochondrial damages but also enhances autophagy. Thus, PPAR-δ may protect against cisplatin-induced kidney injury. METHODS: We examined the protective effects of PPAR-δ activation on cisplatin-induced cellular injury and their detailed mechanisms in a murine renal proximal tubular (mProx) cell line using GW0742, an authentic PPAR-δ activator. Cisplatin-induced cell damages were evaluated by TUNEL assay and immunoblot analyses for p53, 14-3-3, Bax, Bcl2, cytochrome C, and activated caspases. Autophagy status was examined by immunoblot analyses for p62 and LC3. RESULTS: GW0742 suppressed cisplatin-induced apoptosis of mProx cells by reducing the activation of caspase-3 via attenuating the phosphorylation of p53 and 14-3-3, mitochondrial Bax accumulation, cytochrome C release from mitochondria to the cytosol and ensuing cytosolic caspase-9 activation. In contrast, GW0742 did not diminish cisplatin-enhanced activation of caspases-8 or -12 as extrinsic or endothelium reticulum apoptotic pathways, respectively. The inhibitory effect of GW0742 on cisplatin-induced caspase-3 activation was significantly diminished by silencing of the PPAR-δ gene expression. GW0742 itself had no influence on starvation-stimulated or cisplatin-induced autophagy in mProx cells, suggesting that the protective effects were not mediated by autophagy modification. CONCLUSION: Our results indicate that GW0742 may serve as a candidate agent to mitigate cisplatin nephrotoxicity via inhibiting the mitochondrial apoptotic pathway considerably depending on PPAR-δ, without modulating autophagy.

AR420626, a selective agonist of GPR41/FFA3, suppresses growth of hepatocellular carcinoma cells by inducing apoptosis via HDAC inhibition.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major cause of cancer death worldwide and establishment of new chemotherapies for HCC is urgently needed. GPR41 [free fatty acid receptor 3 (FFA3)] is a G protein-coupled receptor for short chain fatty acids, including acetate, propionate, and butyrate. In our previous study, we showed that propionate enhances the cytotoxic effect of cisplatin in HCC cells and that this mechanism is dependent on inhibition of histone deacetylases (HDACs) via GPR41/FFA3. However, the antitumor action of GPR41/FFA3 has not been elucidated. METHODS: In this study, we examined AR420626 as a GPR41-selective agonist in HepG2 and HLE cells. Nude mice were used for HepG2 xenograft studies. The apoptotic effect of AR420626 was evaluated using flow cytometry analysis. Expression of apoptosis-related proteins and HDACs was evaluated by Western immunoblot. Gene silencing of HDAC 3/5/7 and GPR41 was performed using small interfering RNA. Expression of TNF-α mRNA was evaluated by TaqMan real-time polymerase chain reaction. RESULTS: We found that AR420626, a selective GPR41/FFA3 agonist, suppressed growth of HepG2 xenografts and inhibited proliferation of HCC cells by inducing apoptosis. AR420626 induced proteasome activation through mTOR phosphorylation, which reduced HDAC proteins, and then increased expression of TNF-α. CONCLUSION: AR420626, a selective GPR41/FFA3 agonist, may be a candidate as a therapeutic agent for HCC.

Chronic hypoxia exacerbates diabetic glomerulosclerosis through mesangiolysis and podocyte injury in db/db mice.

BACKGROUND: Chronic hypoxia may play a pivotal role in the development of diabetic nephropathy (DN). However, the precise mechanisms underlying progressive hypoxia-induced glomerular injury remain unclear. METHODS: We housed db/db mice in a hypoxia chamber (12% O2) for up to 16 weeks beginning at 8 weeks of age. Various urine, serum and kidney abnormalities and glomerular messenger RNA (mRNA) expression were compared with those in age-matched db/db mice housed under normoxia. RESULTS: Levels of urinary albumin and podocalyxin (PCX) were significantly higher in hypoxic mice early during hypoxia. Ultracentrifugation of urine samples revealed that podocytes in the hypoxic mice shed PCX-positive microparticles into the urine. After 16 weeks of hypoxia, the mice also had higher hematocrits with lower serum glucose and various degrees of mesangiolytic glomerulosclerosis with microaneurysms and the infrequent occurrence of nodular lesions. Immunohistologically, hypoxic mice showed significantly decreased endothelial cell densities early during hypoxia and decreased podocyte densities later. In both hypoxic and normoxic mice, glomerular macrophage and transforming growth factor-ß1 (TGF-ß1) staining significantly increased with aging, without changes in vascular endothelial growth factor or endothelial nitric oxide synthase (eNOS). Glomerular mRNA expression of monocyte chemoattractant protein-1, eNOS and TGF-ß1 was significantly enhanced in the hypoxic mice. CONCLUSIONS: These results indicate that chronic hypoxia induces advanced glomerulosclerosis with accelerated albuminuria triggered by mesangiolysis and podocyte injury in a murine model of DN.

Short-chain fatty acid mitigates adenine-induced chronic kidney disease via FFA2 and FFA3 pathways.

Short-chain fatty acids (SCFAs), including acetate, butyrate, and propionate, are produced when colonic bacteria in the human gastrointestinal tract ferment undigested fibers. Free fatty acid receptor 2 (FFA2) and FFA3 are G-protein-coupled receptors recently identified as SCFA receptors that may modulate inflammation. We previously showed through in vitro experiments that SCFAs activate FFA2 and FFA3, thereby mitigating inflammation in human renal cortical epithelial cells. This study used a murine model of adenine-induced renal failure to investigate whether or not SCFAs can prevent the progression of renal damage. We also examined whether or not these FFA2 and FFA3 proteins have some roles in this protective mechanism in vivo. Immunohistochemical analyses of mouse kidneys showed that FFA2 and FFA3 proteins were expressed mainly in the distal renal tubules and collecting tubules. First, we observed that the administration of propionate mitigated the renal dysfunction and pathological deterioration caused by adenine. Consistent with this, the expression of inflammatory cytokines and fibrosis-related genes was reduced. Furthermore, the mitigation of adenine-induced renal damage by the administration of propionate was significantly attenuated in FFA2-/- and FFA3-/- mice. Therefore, the administration of propionate significantly protects against adenine-induced renal failure, at least in part, via the FFA2 and FFA3 pathways. Our data suggest that FFA2 and FFA3 are potential new therapeutic targets for preventing or delaying the progression of chronic kidney disease.

ß-Hydroxybutyrate enhances the cytotoxic effect of cisplatin via the inhibition of HDAC/survivin axis in human hepatocellular carcinoma cells.

Ketone bodies, including acetoacetate and ß-hydroxybutyrate (ßOHB), are produced from acetyl coenzyme A in the liver and then secreted into the blood. These molecules are a source of energy for peripheral tissues during exercise or fasting. ßOHB has been reported to inhibit histone deacetylases (HDACs) 1, 3, and 4 in human embryonic kidney 293 cells. Thus, ßOHB may regulate epigenetics by modulating HDACs. There have been several reports that the administration of ßOHB or induction of a physiological state of ketosis has an antitumor effect; however, the mechanism remains unclear. The aim of this study was to investigate whether ßOHB enhances cisplatin-induced apoptosis in hepatocellular carcinoma (HCC) cells by modulating activity and/or expression of HDACs. We found that ßOHB significantly enhanced cisplatin-induced apoptosis and cleavage of caspase-3 and -8 in HCC cells. Further, ßOHB significantly decreased the expression of HDCA 3/5/6 and survivin in liver hepatocellular (HepG2) cells. In HDAC3/6 gene silencing, survivin expression was significantly decreased, and cisplatin-induced cleavage of caspase-3 was significantly enhanced compared with control in HepG2 cells. In conclusion, ßOHB enhanced cisplatin-induced apoptosis via HDAC3/6 inhibition/survivin axis in HepG2 cells, which suggests that ßOHB could be a new adjuvant agent for cisplatin chemotherapy.

ß-Hydroxybutyrate, a ketone body, reduces the cytotoxic effect of cisplatin via activation of HDAC5 in human renal cortical epithelial cells.

AIMS: ß-Hydroxybutyrate (ßOHB) is a metabolic intermediate that constitutes about 70% of ketone bodies produced in liver from oxidation of fatty acids released from adipose tissue. A recent study showed that ßOHB inhibits HDAC1, 3 and 4 (classes I and IIa) in human embryonic kidney 293 (HEK293) cells. Therefore, ßOHB could regulate epigenetics via modulating HDACs. However, little is known about the protective effect of ßOHB on renal cells through epigenetics. The aim of this study is to investigate whether ßOHB reduces cisplatin-induced nephrotoxicity in human renal cortical epithelial (HRCE) cells by modulating HDACs. MAIN METHODS: In this study, we used human renal cortical epithelial (HRCE) cells. The anti-apoptotic effect of ßOHB was evaluated using flow cytometry analysis. The expression of apoptosis-related proteins and HDACs was evaluated by western immunoblot. KEY FINDINGS: The results showed that ßOHB significantly reduced cisplatin-induced apoptosis in HRCE cells. Furthermore, ßOHB significantly reduced cisplatin-induced cleavage of caspase-3, acetylation of histone H3, and phosphorylation of AMP-activated kinase. This anti-apoptotic effect of ßOHB was markedly attenuated by an inhibitor of HDAC4/5, and ßOHB-mediated suppression of cleavage of caspase3 was significantly blocked by siRNA-induced gene silencing of HDAC5. SIGNIFICANCE: ßOHB attenuates cisplatin-induced apoptosis by activation of HDAC5 in HRCE cells, suggesting that ßOHB may be a new therapeutic agent for cisplatin nephropathy.

Elevated Levels of Urinary Extracellular Vesicle Fibroblast-Specific Protein 1 in Patients with Active Crescentic Glomerulonephritis.

BACKGROUND/AIMS: Extracellular vesicles (EVs), including exosomes, are present in various bodily fluids, including urine. We and others previously reported that cells expressing fibroblast-specific protein 1 (FSP1) accumulate within damaged glomeruli, and that urinary FSP1, as well as urinary soluble CD163, could potentially serve as a biomarker of ongoing glomerular injury. METHODS: To test that idea, we collected urine samples from 37 patients with glomerular disease; purified the urinary EVs; characterized them using Nanosight, western blotting, and immunoelectron microscopy; and determined FSP1 and soluble CD163 levels using enzyme-linked immunosorbent assays. RESULTS: Deemed to be mainly exosomes based on their size distribution, the EVs in urine contained FSP1, and a portion of the FSP1-positive vesicles was also positive for podocalyxin. FSP1 levels in urinary EVs were (1) positively correlated with rates of biopsy-proven cellular crescent formation (r = 0.562, p < 0.001) and total crescent formation (r = 0.448, p = 0.005) among total glomeruli; (2) significantly higher in patients with cellular crescents affecting 20% or more of their glomeruli than in those with fewer affected glomeruli (p = 0.003); and (3) significantly decreased after glucocorticoid and immunosuppressant therapy (p < 0.05). A positive correlation between FSP1 levels in urinary EVs and urinary soluble CD163 levels was confirmed (r = 0.367, p < 0.05). CONCLUSION: These data suggest that a portion of urinary FSP1 is secreted as EVs originating from podocytes, and that FSP1 levels reflect active and ongoing glomerular injury and disease activity, such as cellular crescent formation.

A short-chain fatty acid, propionate, enhances the cytotoxic effect of cisplatin by modulating GPR41 signaling pathways in HepG2 cells.

Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are generated by microbial fermentation of indigestible fiber by gut flora. SCFAs are ligands of two orphan G protein-coupled receptors, GPR41 and GPR43, that modulate cell proliferation and induce apoptosis. However, it is unclear if SCFAs enhance the effects of chemotherapy in a GPR41- or GPR43-dependent manner. The aim of this study was to investigate whether SCFAs, and particularly propionate, activate GPR41 or GPR43, and thereby enhance the antitumor effects of cisplatin in HepG2 human hepatocellular carcinoma (HCC) cells. The inhibitory effects of propionate and cisplatin on proliferation of HCC cells were determined by MTS assay. Changes in apoptosis rate were analyzed by flow cytometry. The effects of combined propionate and cisplatin on these properties in HCC cells were significantly higher than those of cisplatin alone. With combined treatment, the levels of cleaved caspase-3, active caspase-3 forms, and acetylated histone H3 were enhanced in a GPR41-dependent manner; expression of histone deacetylases (HDAC) 3, 4, 5, 6, 8 proteins was significantly reduced; and induction of TNF-α expression was significantly enhanced. These results suggest that propionate and cisplatin synergistically and significantly induce apoptosis of HepG2 cells by increasing expression of autocrine TNF-α via reduction of HDACs through GPR41 signaling. From clinical and translational perspectives, our data suggest that a combination of propionate with cisplatin may have better therapeutic effects on HCC compared with conventional treatment, and that a selective GPR41 agonist may be a candidate as an adjuvant therapeutic agent for HCC.

Short-chain fatty acids, GPR41 and GPR43 ligands, inhibit TNF-α-induced MCP-1 expression by modulating p38 and JNK signaling pathways in human renal cortical epithelial cells.

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced predominantly by gut microbiota fermentation of dietary fiber. SCFAs are newly identified as endogenous ligands of two orphan G protein-coupled receptors, GPR41 and GPR43, which have the potential to modulate inflammation. Therefore, GPR41 and GPR43 may mediate the link between the gut microbiome status and various disease conditions including renal inflammation. This study aimed at investigating whether SCFAs activate GPR41 and GPR43, and thereby exert anti-inflammatory effects in human renal cortical epithelial cells (HRCEs) as a main component of kidney tissue. Immunohistochemical analyses of human renal biopsy specimens revealed the expression of GPR41 and GPR43 protein in the distal renal tubules and collecting tubules. TNF-α increased the expression of monocyte chemoattractant protein-1 (MCP-1), a potential fibrotic inducer, at least partly via enhancing phosphorylation of p38 and JNK in HRCEs. SCFAs, especially propionate, attenuated TNF-α- stimulated MCP-1 expression by inhibiting the phosphorylation of p38 and JNK. This inhibitory effect was considerably attenuated by an inactivator of the Gi/o-type G protein and a Gßγ (i/o) blocker, but not by a Gα (i/o) blocker. Furthermore, SCFA-mediated inhibition of MCP-1 expression was significantly blocked by siRNA-induced gene silencing of GPR41 and GPR43. In conclusion, SCFAs lowered TNF-α-induced MCP-1 expression by reducing phosphorylation of p38 and JNK in a GPR41/43-dependent manner in HRCEs, suggesting that SCFA modification may be a new therapeutic tool for preventing progression of renal inflammation and fibrosis.

Serum CETP status is independently associated with reduction rates in LDL-C in pitavastatin-treated diabetic patients and possible involvement of LXR in its association.

BACKGROUND: Statins decrease cholesteryl ester transfer protein (CETP) levels, which have been positively associated with hepatic lipid content as well as serum low density lipoproteins-cholesterol (LDL-C) levels. However, the relationship between the CETP status and statin-induced reductions in LDL-C levels has not yet been elucidated in detail. We herein examined the influence of the CETP status on the lipid-reducing effects of pitavastatin in hypercholesterolemic patients with type 2 diabetes mellitus as well as the molecular mechanism underlying pitavastatin-induced modifications in CETP levels. METHODS: Fifty-three patients were treated with 2 mg of pitavastatin for 3 months. Serum levels of LDL-C, small dense (sd) LDL-C, and CETP were measured before and after the pitavastatin treatment. The effects of pitavastatin, T0901317, a specific agonist for liver X receptor (LXR) that reflects hepatic cholesterol contents, and LXR silencing on CETP mRNA expression in HepG2 cells were also examined by a real-time PCR assay. RESULTS: The pitavastatin treatment decreased LDL-C, sdLDL-C, and CETP levels by 39, 42, and 23%, respectively. Despite the absence of a significant association between CETP and LDL-C levels at baseline, baseline CETP levels and its percentage change were an independent positive determinant for the changes observed in LDL-C and sdLDL-C levels. The LXR activation with T0901317 (0.5 µM), an in vitro condition analogous to hepatic cholesterol accumulation, increased CETP mRNA levels in HepG2 cells by approximately 220%, while LXR silencing markedly diminished the increased expression of CETP. Pitavastatin (5 µM) decreased basal CETP mRNA levels by 21%, and this was completely reversed by T0901317. CONCLUSION: Baseline CETP levels may predict the lipid-reducing effects of pitavastatin. Pitavastatin-induced CETP reductions may be partially attributed to decreased LXR activity, predictable by the ensuing decline in hepatic cholesterol synthesis. TRIAL REGISTRATION: UMIN Clinical Trials Registry ID UMIN000019020.

Telmisartan, a possible PPAR-δ agonist, reduces TNF-α-stimulated VEGF-C production by inhibiting the p38MAPK/HSP27 pathway in human proximal renal tubular cells.

Vascular endothelial growth factor-C (VEGF-C) is a main inducer of inflammation-associated lymphangiogenesis in various inflammatory disorders including chronic progressive kidney diseases, for which angiotensin II receptor type 1 blockers (ARBs) are widely used as the main treatment. Although proximal renal tubular cells may affect the formation of lymphatic vessels in the interstitial area by producing VEGF-C, the molecular mechanisms of VEGF-C production and its manipulation by ARB have not yet been examined in human proximal renal tubular epithelial cells (HPTECs). In the present study, TNF-α dose-dependently induced the production of VEGF-C in HPTECs. The TNF-α-induced production of VEGF-C was mediated by the phosphorylation of p38MAPK and HSP27, but not by that of ERK or NFkB. Telmisartan, an ARB that can activate the peroxisome proliferator-activated receptor (PPAR), served as a PPAR-δ activator and reduced the TNF-α-stimulated production of VEGF-C. This reduction was partially attributed to a PPAR-δ-dependent decrease in p38MAPK phosphorylation. Our results indicate that TNF-α induced the production of VEGF-C in HPTECs by activating p38MAPK/HSP27, and this was partially inhibited by telmisartan in a PPAR-δ dependent manner. These results provide a novel insight into inflammation-associated lymphangiogenesis.

Telmisartan activates endogenous peroxisome proliferator-activated receptor-δ and may have anti-fibrotic effects in human mesangial cells.

Telmisartan, an angiotensin II receptor type 1 blocker (ARB), was recently reported to promote lipolysis in mice by acting as a peroxisome proliferator-activated receptor (PPAR)-δ activator, although in clinical studies, it has also been recognized to activate PPAR-γ as a major cause of its pleiotropic actions. The aim of this study was to investigate whether telmisartan activates endogenous PPAR-δ and thereby exerts anti-fibrotic effects in human mesangial cells (HMC). Immunohistochemical analysis of human renal biopsy specimens revealed that PPAR-δ protein was detected in the HMC of glomeruli with moderately proliferative changes. In the HMC, both GW0742, an authentic PPAR-δ agonist, and telmisartan enhanced PPAR response element (PPRE)-luciferase activity dose dependently, and these increases were blunted by GSK0660, a specific PPAR-δ antagonist, but not by GW9662, a PPAR-γ antagonist. Telmisartan also upregulated the expression of PPAR-δ target genes related to fatty acid oxidation; that is, heart type-fatty acid-binding protein and uncoupling protein-2. These effects were inhibited by both PPAR-δ antagonism and PPAR-δ gene silencing. Transforming growth factor-ß1 (TGF-ß1) increased the expression of plasminogen activator inhibitor-1 (PAI-1), TGF-ß1 and collagen IV. The PAI-1 expression was mediated, at least in part by the phosphorylation of extracellular signal-regulated kinases (ERKs). Telmisartan suppressed TGF-ß1-stimulated PAI-1 and collagen IV expression and ERK phosphorylation, and these effects were weakened by PPAR-δ antagonism, whereas eprosartan, a non-PPAR activating ARB, did not affect TGF-ß1-stimulated PAI-1 expression. These results indicate that in HMC telmisartan activates endogenous PPAR-δ and may prevent TGF-ß1-induced fibrotic changes by reducing ERK phosphorylation in a PPAR-δ-dependent manner, and thus, might be useful for treating hypertensive patients with renal and metabolic disorders.

Glucocorticoid enhances hypoxia- and/or transforming growth factor-ß-induced plasminogen activator inhibitor-1 production in human proximal renal tubular cells.

BACKGROUND: Glucocorticoid (GC) treatment reportedly exaggerates renal fibrosis in progressive kidney diseases during which hypoxia occurs as an unavoidable consequence in renal tubular cells. Two major fibrotic factors, hypoxia and transforming growth factor-ß (TGF-ß), upregulate the production of plasminogen activator inhibitor-1 (PAI-1), a fibrosis enhancer. Most recently, we reported that GC increases PAI-1 production in human proximal tubular epithelial cells (HPTEC). However, the detailed interactions that occur between these PAI-1 inducers in HPTEC remain to be clarified. METHODS: Confluent HPTECs were treated with GC and/or TGF-ß for 24 h under normoxia or hypoxia. The mRNA and protein amounts of PAI-1 and GC receptor (GR) were determined by TaqMan quantitative PCR and immunoassays, respectively. GC and hypoxia response element (GRE and HRE) activities were measured by transient transfection of GRE- and HRE-luciferase expression vector. RESULTS: Hypoxia had no influence on dexamethasone (DXA)-enhanced GRE activity, as DXA had no influence on hypoxia-enhanced HRE activity. Hypoxia induced PAI-1 expression. TGF-ß increased basal and hypoxia-stimulated PAI-1 production. Hydrocortisone (HC) and DXA increased hypoxia- or TGF-ß-stimulated production of PAI-1 mRNA and protein. Moreover, DXA enhanced hypoxia plus TGF-ß-stimulated PAI-1 production. The PAI-1-increasing effect of HC under hypoxia was abolished completely by RU-486, a specific inhibitor of the GR, and largely by PP2, a specific inhibitor of the Src family of protein tyrosine kinases. CONCLUSION: Glucocorticoid induces hypoxia- and hypoxia plus TGF-ß-stimulated PAI-1 production via the GR and tyrosine kinase pathways. These actions of GC may partially explain the renal fibrotic changes seen in progressive inflammatory kidney diseases during GC treatment.

Dexamethasone enhances basal and TNF-alpha-stimulated production of PAI-1 via the glucocorticoid receptor regardless of 11beta-hydroxysteroid dehydrogenase 2 status in human proximal renal tubular cells.

BACKGROUND: Long-term treatment with glucocorticoids (GCs) reportedly exaggerates renal fibrosis in chronic progressive inflammatory kidney disease. GCs induce the gene expression of plasminogen activator inhibitor-1 (PAI-1), a fibrosis enhancer in non-renal cells. Tumour necrosis factor-alpha (TNF-alpha) reduces the gene expression of 11beta-hydroxysteroid dehydrogenase (HSD) 2, an inactivator of GCs, and may enhance GC activity. However, the individual and collective effects of adrenal steroids, TNF-alpha and HSD2 status on PAI-1 production are unknown in human proximal renal tubular epithelial cells (HPTECs). METHODS: Confluent HPTECs were treated with adrenal steroids (10 nM to 10 microM) or TNF-alpha (10 ng/ml) for up to 48 h. The mRNA amounts of the target genes were determined by TaqMan quantitative PCR, and the PAI-1 protein amounts were measured by an immunoassay. RESULTS: Dexamethasone (DXA) maximally increased the amounts of PAI-1 mRNA and protein at 100 nM. Aldosterone (Ald) increased PAI-1 expression dose dependently, but the effect was over 100-fold weaker than that of DXA. The PAI-1-increasing effects of DXA and Ald were abolished completely by U-486, a specific inhibitor of the glucocorticoid receptor (GR) but not by spironolactone, a specific inhibitor of the mineralocorticoid receptor. The effect of DXA was also blocked partially by AG1478 and herbimycin A, tyrosine kinase inhibitors. DXA further increased TNF-alpha-stimulated PAI-1 expression via the GR. Although TNF-alpha treatment caused an 80% reduction in the gene expression of HSD2, an inactivator of GCs, HSD2 inhibition did not enhance DXA-induced PAI-1 production. CONCLUSIONS: DXA induces basal and TNF-alpha-stimulated PAI-1 expression via the GR pathway, regardless of HSD2 status in HPTECs. Excess GCs may serve as a pro-fibrotic factor in chronic inflammatory kidney diseases.