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• W2072986997 abstract "Progression of diabetic nephropathy appears directly related to renal tubulointerstitial injury, but the involved genes are incompletely delineated. To identify such genes, DNA microarray analysis was performed with RNA from renal proximal tubules (RPTs) of streptozotocin-induced diabetic Wistar rats, spontaneously diabetic BioBreeding rats, and rat immortalized renal proximal tubular cells (IRPTCs) exposed to high glucose (25 mM) medium for 2 weeks. Osteopontin (OPN) mRNA expression was quantified by real time-quantitative polymerase chain reaction (RT-qPCR) or conventional reverse transcriptase-polymerase chain reaction (RT-PCR). OPN mRNA expression was upregulated (5–70-fold increase) in diabetic rat RPTs and in IRPTCs chronically exposed to high glucose compared to control RPTs and IRPTCs. High glucose, angiotensin II, phorbol 12-myristate 13-acetate and transforming growth factor-beta 1 (TGF-β1) stimulated OPN mRNA expression in IRPTCs in a dose- and time-dependent manner. This effect was inhibited by tiron, taurine, diphenylene iodinium, losartan, perindopril, calphostin C, or LY 379196 but not PD123319. IRPTCs overexpressing dominant-negative protein kinase C-beta 1 (PKC-β1) cDNA or antisense TGF-β1 cDNA prevented the high glucose effect on OPN mRNA expression. We concluded that high glucose-mediated increases in OPN gene expression in diabetic rat RPTs and IRPTCs are mediated, at least in part, via reactive oxygen species generation, intrarenal rennin–angiotensin system activation, TGF-β1 expression, and PKC-β1 signaling. Progression of diabetic nephropathy appears directly related to renal tubulointerstitial injury, but the involved genes are incompletely delineated. To identify such genes, DNA microarray analysis was performed with RNA from renal proximal tubules (RPTs) of streptozotocin-induced diabetic Wistar rats, spontaneously diabetic BioBreeding rats, and rat immortalized renal proximal tubular cells (IRPTCs) exposed to high glucose (25 mM) medium for 2 weeks. Osteopontin (OPN) mRNA expression was quantified by real time-quantitative polymerase chain reaction (RT-qPCR) or conventional reverse transcriptase-polymerase chain reaction (RT-PCR). OPN mRNA expression was upregulated (5–70-fold increase) in diabetic rat RPTs and in IRPTCs chronically exposed to high glucose compared to control RPTs and IRPTCs. High glucose, angiotensin II, phorbol 12-myristate 13-acetate and transforming growth factor-beta 1 (TGF-β1) stimulated OPN mRNA expression in IRPTCs in a dose- and time-dependent manner. This effect was inhibited by tiron, taurine, diphenylene iodinium, losartan, perindopril, calphostin C, or LY 379196 but not PD123319. IRPTCs overexpressing dominant-negative protein kinase C-beta 1 (PKC-β1) cDNA or antisense TGF-β1 cDNA prevented the high glucose effect on OPN mRNA expression. We concluded that high glucose-mediated increases in OPN gene expression in diabetic rat RPTs and IRPTCs are mediated, at least in part, via reactive oxygen species generation, intrarenal rennin–angiotensin system activation, TGF-β1 expression, and PKC-β1 signaling. Renal proximal tubules (RPTs) play a role in tubulointerstitial fibrosis has been addressed recently.1.Gilbert R.E. Cooper M.E. The tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury?.Kidney Int. 1999; 56: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar, 2.Phillips A.O. Steadman R. Diabetic nephropathy: the central role of renal proximal tubular cells in tubulointerstitial injury.Histol Histopathol. 2002; 17: 247-252PubMed Google Scholar, 3.Phillips A.O. The role of renal proximal tubular cells in diabetic nephropathy.Curr Diab Rep. 2003; 3: 491-496Crossref PubMed Scopus (63) Google Scholar A number of cytokine systems such as insulin-like growth factor-1,4.Bach L.A. Jerums G. Effect of puberty on initial kidney growth and rise in kidney IGF-I in diabetic rats.Diabetes. 1990; 39: 557-562Crossref PubMed Scopus (71) Google Scholar epidermal growth factor,5.Gilbert R.E. Cox A. McNally P.G. et al.Increased epidermal growth factor expression in diabetes related kidney growth.Diabetologia. 1997; 40: 778-785Crossref PubMed Scopus (53) Google Scholar endothelin-1,6.Benigni A. Colosio W. Brena C. et al.Unselective inhibition of endothelin receptors reduces renal dysfunction in experimental diabetes.Diabetes. 1998; 47: 450-456Crossref PubMed Scopus (110) Google Scholar transforming growth factor-beta 1 (TGF-β1),7.Sharma K. Ziyadeh F.N. Renal hypertrophy is associated with upregulation of TGF-beta 1 gene expression in diabetic BB rat and NOD mouse.Am J Physiol. 1994; 267: F1094-F1101PubMed Google Scholar and angiotensin II (Ang II)8.Wolf G. Mueller E. Stahl R.A.K. Ziyadeh F.N. Angiotensin II-induced hypertrophy of cultured murine proximal tubular cells is mediated by endogenous transforming growth factor-β.J Clin Invest. 1993; 92: 1366-1372Crossref PubMed Google Scholar have been implicated in tubulointerstitial injury. Renin–angiotensin system (RAS) blockade and neutralizing anti-TGF-β1 antibodies ameliorate renal hypertrophy and suppress matrix protein mRNA expression in murine RPTs,9.Sharma K. Jin Y. Guo J. Ziyadeh F.N. Neutralization of TGF-beta by TGF-beta antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice.Diabetes. 1996; 45: 522-530Crossref PubMed Scopus (0) Google Scholar, 10.Han D.C. Hoffman B.B. Hone S.W. et al.Therapy with antisense TGF-beta 1 oligodeoxynucleotides reduces kidney weight and matrix mRNA in diabetic mice.Am J Physiol. 2000; 278: F628-F634PubMed Google Scholar indicating the importance of these factors in the progression of diabetic nephropathy. Gene profiling studies concerning the development of diabetic nephropathy11.Wada J. Zhang H. Tsuchiyama Y. et al.Gene expression profile in streptozotocin-induced diabetic mice kidneys undergoing glomerulosclerosis.Kidney Int. 2001; 59: 1363-1373Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 12.Goruppi S. Bonventre J.V. Kyriakis J.M. Signaling pathways and late-onset gene induction associated with renal mesangial cell hypertrophy.EMBO J. 2002; 21: 5427-5436Crossref PubMed Scopus (67) Google Scholar, 13.Fan Q. Shike T. Shigihara T. et al.Gene expression profile in diabetic KK/Ta mice.Kidney Int. 2003; 64: 1978-1985Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 14.Wilson K.H. Eckenrode S.E. Li Q.Z. et al.Microarray analysis of gene expression in the kidneys of new- and post-onset diabetic NOD mice.Diabetes. 2003; 52: 2151-2159Crossref PubMed Scopus (55) Google Scholar, 15.Baelde H.J. Eikmans M. Doran P.P. et al.Gene expression profiling in glomeruli from human kidneys with diabetic nephropathy.Am J Kidney Dis. 2004; 43: 636-650Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 16.Susztak K. Bottinger E. Novetsky A. et al.Molecular profiling of diabetic mouse kidney reveals novel genes linked to glomerular disease.Diabetes. 2004; 53: 784-794Crossref PubMed Scopus (123) Google Scholar have identified osteopontin (OPN), as a candidate upregulated in experimental diabetic glomerular disease. In the kidneys, OPN is normally expressed in the descending thin limb of the loop of Henle and collecting ducts of the medulla but is absent from renal cortical tubules.17.Brown L.F. Berse B. Van de Water L. et al.Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces.Mol Biol Cell. 1992; 3: 1169-1180Crossref PubMed Scopus (398) Google Scholar, 18.Mazzali M. Kipari T. Ophascharoensuk V. et al.Osteopontin – a molecule for all seasons.Q J Med. 2002; 95: 3-13Crossref Scopus (331) Google Scholar OPN expression is, however, elevated in established experimental models such as diabetic nephropathy in the rat,19.Gilbert R.E. Progression of tubulointerstitial injury by osteopontin-induced recruitment in advanced diabetic nephropathy of transgenic (mRen-2) 27 rats.Nephrol Dial Transplant. 2002; 17: 985-991Crossref PubMed Scopus (32) Google Scholar, 20.Li C. Yang C.W. Park C.W. et al.Long-term treatment with ramipril attenuates renal osteopontin expression in diabetic rats.Kidney Int. 2003; 63: 454-463Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar models with unilateral ureteral obstruction,21.Diamond J.R. Kriesberg R. Evans R. et al.Regulation of proximal tubular osteopontin in experimental hydronephrosis in the rat.Kidney Int. 1998; 54: 1501-1509Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 22.Ricardo S.D. Franzoni D.F. Roesner C.D. et al.Angiotensinogen and AT(1)R inhibition of osteopontin translation in rat proximal tubular cells.Am J Physiol Renal Physiol. 2000; 278: F708-F716PubMed Google Scholar and renal disease associated with cortical tubulointerstitial fibrosis.23.Giachelli C.M. Pichler R. Lombardi D. et al.Osteopontin expression in angiotensin II-induced tubulointerstitial nephritis.Kidney Int. 1994; 45: 515-524Abstract Full Text PDF PubMed Scopus (217) Google Scholar, 24.Pichler R. Giachelli C.M. Lombardi D. et al.Tubulointerstitial disease in glomerulonephritis: potential role of osteopontin.Am J Pathol. 1994; 144: 915-926PubMed Google Scholar Such observations, together with the apparent role of OPN in inflammation and cell reruitment, suggest that OPN may play a role in the evolution of diabetic nephropathy.25.Denhardt D.T. Noda M. O'Regan A.W. et al.Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival.J Clin Invest. 2001; 107: 1055-1061Crossref PubMed Scopus (915) Google Scholar We performed DNA microarray profiling to identify genes uniquely upregulated within diabetic rat RPTs. We observed that OPN was upregulated both in diabetic rat RPTs and in immortalized renal proximal tubular cells (IRPTCs) chronically exposed to high glucose. We then demonstrated that the high glucose effect on OPN gene expression in IRPTCs is mediated, at least in part, via reactive oxygen species (ROS) generation, RAS activation, protein kinase C-beta 1 (PKC-β1) signaling, and TGF-β1 gene expression. Table 1 showed the physical data. Weight gain was significantly lower after 2 weeks (day 14) of diabetes but was improved with insulin treatment. Animals in both diabetic groups had elevated blood glucose and a higher ratio of kidney weight/body weight as compared to non-diabetic group. Normalization of blood glucose by insulin reversed the ratio of kidney weight/body weight to non-diabetic levels.Table 1Physical parameters of the animalsWistar ratBB ratControlSTZSTZ+InsNon-diabetesDiabetesDiabetes+InsBody weight (g) – day 0257±4.1247±6.5250±11.8222±24.5228±41219±42.4Body weight (g) – day 14342±18.9aP<0.001 when day 14 compared with day 0; n=8 for each group.185±15.6a,P<0.001 when day 14 compared with day 0; n=8 for each group.bP<0.001 when STZ (diabetes) compared with control (non-diabetes); n=8 for each group.331±15.0a,P<0.001 when day 14 compared with day 0; n=8 for each group.cP<0.001 when STZ+Ins (diabetes+Ins) compared with STZ (diabetes); n=8 for each group.367±25.8aP<0.001 when day 14 compared with day 0; n=8 for each group.304±44.1a,P<0.001 when day 14 compared with day 0; n=8 for each group.bP<0.001 when STZ (diabetes) compared with control (non-diabetes); n=8 for each group.343±28.2a,P<0.001 when day 14 compared with day 0; n=8 for each group.cP<0.001 when STZ+Ins (diabetes+Ins) compared with STZ (diabetes); n=8 for each group.Blood glucose (mM) – day 07.9±0.77.8±0.78.0±6.26.9±0.39.1±5.56.8±0.2Blood glucose (mM) – day 147.6±0.733.6±2.0a,P<0.001 when day 14 compared with day 0; n=8 for each group.bP<0.001 when STZ (diabetes) compared with control (non-diabetes); n=8 for each group.6.2±5.96.6±0.531.5±6.6a,P<0.001 when day 14 compared with day 0; n=8 for each group.bP<0.001 when STZ (diabetes) compared with control (non-diabetes); n=8 for each group.5.9±2.5Kidney weight (g)1.1±0.11.1±0.11.2±0.11.1±0.11.3±0.11.2±0.1Ratio of body weight/kidney weight (% of control)100±5.8192±14.8dP<0.01 when STZ compared with control; n=8 for each group.110±8.0eP<0.01 when STZ+Ins compared with STZ; n=8 for each group.100±5.1141±10.4fP<0.05 when diabetes compared with non-diabetes; n=8 for each group.118±10.5gP<0.05 when diabetes+Ins compared with diabetes; n=8 for each group.Ins=insulin; STZ, streptozotocin.a P<0.001 when day 14 compared with day 0; n=8 for each group.b P<0.001 when STZ (diabetes) compared with control (non-diabetes); n=8 for each group.c P<0.001 when STZ+Ins (diabetes+Ins) compared with STZ (diabetes); n=8 for each group.d P<0.01 when STZ compared with control; n=8 for each group.e P<0.01 when STZ+Ins compared with STZ; n=8 for each group.f P<0.05 when diabetes compared with non-diabetes; n=8 for each group.g P<0.05 when diabetes+Ins compared with diabetes; n=8 for each group. Open table in a new tab Ins=insulin; STZ, streptozotocin. RAE230A expression chips analyzed the changes in mRNA levels of 15 925 genes and expressed sequence tags (ESTs). The percentage of these genes present in RPTs isolated from non-diabetic BioBreeding (BB) rats, Wistar rats, and control IRPTCs were 58.7, 57.8, and 28.9%, respectively. ESTs were excluded in the analysis. In RPTs isolated from diabetic BB rats, streptozotocin (STZ)-induced diabetic Wistar rats, and high glucose-treated IRPTCs, 144, 60, and 21 genes were upregulated, respectively (Figure 1a), while 83, 37, and 13 genes were downregulated, respectively (Figure 1b). Only two genes, OPN and CCAAT enhancer binding protein-delta (C/EBP-δ), were commonly upregulated in RPTs from diabetic BB rats, STZ-induced diabetic Wistar rats, and high glucose-treated IRPTCs (Table 2). Tables 3 and 4 identify the genes that most often increased or decreased in RPTs from diabetic BB rats and STZ-induced diabetic Wistar rats, respectively, with or without insulin treatment.Table 2Common genes upregulated in renal proximal tubules (RPTs) of diabetic BioBreeding (BB) and Wistar rats, and in rat immortalized renal proximal tubular cells (IRPTCs) chronically exposed to high glucose for 2 weeksFold changeGenes (mRNA)BBWistarIRPTCsMitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase mRNA51.983.25Insulin-like growth factor-binding protein 1 mRNA12.133.25Bile acid-coenzyme A dehydrogenase: amino-acid n-acyltransferase mRNA9.8510.56Organic anion transporter 5 mRNA9.854.29Cytochrome P450CMF1b mRNA8.002.64Kynurenine aminotransferase II mRNA4.923.48ATP-binding cassette, sub-family B (MDRTAP), member 11 (Abcb11), mRNA4.593.25Deubiquitinating enzyme Ubp69 mRNA4.295.66Pregnancy-induced growth inhibitor OKL38 mRNA4.002.14Rgc32 protein mRNA4.002.00Selenium-binding protein 2 mRNA3.732.00Basic helix–loop–helix domain-containing, class B, 3 mRNA3.482.4617-β hydroxysteroid dehydrogenase type 2 mRNA3.482.46Fibrinogen, an α polypeptide3.252.46Pyruvate dehydrogenase kinase, isoenzyme 4 mRNA3.252.00Kruppel-like factor 15 (kidney) mRNA2.832.64Sulfotransferase SULT1A1 mRNA2.832.64Solute carrier 6, member 6 (taurine transporter) mRNA2.832.14CCAAT enhancer-binding protein (CEBP)-δ mRNA2.462.006.06D site albumin promoter-binding protein2.304.59Osteopontin mRNA2.304.593Solute carrier family 2 A2 (glucose transporter, type 2) mRNA2.142.64Glutamylcysteine-γ synthetase light chain mRNA2.142.30Nuclear receptor Rev-ErbA-β mRNA2.002.30Bold values indicate genes that are commonly regulated in all three groups of BB, Wistar, and IRPTCs. Open table in a new tab Table 3Common genes downregulated in renal proximal tubules (RPTs) of diabetic BioBreeding (BB) and Wistar ratsFold changeGenes (mRNA)BBWistarInsulin-like growth factor-binding protein 5-19.70-3.25Insulin-like growth factor-binding protein 5-3.48-2.14Kidney androgen-regulated protein-13.93-6.06Hemoglobin, α1-4.29-4.59Hemoglobin, α1-3.48-3.48Hemoglobin, α1-2.30-2.14Calpain 6-4.29-2.30Cyclooxygenase 2-4.00-2.14Ly6-B antigen-4.00-2.00Hydroxysteroid dehydrogenase, 11 β type 2-3.73-2.64Stearoyl-coenzyme A desaturase 2-3.25-5.66Hemoglobin, β-2.83-3.03Hemoglobin, β-2.46-3.25Protein associating with small stress protein PASS1-2.64-2.83Thyroxine deiodinase, type I-2.64-2.64Insulin-like growth factor I-2.64-2.64SKR6 gene, a CB1 cannabinoid receptor-2.64-2.14Solute carrier family 34 (sodium phosphate), member 2-2.46-2.00Fatty acid desaturase 1-2.14-2.14Voltage-dependent calcium channel γ subunit-like protein-2.14-2.00Prostaglandin D synthetase-2.00-2.30Tissue-type transglutaminase-2.00-2.30Cadherin EGF LAG seven-pas G-type receptor 2-2.00-2.14Bold values indicate genes that are commonly downregulated in all two groups of BB and Wistar. Open table in a new tab Table 4Common genes regulated by insulin treatmentFold changeBBWistarGenes (mRNA)DiabetesaDiabetic group compared with non-diabetic group.Diabetes+InsulinbDiabetic with insulin treatment group compared with diabetic group.STZaDiabetic group compared with non-diabetic group.STZ+InsulinbDiabetic with insulin treatment group compared with diabetic group.Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase51.98-22.633.25-7.46Insulin-like growth factor-binding protein 112.13-17.153.25-3.73Bile acid-coenzyme A dehydrogenase: amino-acid n-acyltransferase9.85-6.5010.56-8.57Organic anion transporter 59.85-6.064.29Cytochrome P450CMF1b8.00-2.832.64-2.46Kynurenine aminotransferase II4.92-3.033.48-2.64ATP-binding cassette, sub-family B (MDRTAP), member 11 (Abcb11)4.59-7.463.25-2.14Deubiquitinating enzyme Ubp694.29-2.835.66-4.92Pregnancy-induced growth inhibitor OKL384.00-2.142.14Rgc32 protein4.00-2.832.00Selenium-binding protein 23.73-2.002.00Basic helix–loop–helix domain-containing, class B, 33.48-2.832.46-4.9217-β hydroxysteroid dehydrogenase type 23.48-3.252.46Fibrinogen, an α polypeptide3.25-3.732.46Pyruvate dehydrogenase kinase, isoenzyme 43.25-3.732.00Kruppel-like factor 15 (kidney)2.83-2.832.64-2.30Sulfotransferase SULT1A12.83-3.482.64-2.64Solute carrier 6, member 6 (taurine transporter)2.83-2.302.14-2.46CCAAT enhancer-binding protein (CEBP)-δ2.46-3.732.00D site albumin promoter-binding protein2.304.59-3.03Osteopontin2.30-2.464.59-3.03Solute carrier family 2 A2 (glucose transporter, type 2)2.14-2.002.64-2.00Insulin-like growth factor-binding protein 5-19.702.30-3.252.14Kidney androgen-regulated protein-13.9317.15-6.065.28Hemoglobin, α1-4.292.46-4.592.00Calpain 6-4.292.00-2.30Hydroxysteroid dehydrogenase, 11 β type 2-3.73-2.642.46Stearoyl-coenzyme A desaturase 2-3.254.59-5.663.48Protein associating with small stress protein PASS1-2.644.00-2.832.83Thyroxine deiodinase, type I-2.643.73-2.642.46Insulin-like growth factor I-2.64-2.643.48Fatty acid desaturase 1-2.143.03-2.14Voltage-dependent calcium channel γ subunit-like protein-2.142.30-2.002.30Prostaglandin D synthetase-2.002.30-2.302.30Tissue-type transglutaminase-2.002.14-2.302.14Cadherin EGF LAG seven-pas G-type receptor 2-2.002.14-2.142.00BB, bio breeding; STZ, streptozotocin.Bold values indicate genes that are commonly up- or downregulated in two groups of BB and Wistar.a Diabetic group compared with non-diabetic group.b Diabetic with insulin treatment group compared with diabetic group. Open table in a new tab Bold values indicate genes that are commonly regulated in all three groups of BB, Wistar, and IRPTCs. Bold values indicate genes that are commonly downregulated in all two groups of BB and Wistar. BB, bio breeding; STZ, streptozotocin. Bold values indicate genes that are commonly up- or downregulated in two groups of BB and Wistar. We used a specific RT-qPCR for rat OPN mRNA to validate DNA microarray findings. Figure 2a, b, and c illustrate the results of OPN mRNA expression in freshly isolated RPTs from Wistar rats, BB rats, and IRPTCs, respectively. In Wistar rats with STZ-induced diabetes, OPN mRNA expression was increased approximately 70-fold (P<0.001) (n=14) as compared to the controls. This increment was reversed by insulin treatment (P<0.001; n=14). In BB rats, OPN mRNA expression was elevated greater than fivefold in the diabetic group as compared to the non-diabetic controls (P<0.001; n=10). Insulin treatment prevented the increase of OPN mRNA in diabetic BB rats. The basal expression of OPN mRNA in non-diabetic BB rats, however, was twofold higher as compared to non-diabetic Wistar rats (P<0.01) (data not shown). Prolonged exposure of IRPTCs to high glucose (i.e., 25 mM) for 2 weeks also induced a fivefold increase (P<0.001) in OPN mRNA expression compared to controls incubated in normal glucose (5 mM) (n=9). Insulin (10−7 M) also reversed the high glucose effect on OPN mRNA expression in IRPTCs (P<0.001; n=9). Quantification by conventional reverse transcription-polymerase chain reaction (RT-PCR) revealed that 25 mM D-glucose stimulates OPN mRNA expression in IRPTCs in a time-dependent manner (Figure 3a). A significant increase of OPN mRNA expression (≥2-fold) was observed after 24-h incubation and rose progressively thereafter. In contrast, 25 mM D-mannitol had no significant effect, indicating that upregulation of OPN gene expression is not due to increased osmolarity (Figure 3b). Conventional RT-PCR for OPN mRNA was subsequently used in all experiments. To elucidate the underlying pathway of high glucose regulation of OPN gene expression in IRPTCs, inhibitors of several signaling pathways were tested. As illustrated in Figure 4a, diphenylene iodinium (DPI) (1 × 10−6 M), tiron (1 × 10−4 M), taurine (1 × 10−3 M), and Mn (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) (8 × 10−5 M) blocked the effects of high glucose on OPN mRNA expression. These results indicate that ROS generation might mediate, at least in part, the high glucose effect on OPN mRNA expression. Furthermore, high glucose effect on OPN mRNA expression was inhibited in the presence of losartan (1 × 10−6 M), perindopril (1 × 10−5 M), calphostin C (1 × 10−7 M), and LY 379196 (1 × 10−7 M), but not in the presence of PD123319 (an AT2R blocker) (1 × 10−5 M) (Figure 4b). These results suggest that upregulation of OPN gene expression by high glucose might be mediated, at least in part, by intrarenal RAS activation and PKC signaling. Figure 5a reveals that Ang II stimulated OPN mRNA expression in IRPTCs with an optimal effect at 10−9 M. The stimulatory effect of Ang II was inhibited in the presence of losartan (1 × 10−6 M), calphostin C (1 × 10−7 M), LY 379196 (1 × 10−7 M), tiron (1 × 10−4 M), or DPI (1 × 10−6 M) but not in the presence of PD123319 (1 × 10−5 M) or perindopril (1 × 10−5 M) (Figure 5b). These data indicate that the Ang II effect on OPN mRNA expression is mediated via AT1 receptor (AT1R) activation, ROS generation, and PKC signaling but not AT2 receptor (AT2R). To confirm the role of PKC signaling in OPN mRNA expression, phorbol 12-myristate 13-acetate (PMA) was added to the culture media. Figure 6a illustrates that PMA stimulated OPN mRNA expression in a dose-dependent manner (i.e., 10−10–10−8 M). The addition of calphostin C and LY 379196 (a PKC-β inhibitor) reversed the PMA action on OPN mRNA expression (Figure 6b), demonstrating that the PKC-β signaling pathway is important in regulating OPN mRNA expression in IRPTCs. Figure 7a and b reveal that TGF-β1 stimulated OPN mRNA expression in IRPTCs in a dose- and time-dependent manner, respectively. Calphostin C and LY 379196 inhibited the stimulatory action of TGF-β1 on OPN mRNA expression in IRPTCs (Figure 8a). In addition, TGF-β1 had no effect on OPN mRNA expression in IRPTCs stably transfected with dominant-negative PKC-β1 (Figure 8a). Furthermore, IRPTCs stably transfected with sense TGF-β1 displayed significantly higher basal OPN mRNA expression compared to control cells (P<0.005), and high glucose further enhanced OPN mRNA expression in these cells (Figure 8b). In contrast, cells stably transfected with antisense TGF-β1 exhibited significantly lower OPN mRNA expression compared to the controls (P<0.01) and did not respond to high glucose stimulation (Figure 8b).Figure 8Effect of TGF-β1 on rat OPN mRNA expression in IRPTCs. (a) Cells were incubated for 24 h in normal glucose DMEM with/without TGF-β1 (1 ng/ml) in the absence or presence of calphostin C (1 × 10−7 M), LY379196 (1 × 10−7 M) or stably transfected with dominant-negative PKC-β1. (b) Cells stably transfected with pRC/RSV, pRC/RSV-TGF-β1 cDNA in sense or antisense orientation were incubated in normal glucose or 25 mM D-glucose for 48 h. Cells were collected and assayed for rat OPN mRNA levels by conventional RT-PCR. Each point represents the mean±s.d. of four independent experiments performed in duplicate. *P≤0.05; **P≤0.01; ***P≤0.001.View Large Image Figure ViewerDownload (PPT) Figure 9a indicates that high glucose, TGF-β1, and Ang II stimulated OPN mRNA expression but was abolished in IRPTCs that were stably transfected with dominant-negative (DN) PKC-β1. High glucose, TGF-β1,and Ang II also stimulated PKC-β1 phosphorylation in IRPTCs (Figure 9b). Similarly, the stimulatory effect of high glucose, TGF-β1, and Ang II on PKC-β1 phosphorylation was also inhibited in DN PKC-β1 stable clones. These data confirm that PKC-β1 may mediate, at least in part, the stimulatory effect of high glucose, TGF-β1, and Ang II action on OPN mRNA expression (Figure 10).Figure 10Hypothetical signaling pathway of high glucose on OPN gene expression in IRPTCs.View Large Image Figure ViewerDownload (PPT) Among 227 and 97 differentially expressed transcripts in RPTs from respective spontaneously diabetic BB rats and STZ-induced diabetic Wistar rats, only 43 were commonly expressed in RPTs of both experimental diabetes models (19% in BB rat RPTs, 44% in STZ-induced diabetic Wistar rat RPTs). OPN is one of two genes (CCAAT enhancer-binding protein-δ was the other) that is differentially upregulated in diabetic rat RPTs and in IRPTCs chronically exposed to high glucose. RT-qPCR confirmed this observation. OPN mRNA expression levels in diabetic rat RPTs and in IRPTCs chronically exposed to a high glucose milieu were 5–70-fold higher than in control RPTs and IRPTCs. Insulin treatment normalized OPN mRNA expression. These data are in agreement with findings that OPN expression is enhanced in the renal cortex and in mesangial cells of STZ-induced diabetic rat kidneys20.Li C. Yang C.W. Park C.W. et al.Long-term treatment with ramipril attenuates renal osteopontin expression in diabetic rats.Kidney Int. 2003; 63: 454-463Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 26.Fischer J.W. Tschope C. Reinecke A. et al.Upregulation of osteopontin expression in renal cortex of streptozotocin-induced diabetic rats is mediated by bradykinin.Diabetes. 1998; 47: 1512-1518Crossref PubMed Scopus (47) Google Scholar and in human diabetic RPTCs.27.Junaid A. Amara F.M. Osteopontin: correlation with interstitial fibrosis in human diabetic kidney and PI3-kinase-mediated enhancement of expression by glucose in human proximal tubular epithelial cells.Histopathology. 2004; 44: 136-146Crossref PubMed Scopus (40) Google Scholar, 28.Xie Y. Sakatsume M. Nishi S. et al.Expression, roles, receptors, and regulation of osteopontin in the kidney.Kidney Int. 2002; 60: 1645-1657Abstract Full Text Full Text PDF Scopus (279) Google Scholar Surprisingly, no apparent common gene (in comparison to diabetic rat RPTs) appeared to be downregulated by insulin in IRPTCs. While no explanation for this observation is evident, one possible explanation based on our gene chip studies, however, might be that a smaller percentage of genes is expressed in IRPTC as compared to diabetic rat RPTs, that is, 28.9% in IRPTCs versus 58.7 and 57.8% in non-diabetic BB rats and Wistar rats, respectively. High levels of D-glucose upregulated OPN mRNA expression in a time- and dose-dependent manner. This stimulatory effect of high glucose on OPN mRNA expression was inhibited in the presence of DPI, tiron, taurine, MnTBAP, losartan, perindopril, calphostin C, and LY 379196, suggesting that the high glucose effect on OPN expression might be mediated, at least in part, via ROS generation, RAS activation, and PKC-β signaling. At present, the exact molecular linkage of high glucose and ROS on OPN expression is incompletely delineated. One possibility may be that high glucose evokes mitochondrial ROS generation with inhibition of glyceraldehyde-3-phosphate dehydrogenase activity and flux of glucose to the tricarboxylic acid cycle but stimulates the de novo synthesis of diacylglycerol (the polyol pathway) and subsequently activates PKC-β.29.Nishikawa T. Edelstein D. Du X.L. et al.Normalizing mitochondrial superoxide production blocks three pathways of hyperglycemia damage.Nature. 2000; 404: 787-790Crossref PubMed Scopus (3658) Google Scholar, 30.Brownlee M. Biochemistry and molecular biology of diabetic complications.Nature. 2001; 414: 813-820Crossref PubMed Scopus (7030) Google Scholar This possibility is supported by our earlier work that high glucose evokes ROS generation and activates the polyol/PKC pathway with the outcome being angiotensinogen gene expression and intrarenal RAS activation in IRPTCs.31.Zhang S.-L. Filep J.A. Hohman T.C. et al.Molecular mechanisms of glucose action on angiotensinogen gene expression in rat proximal tubular cells.Kidney Int. 1999; 55: 454-464Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 32.Hsieh T.J. Zhang S.L. Filep J.G. et al.High glucose stimulates angiotensinogen gene expression via reactive oxygen species generation in rat kidney proximal tubu" @default.
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