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Phosphoproteomic analysis of AT1 receptor-mediated signaling responses in proximal tubules of angiotensin II-induced hypertensive rats

2011; Elsevier BV; Volume: 80; Issue: 6 Linguagem: Inglês

10.1038/ki.2011.161

ISSN

1523-1755

Autores

Xiao C. Li, Jia L. Zhuo,

Tópico(s)

Ion Transport and Channel Regulation

Resumo

The signaling mechanisms underlying the effects of angiotensin II in proximal tubules of the kidney are not completely understood. Here we measured signal protein phosphorylation in isolated proximal tubules using pathway-specific proteomic analysis in rats continuously infused with pressor or non-pressor doses of angiotensin II over a 2-week period. Of the 38 phosphoproteins profiled, 14 were significantly altered by the pressor dose. This included increased phosphorylation of the protein kinase C isoenzymes, PKCα and PKCβII, and the glycogen synthase kinases, GSK3α and GSK3β. Phosphorylation of the cAMP-response element binding protein 1 and PKCδ were decreased, whereas PKCε remained unchanged. By contrast, the phosphorylation of only seven proteins was altered by the non-pressor dose, which increased that of PKCα, PKCδ, and GSKα. Phosphorylation of MAP kinases, ERK1/2, was not increased in proximal tubules in vivo by the pressor dose, but was in proximal tubule cells in vitro. Infusion of the pressor dose decreased, whereas the non-pressor dose of angiotensin II increased the phosphorylation of the sodium and hydrogen exchanger 3 (NHE-3) in membrane fractions of proximal tubules. Losartan largely blocked the signaling responses induced by the pressor dose. Thus, PKCα and PKCβII, GSK3α and GSK3β, and cAMP-dependent signaling pathways may have important roles in regulating proximal tubular sodium and fluid transport in Ang II-induced hypertensive rats. The signaling mechanisms underlying the effects of angiotensin II in proximal tubules of the kidney are not completely understood. Here we measured signal protein phosphorylation in isolated proximal tubules using pathway-specific proteomic analysis in rats continuously infused with pressor or non-pressor doses of angiotensin II over a 2-week period. Of the 38 phosphoproteins profiled, 14 were significantly altered by the pressor dose. This included increased phosphorylation of the protein kinase C isoenzymes, PKCα and PKCβII, and the glycogen synthase kinases, GSK3α and GSK3β. Phosphorylation of the cAMP-response element binding protein 1 and PKCδ were decreased, whereas PKCε remained unchanged. By contrast, the phosphorylation of only seven proteins was altered by the non-pressor dose, which increased that of PKCα, PKCδ, and GSKα. Phosphorylation of MAP kinases, ERK1/2, was not increased in proximal tubules in vivo by the pressor dose, but was in proximal tubule cells in vitro. Infusion of the pressor dose decreased, whereas the non-pressor dose of angiotensin II increased the phosphorylation of the sodium and hydrogen exchanger 3 (NHE-3) in membrane fractions of proximal tubules. Losartan largely blocked the signaling responses induced by the pressor dose. Thus, PKCα and PKCβII, GSK3α and GSK3β, and cAMP-dependent signaling pathways may have important roles in regulating proximal tubular sodium and fluid transport in Ang II-induced hypertensive rats. Angiotensin II (Ang II) has a critical role in the regulation of sodium and fluid reabsorption in proximal tubules of the kidney in both physiological and diseased states. The evidence supporting the important roles of Ang II in proximal nephrons is quite overwhelming.1.Harris P.J. Young J.A. Dose-dependent stimulation and inhibition of proximal tubular sodium reabsorption by angiotensin II in the rat kidney.Pflugers Arch. 1977; 367: 295-297Crossref PubMed Scopus (322) Google Scholar, 2.Navar L.G. Carmines P.K. Huang W.C. et al.The tubular effects of angiotensin II.Kidney Int Suppl. 1987; 20: S81-S88PubMed Google Scholar, 3.Cogan M.G. Angiotensin II: a powerful controller of sodium transport in the early proximal tubule.Hypertension. 1990; 15: 451-458Crossref PubMed Scopus (234) Google Scholar In isolated proximal tubule preparations or cultured proximal tubule cells, Ang II stimulates the expression or activities of the sodium and hydrogen exchanger-3 (NHE-3),4.Geibel J. Giebisch G. Boron W.F. Angiotensin II stimulates both Na(+)-H+ exchange and Na+/HCO3- cotransport in the rabbit proximal tubule.Proc Natl Acad Sci USA. 1990; 87: 7917-7920Crossref PubMed Scopus (214) Google Scholar, 5.Houillier P. Chambrey R. Achard J.M. et al.Signaling pathways in the biphasic effect of angiotensin II on apical Na/H antiport activity in proximal tubule.Kidney Int. 1996; 50: 1496-1505Abstract Full Text PDF PubMed Scopus (117) Google Scholar the sodium and potassium ATPase (Na+/K+-ATPase),6.Yingst D.R. Massey K.J. Rossi N.F. et al.Angiotensin II directly stimulates activity and alters the phosphorylation of Na-K-ATPase in rat proximal tubule with a rapid time course.Am J Physiol Renal Physiol. 2004; 287: F713-F721Crossref PubMed Scopus (39) Google Scholar, 7.Bharatula M. Hussain T. Lokhandwala M.F. Angiotensin II AT1 receptor/signaling mechanisms in the biphasic effect of the peptide on proximal tubular Na+/K+-ATPase.Clin Exp Hypertens. 1998; 20: 465-480Crossref PubMed Scopus (54) Google Scholar or the sodium and bicarbonate co-transporter (Na+/HCO3−).8.Horita S. Zheng Y. Hara C. et al.Biphasic regulation of Na+-HCO3- cotransporter by angiotensin II type 1A receptor.Hypertension. 2002; 40: 707-712Crossref PubMed Scopus (47) Google Scholar, 9.Zhou Y. Bouyer P. Boron W.F. Role of the AT1A receptor in the CO2-induced stimulation of HCO3- reabsorption by renal proximal tubules.Am J Physiol Renal Physiol. 2007; 293: F110-F120Crossref PubMed Scopus (12) Google Scholar In acute in vivo micropuncture or in vitro microperfusion studies, peritubular and intraluminal Ang II infusion induces biphasic transport responses, with picomolar doses of Ang II stimulate sodium transport while nanomolar doses inhibit sodium transport.1.Harris P.J. Young J.A. Dose-dependent stimulation and inhibition of proximal tubular sodium reabsorption by angiotensin II in the rat kidney.Pflugers Arch. 1977; 367: 295-297Crossref PubMed Scopus (322) Google Scholar, 10.Schuster V.L. Kokko J.P. Jacobson H.R. Angiotensin II directly stimulates sodium transport in rabbit proximal convoluted tubules.J Clin Invest. 1984; 73: 507-515Crossref PubMed Scopus (300) Google Scholar Sustained increases of circulating and tissue Ang II levels in proximal tubules in experimental animals, however, are associated with high blood pressure, sodium retention, and tubulointerstitial injury in the kidney.11.Johnson R.J. Alpers C.E. Yoshimura A. et al.Renal injury from angiotensin II-mediated hypertension.Hypertension. 1992; 19: 464-474Crossref PubMed Scopus (520) Google Scholar, 12.Zhuo J.L. Imig J.D. Hammond T.G. et al.Ang II accumulation in rat renal endosomes during Ang II-induced hypertension: role of AT1 receptor.Hypertension. 2002; 39: 116-121Crossref PubMed Scopus (126) Google Scholar, 13.Muller D.N. Dechend R. Mervaala E.M. et al.NF-kappaB inhibition ameliorates angiotensin II-induced inflammatory damage in rats.Hypertension. 2000; 35: 193-201Crossref PubMed Google Scholar Signaling mechanisms mediating the physiological or pathophysiological effects of Ang II in proximal tubules of the kidney remain incompletely understood. Our current understanding of Ang II-dependent signaling mechanisms is based primarily on studies in cultured proximal tubule cells or in isolated proximal tubules. In vitro, Ang II activates several heterotrimeric G-proteins including Gq/11, Gs, Gi, G12, and G13.14.Touyz R.M. Schiffrin E.L. Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular smooth muscle cells.Pharmacol Rev. 2000; 52: 639-672PubMed Google Scholar, 15.Higuchi S. Ohtsu H. Suzuki H. et al.Angiotensin II signal transduction through the AT1 receptor: novel insights into mechanisms and pathophysiology.Clin Sci (Lond). 2007; 112: 417-428Crossref PubMed Scopus (339) Google Scholar, 16.Zou Y. Komuro I. Yamazaki T. et al.Cell type-specific angiotensin II-evoked signal transduction pathways: critical roles of Gbetagamma subunit, Src family, and Ras in cardiac fibroblasts.Circ Res. 1998; 82: 337-345Crossref PubMed Scopus (133) Google Scholar The activated downstream signaling proteins range from inositol triphosphate, calcium, diacylglycerol, adenylyl cyclase/cAMP, and protein kinase C (PKC) to other receptor and non-receptor tyrosine kinases and serine/threonine kinases, such as the MAP kinase family (ERK, JNK, and p38 MAPK).15.Higuchi S. Ohtsu H. Suzuki H. et al.Angiotensin II signal transduction through the AT1 receptor: novel insights into mechanisms and pathophysiology.Clin Sci (Lond). 2007; 112: 417-428Crossref PubMed Scopus (339) Google Scholar, 17.Griendling K.K. Ushio-Fukai M. Lassegue B. et al.Angiotensin II signaling in vascular smooth muscle. New concepts.Hypertension. 1997; 29: 366-373Crossref PubMed Google Scholar However, it is not clear whether similar signaling mechanisms may be replicated in an integrative physiological or pathophysiological setting. The present study used a novel pathway-specific proteomic approach to study the responses of 38 major signaling phosphoproteins in proximal tubules of the rats treated with 2-week infusion of a pressor or a non-pressor dose of Ang II. Ang II-infused rats were treated with the AT1 receptor blocker losartan to determine the role of AT1 receptors. In order to exclude the contamination of signaling proteins from adjacent glomeruli, blood vessels, or cortical collecting ducts, we isolated fresh proximal tubules specifically from the superficial cortex of the kidney for proteomic analysis of signaling responses to Ang II. Infusion of Ang II (60 ng/min, s.c.) for 2 weeks markedly increased systolic blood pressure in Ang II-infused rats (Table 1; P<0.01). Compared with control, Ang II moderately increased urinary water and sodium excretion and decreased urine osmolality. Urinary albumin-to-creatinine ratio was increased by threefold in Ang II-infused rats. These responses to the pressor dose of Ang II infusion were normalized by losartan treatment.Table 1Effects of the pressor dose of Ang II infusion and concurrent losartan treatment for 2 weeks on body and kidney weights, systolic blood pressure, 24 h urinary excretion of water, and electrolytes in Ang II-infused ratsResponseControlAng IIAng II+losartanBody weight, g322±8316±7300±5Kidney weight, g2.5±0.32.4±0.32.4±0.2Kidney weight/body weight ratio, × 1000.76±0.020.79±0.030.79±0.02SBP, mm Hg118±5175±7**P<0.01 versus control.126±8P<0.01 versus Ang II-infused hypertensive rats.Urine, ml/24 h12.7±0.520.1±0.6**P<0.01 versus control.13.5±0.72P<0.01 versus Ang II-infused hypertensive rats.UNaV, mmol/24 h1.67±0.052.17±0.09*P<0.05 or1.64±0.09P<0.01 versus Ang II-infused hypertensive rats.UKV, mmol/24 h2.32±0.162.61±0.222.78±0.31Urine osmolality, mOsm/kg H2O1535±28943±26*P<0.05 or1162±32*P<0.05 or†P<0.05 orUrine albumin/creatinine ratio, mg/g21.6±2.879.1±11.9**P<0.01 versus control.26.8±5.8P<0.01 versus Ang II-infused hypertensive rats.Abbreviations: Ang, angiotensin; SBP, systolic blood pressure; UKV, urinary potassium excretion; UNaV, urinary sodium excretion.* P<0.05 or** P<0.01 versus control.† P<0.05 ors P<0.01 versus Ang II-infused hypertensive rats. Open table in a new tab Abbreviations: Ang, angiotensin; SBP, systolic blood pressure; UKV, urinary potassium excretion; UNaV, urinary sodium excretion. Infusion of the pressor dose of Ang II for 2 weeks significantly increased plasma and whole kidney Ang II levels in rats (Figure 1). Plasma Ang II was increased by 2.8-fold in Ang II-infused rats (control: 115.5±24.1 fmol/ml versus Ang II-infused: 323±34.4 fmol/ml; P<0.01). Concurrent losartan treatment in Ang II-infused rats further elevated plasma Ang II levels above those of control and Ang II-infused rats because of AT1 receptor occupancy by losartan (421.6±32.3 fmol/ml; P<0.01). Kidney Ang II was increased by twofold (control: 172.6±16.2 pg/g versus Ang II-infused: 336.5±25.7 pg/g kidney weight; P<0.01), which was blocked by losartan in Ang II-infused rats (205.1±19.3 pg/g kidney weight, Figure 1). Proximal tubular Ang II was also increased by 2.6-fold (control: 61.7±8.9 versus Ang II-infused: 162.3±34.9 pg/mg proteins; P<0.01), which was decreased by losartan in Ang II-infused rats (55.7±9.7 pg/mg proteins, P 25% in proximal tubules of the Ang II-infused rats for 2 weeks (Figure 3). Phosphoproteins that were not altered by Ang II are listed in Table 2. Losartan largely, but not completely, blocked the signaling responses to Ang II and restored the phosphoproteins to untreated levels.Figure 3The Kinetwork multi-immunoblot and semi-quantitative analyses identified 14 signaling phosphoproteins that were altered in proximal tubules by 2-week infusion of the pressor dose of angiotensin (Ang) II in rats. The relative changes in the enhanced chemiluminescence (ECL) western signal intensity are expressed as c.p.m., which is the trace quantity of the band corrected to a scan time of 60 s (see online Expanded Methods section).View Large Image Figure ViewerDownload (PPT)Table 2Signaling phosphoproteins that were not significantly altered by the pressor dose of Ang II-infusion or concurrent treatment with losartan in Ang II-infused rats (<25% increase or decrease)ProteinsAbbreviationLaneRecognized epitope(s)Epitope(s)Adducin αAdducin α3S726Adducin γAdducin γ3S693B23 (nucleophosmin, numatrin, nucleolar protein NO38)B23 (NPM)19S4Cyclin-dependent protein-serine kinase 1/2CDK1/23Y15Double-stranded RNA-dependent protein-serine kinasePKR116T451Extracellular-regulated protein-serine kinase 2ERK28T185+Y187Jun N-terminus protein-serine kinaseJNK6T183+Y185Stress-activated protein kinase (SAPK)Jun proto-oncogene-encoded AP1 transcription factorJun11S73MAPK/ERK protein-serine kinase 1/2MEK1/219S218+S222Mitogen-activated protein-serine kinase p38 alphap38α MAPK18T180+Y182N-methyl-D-aspartate (NMDA) glutamate receptor 1NR12S896Protein-serine kinase B alpha (Akt1)PKBa (Akt1)13S473Protein-serine kinase C epsilonPKCε9S729Ribosomal S6 protein-serine kinase 1/3RSK1/36T359+S363/T356+S360Signal transducer and activator of transcription 1STAT112Y701Signal transducer and activator of transcription 5STAT54Y694 Open table in a new tab Phospho-CREB1 immunofluorescence staining was much weaker in proximal tubules of Ang II-infused rats than in control rats, and the response was reversed by losartan (Figure 4a-c). Ang II decreased phosphorylated CREB1 [S133] proteins in proximal tubules by 56±7% (P<0.01). Concurrent treatment with losartan restored phospho-CREB1 proteins to the control level in Ang II-infused rats (P<0.01). Angiotensin II markedly increased phospho-PKCα [S657] immunofluorescence staining in proximal tubules of Ang II-infused rats, which was reversed by losartan (Figure 5a–c). Western blot analysis confirmed that phospho-PKCα [S657] was increased by 86±9% in membrane fractions, but was decreased in cytosolic fractions (P<0.01, Figure 5d). Activation of PKCα [S657] in proximal tubules by Ang II was blocked by losartan. Phospho-PKCα/βII [T638/T641] was increased by 122±13% in Ang II-infused rats (P<0.01), which was also blocked by losartan (seeSupplementary Figure S1 online). By contrast, PKCδ [T507] phosphoproteins were decreased by 53±9% in proximal tubules by Ang II (P<0.01), and the response was blocked by losartan (P<0.01 versus Ang II-infused) (see Supplementary Figure S1 online). PKCε [S729] phosphoproteins were not altered in Ang II-infused rats with or without losartan treatment. Download .pdf (1.52 MB) Help with pdf files Supplementary Figures S1–S10 Figure 6 shows that glycogen synthase kinase 3 (GSK3) (α+β)/[Y216+Y279] phosphoproteins were increased by 56±9% (P<0.01) and 140±11% (P<0.01), respectively, in proximal tubules of Ang II-infused rats. Again, losartan completely blocked these responses. MAP kinases ERK1/ERK2 [T202+Y204] phosphoproteins were decreased by 44±5% in proximal tubules of Ang II-infused rat kidneys (P<0.05 versus control). Losartan treatment blocked these responses (see Supplementary Figure S2 online). Specific [125I]Val5-Ang II receptor binding was localized in the glomeruli and proximal tubules of the cortex and in the inner stripe of the outer medulla (Figure 7). Ang II infusion decreased [125I]Val5-Ang II binding by 50% in the cortex (control: 183±8 dpm/mm2 versus Ang II: 90±12 dpm/mm2; P<0.01) and in the inner stripe of the outer medulla (control: 276±13 dpm/mm2 versus Ang II: 128±6 dpm/mm2; P 90% of [125I]Val5-Ang II binding in the kidney (15±3 dpm/mm2, P<0.01). AT1 protein was measured by western blot analysis in membrane fractions of isolated proximal tubules, which was decreased in Ang II-infused rats (control: 0.24±0.02 versus Ang II: 0.09±0.02 AT1/actin ratio, P<0.01) and restored by losartan (Figure 7e). In untreated rats, phospho-NHE-3 immunofluorescence staining was distributed throughout the wall of proximal tubules (Figure 8a), which was much weaker in Ang II-infused rats (Figure 8b). Concurrent losartan treatment largely restored phospho-NHE-3 immunofluorescence staining to the control level (Figure 8c). In membrane fractions of freshly isolated proximal tubules, phospho-NHE-3 proteins were decreased in Ang II-infused rats (control: 0.24±0.02 versus Ang II: 0.10±0.03 NHE-3/actin ratio, P<0.01). Losartan again restored membrane phospho-NHE-3 proteins to the control level (Figure 8d). Interestingly, NHE-3 mRNA expression was increased in proximal tubules of the rats treated with the pressor dose of Ang II infusion, probably because of the feedback response to the decreased membrane (or total) phospho-NHE-3 proteins in proximal tubules (see Supplementary Figure S3 online). Again, losartan reversed the NHE-3 mRNA response to the pressor dose of Ang II infusion (see Supplementary Figure S3 online). Table 3 summarizes blood pressure and renal electrolyte responses to infusion of the non-pressor dose of Ang II (15 ng/min, s.c.) and concurrent losartan treatment for 2 weeks. Systolic blood pressure was not changed by Ang II, but it was decreased by losartan. Twenty-four hour urine excretion was also unaltered, accompanied by a small decrease in 24-h urinary sodium excretion. AT1 receptor proteins were increased by Ang II (control: 0.26±0.03 versus Ang II: 0.42±0.03 AT1/actin ratio, P<0.01), which was blocked by losartan (see Supplementary Figure S4 online). Compared with control (Figure 9a), phospho-NHE-3 immunofluorescence staining was stronger in proximal tubules of Ang II-infused rats (Figure 9b), which was reversed by losartan (Figure 9c). Furthermore, phospho-NHE-3 proteins were increased in membrane fractions of proximal tubules by the non-pressor dose of Ang II (control: 0.18±0.03 versus Ang II: 0.38±0.06 NHE-3/actin ratio, P<0.01) (Figure 9d). Interestingly, PKCα [S657] and PKCδ [T507], but not PKCβII or PKCε, and GSK3α [Y279], but not GSK3β [Y216], were activated by the non-pressor dose of Ang II (Figure 10a–d).Table 3Effects of the non-pressor dose of Ang II infusion and concurrent losartan treatment for 2 weeks on body and kidney weights, systolic blood pressure, 24 h urinary excretion of water, and electrolytes in Ang II-infused ratsResponseControlNon-pressor Ang IINon-pressor Ang II + LosBody weight, g314±11328±9298±7Kidney weight, g2.4±0.072.6±0.12.37±0.03Kidney weight/0.77±0.020.78±0.020.79±0.01body weight ratio, × 100SBP, mm Hg115±4116±3105±6Urine, ml/24 h15.5±0.517.8±0.919.1±3.0UNaV, mmol/24 h2.35±0.21.93±0.21.98±0.25UKV, mmol/24 h3.2±0.64.8±0.22**P<0.01 versus control.2.73±0.36†P<0.05 versus the non-pressor Ang II-treated group.Urine osmolality, mOsm/kg H2O1418±351084±56*P<0.05 or1028±151*P<0.05 orAbbreviations: Ang, angiotensin; SBP, systolic blood pressure; UKV, urinary potassium excretion; UNaV, urinary sodium excretion.* P<0.05 or** P<0.01 versus control.† P<0.05 versus the non-pressor Ang II-treated group. Open table in a new tab Figure 10The Kinetwork multi-immunoblot comparison analyses identified seven signaling phosphoproteins that were altered in proximal tubules of the rat kidneys by 2-week infusion of the non-pressor dose of Ang II. (a) A multi-immunoblot from pooled control proximal tubule samples. (b) A multi-immunoblot from pooled Ang II-infused proximal tubule samples. (c) The colored overlay of multi-immunoblots of a and b. (d) The summary of the Kinetwork multi-immunoblot, semi-quantitative analysis results. The relative changes in the enhanced chemiluminescence (ECL) western signal intensity are expressed as c.p.m., which is the trace quantity of the band corrected to a scan time of 60 sec (see online Expanded Methods section). ND, not determined.View Large Image Figure ViewerDownload (PPT) Abbreviations: Ang, angiotensin; SBP, systolic blood pressure; UKV, urinary potassium excretion; UNaV, urinary sodium excretion. The lack of effects of the pressor dose of Ang II infusion on ERK1/2 signaling phosphoproteins in proximal tubules by Kinetwork multi-immunoblot analysis prompted us to test whether Ang II activates ERK/1/2 in wild type and AT1a-KO mouse proximal tubule cells with the knock-in of a wild-type AT1a receptor (Figure 11). In wild-type cells, Ang II induced dose- and time-dependent increases in phospho-ERK1/2 with peak responses at 10 nmol/l (Figure 11a), and at 5 to 10 min, respectively, (Figure 11b). Ang II (10 nmol/l) increased phospho-ERK1/2 by 2.6-fold (control: 0.28±0.05 versus Ang II: 0.73±0.12 p-ERK1/2 to t-ERK1/2 ratio, P<0.01), which was blocked by losartan (0.46±0.06 p-ERK1/2 to t-ERK1/2 ratio, P<0.01 versus Ang II) (Figure 11). No biphasic p-ERK1/2 responses to Ang II were observed. In AT1a-KO cells, Ang II had little effect on p-ERK1/2 (not shown), but knock-in of the AT1a receptor in these cells restored the p-ERK1/2 response to Ang II (control: 0.16±0.05 versus Ang II: 0.62±0.08, p-ERK1/2 to t-ERK1/2 ratio, P<0.01). Losartan blocked the effect of Ang II on p-ERK1/2 in AT1a-KO cells with the knock-in of the AT1a receptor (0.36±0.06 p-ERK1/2 to t-ERK1/2 ratio, P<0.01 versus Ang II) (Figure 11). We determined whether activation of PKCα signaling phosphoproteins is actually involved in the activation or phosphorylation of NHE-3 proteins in wild-type mouse proximal tubule cells. Ang II induced both dose- and time-dependent increases in phospho-NHE-3 proteins with peak responses at 1 to 10 nmol/l (Figure 12a), and at 5 to 10 min after Ang II stimulation (Figure 12b). The effect of Ang II (1 nmol/l) on activation of NHE-3 proteins was inhibited by knocking down the expression of PKCα signaling proteins with a specific PKCα siRNA, but not with a scrambled siRNA control (Figure 12c). Although proteomic approaches are increasingly used to identify novel proteins in target tissues, few studies have used these techniques to profile pathway-specific signaling responses to 2 weeks of Ang II infusion specifically in proximal tubules of the kidney.18.Janech M.G. Raymond J.R. Arthur J.M. Proteomics in renal research.Am J Physiol Renal Physiol. 2007; 292: F501-F512Crossref PubMed Scopus (51) Google Scholar Leong et al.19.Leong P.K. Devillez A. Sandberg M.B. et al.Effects of ACE inhibition on proximal tubule sodium transport.Am J Physiol Renal Physiol. 2006; 290: F854-F863Crossref PubMed Scopus (50) Google Scholar used one-dimensional SDS-PAGE and MALDI-MS analyses to study the trafficking of renal cortical membrane transporter proteins in response to acute angiotensin-converting enzyme inhibition with captopril in rats. Using these techniques, Leong et al.19.Leong P.K. Devillez A. Sandberg M.B. et al.Effects of ACE inhibition on proximal tubule sodium transport.Am J Physiol Renal Physiol. 2006; 290: F854-F863Crossref PubMed Scopus (50) Google Scholar were able to characterize the patterns of redistribution of NHE-3, NaPi2, and vacuolar H+-ATPases in the rat renal cortex after acute angiotensin-converting enzyme inhibition. de Borst et al.20.de Borst M.H. Diks S.H. Bolbrinker J. et al.Profiling of the renal kinome: a novel tool to identify protein kinases involved in angiotensin II-dependent hypertensive renal damage.Am J Physiol Renal Physiol. 2007; 293: F428-F437Crossref PubMed Scopus (16) Google Scholar used novel peptide array chips to profile protein kinase substrates and/or protein kinase activities in the renal cortex of homozygous Ren2 rats, a model of Ang II-dependent hypertension. The notable observations in the latter study are the activation of p38 MAP kinase and the platelet-derived growth factor receptor-β, which were increased in Ren2 and reversed by ramipril.20.de Borst M.H. Diks S.H. Bolbrinker J. et al.Profiling of the renal kinome: a novel tool to identify protein kinases involved in angiotensin II-dependent hypertensive renal damage.Am J Physiol Renal Physiol. 2007; 293: F428-F437Crossref PubMed Scopus (16) Google Scholar The present study differs from the abovementioned studies in three unique ways. First, only freshly isolated proximal tubules were used for analysis of signaling responses, which exclude the contamination of signaling proteins from other cellular structures. Second, we used the pathway-specific, multi-immunoblotting approach for proteomic analysis of signaling responses to 2 weeks of pressor or non-pressor dose of Ang II infusion with or without hypertension. Finally, we used antibodies that target specific phosphorylation sites of the proteins of interest. This pathway-specific multi-immunoblotting analysis revealed several key signaling responses in proximal tubules of rats infused with the pressor or the non-pressor dose of Ang II. Ang II regulates proximal tubule sodium and bicarbonate reabsorption in part by inhibiting adenylate cyclase and cAMP.21.Douglas J.G. Angiotensin receptor subtypes of the kidney cortex.Am J Physiol. 1987; 253: F1-F7PubMed Google Scholar, 22.Liu F.Y. Cogan M.G. Angiotensin II stimulates early proximal bicarbonate absorption in the rat by decreasing cyclic adenosine monophosphate.J Clin Invest. 1989; 84: 83-91Crossref PubMed Scopus (169) Google Scholar Although it remains controversial, there is evidence that Ang II inhibits cAMP production in proximal tubule cells.23.Schelling J.R. Singh H. Marzec R. et al.Angiotensin II-dependent proximal tubule sodium transport is mediated by cAMP modulation of phospholipase C.Am J Physiol. 1994; 267: C1239-C1245PubMed Google Scholar, 24.Thekkumkara T. Linas S.L. Role of internalization in AT1A receptor function in proximal tubule epithelium.Am J Physiol Renal Physiol. 2002; 282: F623-F629Crossref PubMed Google Scholar, 25.Li X.C. Carretero O.A. Navar L.G. et al.AT1 receptor-mediated accumulation of extracellular angiotensin II in proximal tubule cells: role of cytoskeleton microtubules and tyrosine phosphatases.Am J Physiol Renal Physiol. 2006; 291: F375-F383Crossref PubMed Scopus (52) Google Scholar Conversely, increases in cAMP and activation of cAMP-dependent PKA inhibit Ang II-induced acidification of proximal tubule fluid or apical Na+/H+ exchange in isolated perfused proximal tubules.22.Liu F.Y. Cogan M.G. Angiotensin II stimulates early proximal bicarbonate absorption in the rat by decreasing cyclic adenosine monophosphate.J Clin Invest. 1989; 84: 83-91Crossref PubMed Scopus (169) Google Scholar, 23.Schelling J.R. Singh H. Marzec R. et al.Angiotensin II-dependent proximal tubule sodium transport is mediated by cAMP modulation of phospholipase C.Am J Physiol. 1994; 267: C1239-C1245PubMed Google Scholar Phospho-CREB1 [S133] is the major downstream signaling protein in response to activation of cAMP-dependent PKA and was measured as a function of cAMP activation in the present study. We found a ∼60% decrease in phospho-CREB1 [S133] proteins in proximal tubules of rats infused with the pressor dose Ang II and the effect was blocked by losartan treatment. These responses were confirmed in phospho-CREB1-immunofluorescence staining in proximal tubules of Ang II-infused rats treated with or without losartan. However, we did not observe similar changes in phospho-CREB1 [S133] proteins in proximal tubules of rats infused with the non-pressor dose of Ang II. This raises the possibility that the response of phospho-CREB1 [S133] proteins to the pressor dose of Ang II in proximal tubules may be pressure-dependent. The phospholipase C-dependent PKC signaling pathway represents a major cellular mechanism underlying Ang II-induced transport responses in proximal tubules.5.Houillier P. Chambrey R. Achard J.M. et al.Signaling pathways in the biphasic effect of angiotensin II on apical Na/H antiport activity in proximal tubule.Kidney Int. 1996; 50: 1496-1505Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 26.Du Z. Ferguson W.

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