Superoxide and hydrogen peroxide counterregulate myogenic contractions in renal afferent arterioles from a mouse model of chronic kidney disease
2017; Elsevier BV; Volume: 92; Issue: 3 Linguagem: Inglês
10.1016/j.kint.2017.02.009
ISSN1523-1755
AutoresLingli Li, En Yin Lai, Zaiming Luo, Glenn Solis, Kathy K. Griendling, W. Robert Taylor, Pedro A. José, Anton Wellsten, William J. Welch, Christopher S. Wilcox,
Tópico(s)Cardiovascular and Diving-Related Complications
ResumoMyogenic contractions protect kidneys from barotrauma but are impaired in chronic kidney disease (CKD). Since myogenic contractions are enhanced by superoxide but impaired by hydrogen peroxide, we tested the hypothesis that they are counterregulated by superoxide and H2O2 from NOX2/p47phox and/or NOX4/POLDIP2 in CKD. Myogenic contraction in isolated perfused afferent arterioles from mice with surgical 5/6 nephrectomy or sham operations fed a 6% sodium chloride diet was measured directly while superoxide and H2O2 were measured by fluorescence microscopy. Compared to sham-operated animals, an increase in perfusion pressure of arterioles from CKD mice doubled superoxide (21 versus 11%), increased H2O2 seven-fold (29 versus 4%), and reduced myogenic contractions profoundly (-1 versus -14%). Myogenic contractions were impaired further by PEG-superoxide dismutase or in arterioles from p47phox-/- (versus wild type) mice but became supra-normal by PEG-catalase or in mice with transgenic expression of catalase in vascular smooth muscle cells (-11 versus -1%). Single arterioles from mice with CKD expressed over 40% more mRNA and protein for NOX4 and POLDIP2. Myogenic responses in arterioles from POLDIP2 +/- (versus wild type) mice with CKD had over an 85% reduction in H2O2, but preserved superoxide and a normal myogenic response. Tempol administration to CKD mice for 3 months decreased afferent arteriolar superoxide and H2O2 and maintained myogenic contractions. Thus, afferent arteriolar superoxide generated by NOX2/p47phox opposes H2O2 generated by NOX4/POLDIP2 whose upregulation in afferent arterioles from mice with CKD accounts for impaired myogenic contractions. Myogenic contractions protect kidneys from barotrauma but are impaired in chronic kidney disease (CKD). Since myogenic contractions are enhanced by superoxide but impaired by hydrogen peroxide, we tested the hypothesis that they are counterregulated by superoxide and H2O2 from NOX2/p47phox and/or NOX4/POLDIP2 in CKD. Myogenic contraction in isolated perfused afferent arterioles from mice with surgical 5/6 nephrectomy or sham operations fed a 6% sodium chloride diet was measured directly while superoxide and H2O2 were measured by fluorescence microscopy. Compared to sham-operated animals, an increase in perfusion pressure of arterioles from CKD mice doubled superoxide (21 versus 11%), increased H2O2 seven-fold (29 versus 4%), and reduced myogenic contractions profoundly (-1 versus -14%). Myogenic contractions were impaired further by PEG-superoxide dismutase or in arterioles from p47phox-/- (versus wild type) mice but became supra-normal by PEG-catalase or in mice with transgenic expression of catalase in vascular smooth muscle cells (-11 versus -1%). Single arterioles from mice with CKD expressed over 40% more mRNA and protein for NOX4 and POLDIP2. Myogenic responses in arterioles from POLDIP2 +/- (versus wild type) mice with CKD had over an 85% reduction in H2O2, but preserved superoxide and a normal myogenic response. Tempol administration to CKD mice for 3 months decreased afferent arteriolar superoxide and H2O2 and maintained myogenic contractions. Thus, afferent arteriolar superoxide generated by NOX2/p47phox opposes H2O2 generated by NOX4/POLDIP2 whose upregulation in afferent arterioles from mice with CKD accounts for impaired myogenic contractions. Chronic kidney disease (CKD) can be modeled experimentally by surgically reduced renal mass (RRM).1Anderson S. Meyer T.W. Rennke H.G. Brenner B.M. Control of glomerular hypertension limits glomerular injury in rats with reduced renal mass.J Clin Invest. 1985; 76: 612-619Crossref PubMed Scopus (846) Google Scholar The progression of CKD remains poorly understood but has been related to reactive oxygen species (ROS),2Wang D. Strandgaard S. Borresen M.L. et al.Asymmetric dimethylarginine and lipid peroxidation products in early autosomal dominant polycystic kidney disease.Am J Kidney Dis. 2008; 51: 184-191Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 3Lai E.Y. Luo Z. Onozato M.L. et al.Effects of the antioxidant drug tempol on renal oxygenation in mice with reduced renal mass.Am J Physiol Renal Physiol. 2012; 303: F64-F74Crossref PubMed Scopus (30) Google Scholar hypertension,4Bidani A.K. Griffin K.A. Williamson G. et al.Protective importance of the myogenic response in the renal circulation.Hypertension. 2009; 54: 393-398Crossref PubMed Scopus (134) Google Scholar and impaired renal autoregulation.5Griffin K.A. Picken M. Bidani A.K. Method of renal mass reduction is a critical modulator of subsequent hypertension and glomerular injury.J Am Soc Nephrol. 1994; 4: 2023-2031Crossref PubMed Google Scholar A combination of hypertension and impaired autoregulation can increase the pressure in the glomerular capillaries and renal parenchyma and lead to barotrauma that accelerates the loss of kidney function.1Anderson S. Meyer T.W. Rennke H.G. Brenner B.M. Control of glomerular hypertension limits glomerular injury in rats with reduced renal mass.J Clin Invest. 1985; 76: 612-619Crossref PubMed Scopus (846) Google Scholar, 4Bidani A.K. Griffin K.A. Williamson G. et al.Protective importance of the myogenic response in the renal circulation.Hypertension. 2009; 54: 393-398Crossref PubMed Scopus (134) Google Scholar, 5Griffin K.A. Picken M. Bidani A.K. Method of renal mass reduction is a critical modulator of subsequent hypertension and glomerular injury.J Am Soc Nephrol. 1994; 4: 2023-2031Crossref PubMed Google Scholar Renal autoregulation is mediated by the tubuloglomerular feedback response but primarily by a rapid myogenic contraction of the vascular smooth muscle cells of the afferent arteriole in response to an increase in perfusion pressure.6Carlstrom M. Wilcox C.S. Arendshorst W.J. Renal autoregulation in health and disease.Physiol Rev. 2015; 95: 405-511Crossref PubMed Scopus (274) Google Scholar, 7Just A. Mechanisms of renal blood flow autoregulation: dynamics and contributions.Am J Physiol Regul Integr Comp Physiol. 2007; 292: R1-R17Crossref PubMed Scopus (140) Google Scholar Therefore, this study focused on the mechanisms of impaired myogenic responses of afferent arterioles from the kidneys in a model of CKD. An increase in perfusion pressure of individual afferent arterioles isolated from normal mouse kidneys increases vascular superoxide (O2.−) from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase8Lai E.Y. Solis G. Luo Z. et al.P47(phox) is required for afferent arteriolar contractile responses to angiotensin II and perfusion pressure in mice.Hypertension. 2012; 59: 415-420Crossref PubMed Scopus (44) Google Scholar that mediates much of the normal myogenic contraction.8Lai E.Y. Solis G. Luo Z. et al.P47(phox) is required for afferent arteriolar contractile responses to angiotensin II and perfusion pressure in mice.Hypertension. 2012; 59: 415-420Crossref PubMed Scopus (44) Google Scholar, 9Lai E.Y. Wellstein A. Welch W.J. Wilcox C.S. Superoxide modulates myogenic contractions of mouse afferent arterioles.Hypertension. 2011; 58: 650-656Crossref PubMed Scopus (45) Google Scholar, 10Li L. Lai E.Y. Wellstein A. et al.Differential effects of superoxide and hydrogen peroxide on myogenic signaling, membrane potential, and contractions of mouse renal afferent arterioles.Am J Physiol Renal Physiol. 2016; 310: F1197-F1205Crossref PubMed Scopus (24) Google Scholar However, interventions that generate ROS are reported to have variable effects on myogenic contractions and autoregulation. Thus, afferent arterioles from spontaneously hypertensive rats have exaggerated myogenic contractions that are dependent on O2.− from NADPH oxidase.11Ren Y. D'Ambrosio M.A. Liu R. et al.Enhanced myogenic response in the afferent arteriole of spontaneously hypertensive rats.Am J Physiol Heart Circ Physiol. 2010; 298: H1769-H1775Crossref PubMed Scopus (52) Google Scholar However, metabolism of ROS by tempol paradoxically increases the myogenic contractions of afferent arterioles from rats with oxidative stress from transforming growth factor-β.12Sharma K. Cook A. Smith M. et al.TGF-beta impairs renal autoregulation via generation of ROS.Am J Physiol Renal Physiol. 2005; 288: F1069-F1077Crossref PubMed Scopus (81) Google Scholar Moreover, renal autoregulation and myogenic responses of afferent arterioles are impaired in mice with RRM13Lai E.Y. Onozato M.L. Solis G. et al.Myogenic responses of mouse isolated perfused renal afferent arterioles: effects of salt intake and reduced renal mass.Hypertension. 2010; 55: 983-989Crossref PubMed Scopus (30) Google Scholar despite considerable oxidative stress.3Lai E.Y. Luo Z. Onozato M.L. et al.Effects of the antioxidant drug tempol on renal oxygenation in mice with reduced renal mass.Am J Physiol Renal Physiol. 2012; 303: F64-F74Crossref PubMed Scopus (30) Google Scholar We reported recently that exogenous H2O2 dose-dependently impaired myogenic contractions and blunted the effects of O2.− in normal mouse afferent arterioles.10Li L. Lai E.Y. Wellstein A. et al.Differential effects of superoxide and hydrogen peroxide on myogenic signaling, membrane potential, and contractions of mouse renal afferent arterioles.Am J Physiol Renal Physiol. 2016; 310: F1197-F1205Crossref PubMed Scopus (24) Google Scholar However, H2O2 was not produced by increasing the perfusion pressure of normal arterioles. The renal afferent arterioles of normal mice express the genes for p22phox that act primarily to direct neutrophil oxidase (NOX) to the cell membrane. NOX2 with its cytoplasmic activator p47phox is expressed in renal afferent arterioles. Arterioles also express NOX4 and its regulator POLDIP2 (polymerase [DNA-directed] delta-interacting protein 2).14Lassegue B. Griendling K.K. NADPH oxidases: functions and pathologies in the vasculature.Arterioscler Thromb Vasc Biol. 2010; 30: 653-661Crossref PubMed Scopus (491) Google Scholar, 15Lyle A.N. Deshpande N.N. Taniyama Y. et al.Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells.Circ Res. 2009; 105: 249-259Crossref PubMed Scopus (350) Google Scholar NOX1 is produced primarily in larger vessels, and NOX5 is not expressed in rodents. We have reported that p47phox −/− mice generate less O2.− in their afferent arterioles and have enfeebled myogenic contractions.8Lai E.Y. Solis G. Luo Z. et al.P47(phox) is required for afferent arteriolar contractile responses to angiotensin II and perfusion pressure in mice.Hypertension. 2012; 59: 415-420Crossref PubMed Scopus (44) Google Scholar However, NOX4 may generate H2O2 directly,14Lassegue B. Griendling K.K. NADPH oxidases: functions and pathologies in the vasculature.Arterioscler Thromb Vasc Biol. 2010; 30: 653-661Crossref PubMed Scopus (491) Google Scholar but its effects on afferent arterioles are not known. C57BL/6 mice with RRM have an adaptive increase in glomerular filtration rate of residual nephrons but develop only modest glomerular damage or tubulointerstitial fibrosis, likely because of the lack of hypertension. However, these mice have severely impaired myogenic contractions of their afferent arterioles.13Lai E.Y. Onozato M.L. Solis G. et al.Myogenic responses of mouse isolated perfused renal afferent arterioles: effects of salt intake and reduced renal mass.Hypertension. 2010; 55: 983-989Crossref PubMed Scopus (30) Google Scholar Thus, this is a convenient model with which to study the functional adaptation to RRM without the complications of uremia, hypertension, or extensive glomerulosclerosis and arteriolar damage. The RRM mouse model of CKD has a 6-fold increase in O2.−, as indicated by 8-isoprostane excretion, a tripling of H2O2 excretion, and a 28% reduction in myogenic responses.3Lai E.Y. Luo Z. Onozato M.L. et al.Effects of the antioxidant drug tempol on renal oxygenation in mice with reduced renal mass.Am J Physiol Renal Physiol. 2012; 303: F64-F74Crossref PubMed Scopus (30) Google Scholar A high salt diet for 3 months did not significantly affect the myogenic responses of normal mice yet increased 8-isoprostane and H2O2 excretion and renal NADPH oxidase activity16Kitiyakara C. Chabrashvili T. Chen Y. et al.Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase.J Am Soc Nephrol. 2003; 14: 2775-2782Crossref PubMed Scopus (275) Google Scholar and further worsened myogenic contractions in afferent arterioles from mice with RRM.13Lai E.Y. Onozato M.L. Solis G. et al.Myogenic responses of mouse isolated perfused renal afferent arterioles: effects of salt intake and reduced renal mass.Hypertension. 2010; 55: 983-989Crossref PubMed Scopus (30) Google Scholar Thus, NaCl in the diet does not itself regulate myogenic contractions but enhances the effects of RRM. Therefore, mice were fed a 6% NaCl diet to further enhance the oxidative stress and defective myogenic responses associated with RRM. Myogenic contractions were enhanced at 3 days after RRM, but were reduced at 3 weeks and were absent at 3 months.13Lai E.Y. Onozato M.L. Solis G. et al.Myogenic responses of mouse isolated perfused renal afferent arterioles: effects of salt intake and reduced renal mass.Hypertension. 2010; 55: 983-989Crossref PubMed Scopus (30) Google Scholar Therefore, this study was limited to mice 3 months after RRM or sham operation. To test the hypothesis that H2O2 from NOX4 opposes NOX2-derived, O2.−-induced myogenic contraction, afferent arterioles from p47phox or POLDIP2 gene deleted mice were used. To test the hypothesis that reduced myogenic contractions in mice with RRM derived H2O2, afferent arterioles from mice with vascular smooth muscle cell transgenic for catalase (Tgcat-SMC) were used. Tempol was administered for 3 months to groups of wild-type mice to normalize excessive excretion of 8-isoprostane and H2O2 in those with RRM.3Lai E.Y. Luo Z. Onozato M.L. et al.Effects of the antioxidant drug tempol on renal oxygenation in mice with reduced renal mass.Am J Physiol Renal Physiol. 2012; 303: F64-F74Crossref PubMed Scopus (30) Google Scholar These studies have potential clinical impact because several antioxidant strategies are under investigation yet none is targeted to a specific ROS or NOX isoform. The figures represent myogenic and fluorescence data for arterioles perfused at 40 and 80 mm Hg, whereas Table 1 shows basal data at 40 mm Hg and the myogenic response that was calculated by the slope of the regression of active wall tension on perfusion pressure across the full range of perfusion pressure from 40 to 134 mm Hg because the relationship was linear across this range.Table 1Basal afferent arteriolar diameter and myogenic responses 3 months after reduced renal mass or sham operations in mice given vehicle or oral tempolGroupNBasal diameter for myogenic studies (μm)Myogenic response (dynes·cm−1·mm Hg−1)WT sham + vehicle510.4 ± 0.63.90 ± 0.38WT RRM + vehicle710.0 ± 1.01.63 ± 0.25aWT sham + tempol510.2 ± 0.62.70 ± 0.28bWT RRM + tempol79.0 ± 0.42.22 ± 0.19WT sham + PEG-SOD56.2 ± 0.61.43 ± 0.17aWT sham + PEG-catalase610.6 ± 1.13.0 ± 0.21WT RRM + PEG-SOD49.0 ± 2.22.07 ± 0.78WT RRM + PEG-catalase711.1 ± 1.03.83 ± 0.27cp47Phox−/− RRM510.0 ± 1.31.03 ± 0.38TgCat-SMC RRM58.6 ± 0.63.05 ± 0.36cPOLDIP2+/− sham58.5 ± 0.62.69 ± 0.28bPOLDIP2+/− RRM711.1 ± 0.62.87 ± 0.12cPEG-SOD, polyethylene glycol–superoxide dismutase; RRM, reduced renal mass; TgCat-SMC, vascular smooth muscle cell transgenic for catalase; WT, wild-type.Values shown are mean ± SEM. Basal diameter was measured at 40-mm Hg perfusion pressure. Myogenic response was calculated as the slope of regression of active wall tension on perfusion pressure.Compared with WT sham: aP < 0.005 and bP < 0.05 compared with WT RRM, cP < 0.05. Open table in a new tab PEG-SOD, polyethylene glycol–superoxide dismutase; RRM, reduced renal mass; TgCat-SMC, vascular smooth muscle cell transgenic for catalase; WT, wild-type. Values shown are mean ± SEM. Basal diameter was measured at 40-mm Hg perfusion pressure. Myogenic response was calculated as the slope of regression of active wall tension on perfusion pressure. Compared with WT sham: aP < 0.005 and bP < 0.05 compared with WT RRM, cP < 0.05. The basal diameters of afferent arterioles perfused at 40 mm Hg differed among groups (Table 1). Therefore, we presented the percentage of change in arteriolar luminal diameter. Increasing perfusion pressure of afferent arterioles from sham-operated mice from 40 to 80 mm Hg reduced the luminal diameter substantially (−14 ± 4%) (Figure 1). In contrast, afferent arterioles from mice after RRM did not contract significantly with increasing perfusion pressure from 40 to 80 mm Hg (−1 ± 4%) (Figure 1). Despite severely impaired myogenic contractions, arterioles from mice with RRM generated twice as much of O2.− with increased perfusion pressure from 40 to 80 mm Hg (RRM: 21 ± 2% vs. sham: 11 ± 3%; P < 0.01) (Figure 1a). Incubation with polyethylene glycol–superoxide dismutase (PEG-SOD) to metabolize O2.− reduced myogenic contractions of arterioles from both sham and RRM groups (Figure 1b). An increase in perfusion pressure of arterioles from mice with RRM incubated with PEG-SOD paradoxically increased their diameter (RRM + PEG-SOD: +3 ± 1% vs. RRM: −1 ± 4%, P < 0.05) (Figure 1b). Genetic deletion of p47phox largely prevented arteriolar O2.− generation (Figure 1c) and had similar effects as incubation with PEG-SOD on impairing myogenic contractions (RRM + p47Phox −/−: +2 ± 1% vs. RRM: −1 ± 4%, P < 0.05) (Figure 1d). Arterioles from mice with RRM had a 7-fold greater increase in H2O2 with increased perfusion pressure (RRM: 29 ± 4% vs. sham: 4 ± 0.4%, P < 0.005) (Figure 2a). Remarkably, the severely impaired myogenic contractions of arterioles from mice with RRM were increased even above those from sham-operated mice by metabolism of H2O2 with PEG-catalase (changes in diameter: RRM + PEG-catalase: −19 ± 2% vs. RRM: −1 ± 4%, P < 0.005; myogenic responses: 3.83 ± 0.27 vs. 1.63 ± 0.25 [dynes·cm−1·mm Hg−1], respectively, P < 0.005) (Figure 2d and Table 1). Increasing the perfusion pressure of arterioles from sham-operated mice increased O2.− (Figure 1a), but had no detectable effect on H2O2 (Figure 2a). Correspondingly, incubation of arterioles from sham-operated mice with PEG-SOD reduced myogenic contractions, whereas incubation with PEG-catalase had no effect. Transgenic overexpression of Tgcat-SMC of sham-operated mice had no effects on the basal O2.− and H2O2 generation and myogenic contraction. In contrast, arterioles from Tgcat-SMC mice with RRM had slightly reduced production of O2.− (Figure 2c), yet greatly reduced H2O2 generation (Figure 2b) and normalized myogenic contractions (changes in diameter: RRM + vascular smooth muscle cell transgenic for catalase, −11 ± 1% vs. sham: −14 ± 4%; P = not significant [NS]; myogenic responses: 3.05 ± 0.36 vs. 3.90 ± 0.38 [dynes·cm−1·mm Hg−1], respectively, P = NS) (Figure 2e and Table 1). The mRNA expression of POLDIP2 and NOX4 in individual afferent arterioles was increased by 40% to 50% (Figure 3a and b) and accompanied by a corresponding increase in their protein expression (Figure 3c and d). Genetic reduction of 2 in mice with RRM did not alter arteriolar production of O2.− (Figure 4a), but prevented the excessive H2O2 generation (Figure 4b). Myogenic contractions of arterioles from POLDIP2+/+ mice after sham or RRM operations were similar to their C57BL/6 controls (Figure 4c). However, arterioles from POLDIP2+/− mice with RRM had maintained myogenic contractions that were similar to POLDIP2+/− sham-operated mice (changes in diameter: POLDIP2+/−, RRM: −8 ± 1% vs. POLDIP2+/−, sham: −8 ± 1%, P = NS; myogenic responses: 2.87 ± 0.12 vs. 2.69 ± 0.28 [dynes·cm−1 · mm Hg−1], respectively, P = NS) (Figure 4c and Table 1).Figure 4Mean ± SEM values (N = 5–6) for changes in O2.−, H2O2, and myogenic responses of afferent arterioles in mice after 3 months POLDIP2 +/+ with sham, POLDIP2 +/− with sham, POLDIP2 +/+ RRM, and POLDIP-2 +/− RRM mice. Data are shown for changes in ethidium:dihydroethidium ratio (a), changes in 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence (b), and changes in diameter (c) with increased perfusion from 40 to 80 mm Hg. ANOVA, analysis of variance; NS, not significant; RRM, reduced renal mass.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Tempol given for 3 months reduced the generation of O2.− and H2O2 in arterioles from sham-operated mice and mice with RRM (Figure 5a and b) and normalized their myogenic contractions (changes in diameter: RRM + tempol: −12 ± 2% vs. sham: −14 ± 4%; P = NS; myogenic responses: 2.22 ± 0.19 vs. 3.90 ± 0.38 [dynes·cm−1·mm Hg−1], respectively, P = NS) (Figure 5c and Table 1). We confirm that afferent arterioles from mice with prolonged RRM fed a high salt diet have severely impaired myogenic contractions.13Lai E.Y. Onozato M.L. Solis G. et al.Myogenic responses of mouse isolated perfused renal afferent arterioles: effects of salt intake and reduced renal mass.Hypertension. 2010; 55: 983-989Crossref PubMed Scopus (30) Google Scholar The main new findings are that these arterioles generated much more O2.− and H2O2 during increases in perfusion pressure. The impaired myogenic contractions after RRM were weakened further by reduction of arteriolar O2.− by metabolism with exogenous superoxide dismutase (PEG-SOD) or by reduced generation of O2.− by genetic deletion of p47phox,8Lai E.Y. Solis G. Luo Z. et al.P47(phox) is required for afferent arteriolar contractile responses to angiotensin II and perfusion pressure in mice.Hypertension. 2012; 59: 415-420Crossref PubMed Scopus (44) Google Scholar but were increased even above those of sham-operated mice by metabolism of arteriolar H2O2 with exogenous catalase (PEG-catalase) or transgenic overexpression of catalase in SMCs. Individual afferent arterioles from mice with RRM had overexpressed mRNA and proteins for Polidip-2 and NOX4. Genetic deletion of POLDIP2 (+/−) corrected the increased generation of H2O2 and normalized the myogenic contractions in arterioles from mice with RRM. Administration of the antioxidant drug tempol for 3 months in mice with RRM largely prevented the excessive H2O2 and the loss of myogenic contractions. The changes in diameter with perfusion pressure were modest yet more than sufficient to account for almost perfect autoregulation because in Poiseuille's equation, flow is proportional to the pressure difference and the fourth power of the radius. Thus, although the reduction in diameter was only −14% as pressure doubled from 40 to 80 mm Hg, the calculated increase in flow was only 7%. Thus, this change in diameter from the myogenic response alone is sufficient to account for almost perfect autoregulation. In vivo, the macula densa and connecting tubule tubuloglomerular feedback response and the third and fourth mechanisms further contribute to autoregulation and likely interact with the myogenic response to achieve the almost perfect autoregulation.6Carlstrom M. Wilcox C.S. Arendshorst W.J. Renal autoregulation in health and disease.Physiol Rev. 2015; 95: 405-511Crossref PubMed Scopus (274) Google Scholar Resistance arterioles express predominately NOX2 and NOX4.17Araujo M. Wilcox C.S. Oxidative stress in hypertension: role of the kidney.Antioxid Redox Signal. 2014; 20: 74-101Crossref PubMed Scopus (136) Google Scholar Renal afferent arterioles express p22phox that is a chaperone protein required for membrane location of NOXs.17Araujo M. Wilcox C.S. Oxidative stress in hypertension: role of the kidney.Antioxid Redox Signal. 2014; 20: 74-101Crossref PubMed Scopus (136) Google Scholar, 18Modlinger P. Chabrashvili T. Gill P.S. et al.RNA silencing in vivo reveals role of p22phox in rat angiotensin slow pressor response.Hypertension. 2006; 47: 238-244Crossref PubMed Scopus (116) Google Scholar The NOX2 subunit of NADPH oxidase is activated by p47phox to generate primarily O2.−.19Griendling K.K. Sorescu D. Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease.Circ Res. 2000; 86: 494-501Crossref PubMed Scopus (2610) Google Scholar, 20Gill P.S. Wilcox C.S. NADPH oxidases in the kidney.Antioxid Redox Signal. 2006; 8: 1597-1607Crossref PubMed Scopus (406) Google Scholar Arterioles from p47phox −/− mice8Lai E.Y. Solis G. Luo Z. et al.P47(phox) is required for afferent arteriolar contractile responses to angiotensin II and perfusion pressure in mice.Hypertension. 2012; 59: 415-420Crossref PubMed Scopus (44) Google Scholar and from p47phox −/− mice with RRM in this study, generated ∼80% less O2.− and had diminished myogenic contractions. Indeed, increasing the perfusion pressure of arterioles from p47phox −/− mice with RRM paradoxically increased their luminal diameter. The mRNAs and proteins for NOX4 and POLDIP2 were expressed in afferent arterioles. POLDIP2 enhances NOX4/NADPH oxidase activity to generate primarily H2O2.15Lyle A.N. Deshpande N.N. Taniyama Y. et al.Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells.Circ Res. 2009; 105: 249-259Crossref PubMed Scopus (350) Google Scholar Aortas from POLDIP2+/− mice (POLDIP2 −/− mice do not survive21Brown D.I. Lassegue B. Lee M. et al.Poldip2 knockout results in perinatal lethality, reduced cellular growth and increased autophagy of mouse embryonic fibroblasts.PLoS One. 2014; 9: e96657Crossref PubMed Scopus (32) Google Scholar) were reported to be incompletely developed, to generate less ROS, and have impaired responses to phenylephrine and potassium chloride.22Sutliff R.L. Hilenski L.L. Amanso A.M. et al.Polymerase delta interacting protein 2 sustains vascular structure and function.Arterioscler Thromb Vasc Biol. 2013; 33: 2154-2161Crossref PubMed Scopus (45) Google Scholar However, myogenic contractions in microvascular arterioles from sham-operated POLDIP2+/− mice compared with sham-operated POLDIP2+/+ mice in this study were well maintained. Importantly, the generation of H2O2 by arterioles from POLDIP2+/− mice with RRM was reduced ∼80%, and their myogenic contractions were increased and became similar to arterioles from sham-operated POLDIP2+/− mice. Thus, the increased mRNA and protein expression of POLDIP2/NOX4 in arterioles from mice with RRM is a source of increased arteriolar H2O2 that impairs myogenic contractions. Tempol is a cell-permeable redox cycling nitroxide that has been used widely to metabolize ROS in tissues and animals.23Wilcox C.S. Effects of tempol and redox-cycling nitroxides in models of oxidative stress.Pharmacol Ther. 2010; 126: 119-145Crossref PubMed Scopus (337) Google Scholar, 24Wilcox C.S. Pearlman A. Chemistry and antihypertensive effects of tempol and other nitroxides.Pharmacol Rev. 2008; 60: 418-469Crossref PubMed Scopus (301) Google Scholar Afferent arteriolar O2.− enhances myogenic contractions,10Li L. Lai E.Y. Wellstein A. et al.Differential effects of superoxide and hydrogen peroxide on myogenic signaling, membrane potential, and contractions of mouse renal afferent arterioles.Am J Physiol Renal Physiol. 2016; 310: F1197-F1205Crossref PubMed Scopus (24) Google Scholar, 25Li L. Feng D. Luo Z. et al.Remodeling of afferent arterioles from mice with oxidative stress does not account for increased contractility but does limit excessive wall stress.Hypertension. 2015; 66: 550-556Crossref PubMed Scopus (17) Google Scholar and tempol is a potent SOD mimetic.24Wilcox C.S. Pearlman A. Chemistry and antihypertensive effects of tempol and other nitroxides.Pharmacol Rev. 2008; 60: 418-469Crossref PubMed Scopus (301) Google Scholar Indeed, administration of tempol in sham-operated mice reduces O2.− and myogenic contractions.11Ren Y. D'Ambrosio M.A. Liu R. et al.Enhanced myogenic response in the afferent arteriole of spontaneously hypertensive rats.Am J Physiol Heart Circ Physiol. 2010; 298: H1769-H1775Crossref PubMed Scopus (52) Google Scholar However, it was interesting that 3 months of tempol administration in mice with RRM, which prevented the excessive arteriolar generation of O2.−, increased and in fact normalized the myogenic contractions. We reported recently that incubation of afferent arterioles with H2O2 blunted or prevented myogenic contractions and blocked much of the effects of O2.− to enhance contractions.10Li L. Lai E.Y. Wellstein A. et al.Differential effects of superoxide and hydrogen peroxide on myogenic signaling, membrane potential, and contractions of mouse renal afferent arterioles.Am J Physiol Renal Physiol. 2016; 310: F1197-F1205Crossref PubMed Scopus (24) Google Scholar Tempol also has catalase-mimetic action in vivo.24Wilcox C.S. Pearlman A. Chemistry and antihypertensive effects of tempol and other nitroxides.Pharmacol Rev. 2008; 60: 418-469Crossref PubMed Scopus (301) Google Scholar Indeed, afferent arterioles from mice with RRM given tempol for 3 months produced much less H2O2. Thus, metabolism of H2O2 by tempol may account in part for its effect of preserving myogenic contractions in arterioles from mice with RRM, despite a reduction in arteriolar O2.−. We acknowledge some limitations. Blood pressure, renal function, and renal morphology were not studied. However, in a previous study, continuous telemetric blood pressure recording revealed no effects of RRM or of high salt intake on mean arterial pressure (after an initial increase in the first 3 weeks). Moreover, there were no effects of RRM on the glomerular filtration rate per gram of kidney weight and no effects of high salt intake on renal vascular resistance, renal blood flow, glomerular volume, or the mild changes of glomerulosclerosis. Therefore, these parameters were not evaluated further in this study. In summary, myogenic contractions of afferent arterioles were impaired severely in mice with RRM by H2O2, generated largely by enhanced arteriolar expression of POLDIP2/NOX4. The increase in H2O2 and reduction in myogenic contractions in mice with RRM were corrected by 3 months of oral tempol. Male C57Bl/6 mice 2 to 3 months of age weighing 25 to 31 g (Charles River Inc., Germantown, MD) were randomized to surgical RRM or sham operation after which both groups were fed 6% NaCl diet (Teklad; Harlan, Indianapolis, Ind). Similar randomization was performed for p47Phox−/−, TgCat-SMC, and POLDIP2+/− mice that all are in a C56/Bl/6 background. POLDIP2+/− mice were used because POLDIP2−/− mice do not survive.21Brown D.I. Lassegue B. Lee M. et al.Poldip2 knockout results in perinatal lethality, reduced cellular growth and increased autophagy of mouse embryonic fibroblasts.PLoS One. 2014; 9: e96657Crossref PubMed Scopus (32) Google Scholar All procedures conformed to the Guide for Care and Use of Laboratory Animals prepared by The Institute for Laboratory Animal Research. Studies were approved by the Georgetown University Animal Care and Use Committee. A 2-step surgical 5/6 nephrectomy procedure was used to create RRM under inhalational anesthesia with 2% isoflurane and oxygen mixed with room air in a vaporizer as described.13Lai E.Y. Onozato M.L. Solis G. et al.Myogenic responses of mouse isolated perfused renal afferent arterioles: effects of salt intake and reduced renal mass.Hypertension. 2010; 55: 983-989Crossref PubMed Scopus (30) Google Scholar Sham-operated control mice (sham) were subject to a similar 2-stage procedure without removal of kidney tissue. After 1 week for recovery, wild-type mice in both groups were randomized to receive vehicle (water) or tempol (2 mmol·l−1) in the drinking water. After 3 months, groups (N = 5–12 per group) of mice were killed, and renal afferent arterioles were dissected, mounted, and perfused via a pipette whose pressure at its tip was recorded by a calibrated micropipette within its lumen.13Lai E.Y. Onozato M.L. Solis G. et al.Myogenic responses of mouse isolated perfused renal afferent arterioles: effects of salt intake and reduced renal mass.Hypertension. 2010; 55: 983-989Crossref PubMed Scopus (30) Google Scholar The experiments were recorded by a video system, digitized, and monitored in real time to measure the perfusion pressure and arteriolar luminal diameter. The perfusion pressure was measured at the tip of the perfusion pipette using a closed chamber connected to a model DPM-1B pneumatic transducer calibrator (Bio-Tek Instruments, Inc., Winooski, VT). Myogenic contractions were calculated from the reduction in diameter with increased perfusion pressure that was increased in steps of 20 mm Hg, as previously described.13Lai E.Y. Onozato M.L. Solis G. et al.Myogenic responses of mouse isolated perfused renal afferent arterioles: effects of salt intake and reduced renal mass.Hypertension. 2010; 55: 983-989Crossref PubMed Scopus (30) Google Scholar A set of pressure steps from 40 to 134 mm Hg was undertaken in each arteriole in physiologic solution and in a perfusate without Ca2+ and containing 5 × 10−3 mol·l−1 of ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (Sigma-Aldrich, St. Louis, MO) to abolish active tone. The active wall tension was calculated as the difference between the tensions measured during perfusion with these 2 solutions. Because active wall tension increased linearly with perfusion pressure greater than ∼40 mm Hg, the myogenic response of each arteriole was calculated as the slope of the regression of active wall tension on perfusion pressure. We reported recently that incubation of afferent arterioles with paraquat to generate O2.− increased the fluorescence ratio of ethidium:dihydroethidium and that this was >80% prevented by incubation with PEG-SOD. Therefore, PEG-SOD–inhibitable ethidium:dihydroethidium fluorescence was selected as a measure of arteriolar O2.−. We reported further that incubation with H2O2 increased the fluorescence of H2DCFDA and that this was >90% prevented by incubation with PEG catalase. Therefore, PEG-catalase–inhibitable H2DCFDA fluorescence was selected as a measure of H2O2.10Li L. Lai E.Y. Wellstein A. et al.Differential effects of superoxide and hydrogen peroxide on myogenic signaling, membrane potential, and contractions of mouse renal afferent arterioles.Am J Physiol Renal Physiol. 2016; 310: F1197-F1205Crossref PubMed Scopus (24) Google Scholar, 25Li L. Feng D. Luo Z. et al.Remodeling of afferent arterioles from mice with oxidative stress does not account for increased contractility but does limit excessive wall stress.Hypertension. 2015; 66: 550-556Crossref PubMed Scopus (17) Google Scholar PEG-SOD (#S9549) and PEG-catalase (#C4963) were purchased from Sigma-Aldrich. Individual renal afferent arterioles were dissected and isolated from mouse kidney as previously described.13Lai E.Y. Onozato M.L. Solis G. et al.Myogenic responses of mouse isolated perfused renal afferent arterioles: effects of salt intake and reduced renal mass.Hypertension. 2010; 55: 983-989Crossref PubMed Scopus (30) Google Scholar Briefly, ∼15 isolated afferent arterioles from each mouse were placed in 2 ml of a lysing matrix D tube (MP Biomedicals, Santa Ana, CA) containing QIAzol lysis reagent and homogenized by MP Fast Prep. Total RNA was extracted with RNeasy Mini Kit (Qiagen Inc., Valencia, CA). Primers and probes for mouse POLDIP2 (ID: Mm00458936_m1), NOX4 (ID: Mm00479246_m1), and 18s rRNA (ID: Mm03928990_g1) were used to quantitate the mRNA expression (Applied Biosystems, Foster City, CA) as previously described.26Chabrashvili T. Tojo A. Onozato M.L. et al.Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney.Hypertension. 2002; 39: 269-274Crossref PubMed Scopus (321) Google Scholar, 27Luo Z. Aslam S. Welch W.J. Wilcox C.S. Activation of nuclear factor erythroid 2-related factor 2 coordinates dimethylarginine dimethylaminohydrolase/PPAR-γ/endothelial nitric oxide synthase pathways that enhance nitric oxide generation in human glomerular endothelial cells.Hypertension. 2015; 65: 896-902Crossref PubMed Scopus (38) Google Scholar The protein expression in individual renal afferent arterioles from mice was quantitated in a recently developed, automated, highly sensitive capillary electrophoresis-based immunodetection system (Wes, Simple Western, San Jose, CA) designed for small samples and optimized for high resolution, sensitivity, and reproducibility.28Chan Y.C. Roy S. Huang Y. et al.The microRNA miR-199a-5p down-regulation switches on wound angiogenesis by derepressing the v-ets erythroblastosis virus E26 oncogene homolog 1-matrix metalloproteinase-1 pathway.J Biol Chem. 2012; 287: 41032-41043Crossref PubMed Scopus (57) Google Scholar, 29Gentalen E.T. Proctor J.M. Using the Peggy Simple Western system for fine needle aspirate analysis.Methods Mol Biol. 2015; 1219: 139-155Crossref PubMed Scopus (5) Google Scholar Mouse afferent arterioles were isolated3Lai E.Y. Luo Z. Onozato M.L. et al.Effects of the antioxidant drug tempol on renal oxygenation in mice with reduced renal mass.Am J Physiol Renal Physiol. 2012; 303: F64-F74Crossref PubMed Scopus (30) Google Scholar and placed in 1.5-ml tubes on ice, suspended in 150 μl of RIPA lysis buffer (ProteinSimple, San Jose, CA), sonicated with a Microson sonifier XL-2000 (Misonix, Farmingdale, NY), and the protein was extracted and quantitated as described.27Luo Z. Aslam S. Welch W.J. Wilcox C.S. Activation of nuclear factor erythroid 2-related factor 2 coordinates dimethylarginine dimethylaminohydrolase/PPAR-γ/endothelial nitric oxide synthase pathways that enhance nitric oxide generation in human glomerular endothelial cells.Hypertension. 2015; 65: 896-902Crossref PubMed Scopus (38) Google Scholar All materials were purchased from ProteinSimple except the primary antibodies (anti-POLDIP2 and anti-NOX4 from Abcam [Cambridge, UK], anti–β-actin from Sigma-Aldrich). Briefly, 3 μg of protein sample was mixed with 0.1-fold sample buffer and 5-fold master mix (200 mM dithiothreitol, 5-fold sample buffer, 5-fold fluorescent molecular weight markers), and heated to 95 °C for 5 minutes. The denatured samples and biotinylated ladder (5 μl of each), blocking reagent, diluted primary antibodies (anti-POLDIP2, 1:50 and anti-NOX4, 1:50 from Abcam; anti-β-actin, 1:100 from Sigma-Aldrich), horseradish peroxidase–conjugated secondary antibodies, chemiluminescent substrate, and washing buffer were dispensed into wells in a prefilled microplate. Loaded protein samples were separated and immunodetected automatically in the capillary system. The size-based proteins were analyzed with Compass software (ProteinSimple) and normalized to β-actin. Data are expressed as mean ± SEM. A 2 × 2 analyses of variance was used to compare the effects of RRM, genotype, or tempol and the interaction (i.e., the effect of genotype or tempol on the response to RRM). When appropriate, these calculations were followed by Bonferroni post hoc Student t tests. Changes were analyzed using nonparametric statistics (GraphPad Prism, GraphPad, La Jolla, CA). P < 0.05 was considered statistically significant. The afferent arterioles of rats with RRM have a substantial reduction in resistance despite hypertension, which would be anticipated to increase myogenic tone.30Hostetter T.H. Olson J.L. Rennke H.G. et al.Hyperfiltration in remnant nephrons: a potentially adverse response to renal ablation.Am J Physiol. 1981; 241: F85-F93PubMed Google Scholar This is consistent with the current findings of impaired myogenic responses and with previous reports of impaired renal autoregulation in rodent models of RRM.31Bidani A.K. Schwartz M.M. Lewis E.J. Renal autoregulation and vulnerability to hypertensive injury in remnant kidney.Am J Physiol. 1987; 252: F1003-F1010PubMed Google Scholar Because these changes were accompanied by increased ROS and corrected by tempol, our findings relate the changes in microvascular function in prolonged RRM to excessive ROS. They identify specifically excess H2O2 that is generated from overactivated POLDIP2/NOX4. A return of myogenic responses of these arterioles, accompanied by a reduction in H2O2, could be important in preventing renal barotrauma in CKD and perhaps in preventing the progressive loss of their glomerular filtration rate. All the authors declared no competing interests. This work was supported by grants from the National Institute for Diabetes, Digestive Disorders, and Kidney Disease (DK49870 and DK36079) and the Heart, Lung, and Blood Institute (HL68086) of the National Institutes of Health, and by funds from the George E. Schreiner Chair of Nephrology, the Smith-Kogod Family Foundation and the Georgetown University Hypertension, Kidney, and Vascular Research Center, and by grants to En Yin Lai from National Nature Science Foundation of China (31471100 and 31671193). Li L, Lai EY, Luo Z, et al. Superoxide and hydrogen peroxide counterregulate myogenic contractions in renal afferent arterioles from a mouse model of chronic kidney disease. Kidney Int. 2017;92:625–633Kidney InternationalVol. 92Issue 6PreviewIn the above-mentioned article, Anton Wellstein's name was misspelled. The authors regret the error. Full-Text PDF Open Archive
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