Deletion of LOX-1 attenuates renal injury following angiotensin II infusion
2009; Elsevier BV; Volume: 76; Issue: 5 Linguagem: Inglês
10.1038/ki.2009.234
ISSN1523-1755
AutoresChang-Ping Hu, Bum‐Yong Kang, Judit Megyesi, Gur P. Kaushal, Robert L. Safirstein, Jawahar L. Mehta,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoAngiotensin II upregulates the expression of LOX-1, a recently identified oxidized low-density lipoprotein receptor controlled by redox state which in turn upregulates angiotensin II activity on its activation. To test whether interruption of this positive feedback loop might reduce angiotensin II-induced hypertension and subsequent renal injury, we studied LOX-1 knockout mice. After infusion with angiotensin II for 4 weeks systolic blood pressure gradually increased in the wild-type mice; this rise was significantly attenuated in the LOX-1 knockout mice. Along with the rise in systolic blood pressure, renal function (blood urea nitrogen and creatinine) decreased in the wild-type mice, but the deterioration of function was significantly less in the LOX-1 knockout mice. Glomerulosclerosis, arteriolar sclerosis, tubulointerstitial damage, and renal collagen accumulation were all significantly less in the LOX-1 knockout mice. The reduction in collagen formation was accompanied by a decrease in connective tissue growth factor mRNA, angiotensin type 1 receptor expression, and phosphorylation of p38 and p44/42 mitogen-activated protein kinases. Expression of endothelial nitric oxide synthase was increased in the kidneys of the LOX-1 knockout mice compared to the wild-type mice. Overall, our study suggests that LOX-1 is a key modulator in the development of angiotensin II-induced hypertension and subsequent renal damage. Angiotensin II upregulates the expression of LOX-1, a recently identified oxidized low-density lipoprotein receptor controlled by redox state which in turn upregulates angiotensin II activity on its activation. To test whether interruption of this positive feedback loop might reduce angiotensin II-induced hypertension and subsequent renal injury, we studied LOX-1 knockout mice. After infusion with angiotensin II for 4 weeks systolic blood pressure gradually increased in the wild-type mice; this rise was significantly attenuated in the LOX-1 knockout mice. Along with the rise in systolic blood pressure, renal function (blood urea nitrogen and creatinine) decreased in the wild-type mice, but the deterioration of function was significantly less in the LOX-1 knockout mice. Glomerulosclerosis, arteriolar sclerosis, tubulointerstitial damage, and renal collagen accumulation were all significantly less in the LOX-1 knockout mice. The reduction in collagen formation was accompanied by a decrease in connective tissue growth factor mRNA, angiotensin type 1 receptor expression, and phosphorylation of p38 and p44/42 mitogen-activated protein kinases. Expression of endothelial nitric oxide synthase was increased in the kidneys of the LOX-1 knockout mice compared to the wild-type mice. Overall, our study suggests that LOX-1 is a key modulator in the development of angiotensin II-induced hypertension and subsequent renal damage. Glomerulosclerosis is thought to have a central pathogenetic role in the progression from chronic glomerulopathies to end-stage renal disease.1.Klahr S. Schreiner G. Ichikawa I. The progression of renal disease.N Engl J Med. 1988; 318: 1657-1666Crossref PubMed Scopus (734) Google Scholar It is characterized by relentless renal fibrosis. Stress states, such as sustained hypertension, result in glomerulosclerosis. Oxidant stress has been implicated in atherosclerosis2.Bonomini F. Tengattini S. Fabiano A. et al.Atherosclerosis and oxidative stress.Histol Histopathol. 2008; 23: 381-390PubMed Google Scholar and glomerulosclerosis.3.Modlinger P.S. Wilcox C.S. Aslam S. Nitric oxide, oxidative stress, and progression of chronic renal failure.Semin Nephrol. 2004; 24: 354-365Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar LOX-1 has recently been identified as an oxidized low-density lipoprotein (ox-LDL) receptor that is upregulated by redox state4.Mehta J.L. Chen J. Hermonat P.L. et al.Lectin-like, oxidized low-density lipoprotein receptor-1 (LOX-1): a critical player in the development of atherosclerosis and related disorders.Cardiovasc Res. 2006; 69: 36-45Crossref PubMed Scopus (386) Google Scholar and is abundantly expressed in vascular endothelial cells and the renal tissues.5.Nagase M. Kaname S. Nagase T. et al.Expression of LOX-1, an oxidized low-density lipoprotein receptor, in experimental hypertensive glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 1826-1836PubMed Google Scholar LOX-1 is upregulated in the kidneys of salt-loaded Dahl salt-sensitive rats and chronic renal failure rats and parallels glomerulosclerotic changes and renal dysfunction, suggesting a possible link between LOX-1 and the progression to glomerulosclerosis and renal failure.5.Nagase M. Kaname S. Nagase T. et al.Expression of LOX-1, an oxidized low-density lipoprotein receptor, in experimental hypertensive glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 1826-1836PubMed Google Scholar The renin–angiotensin system and its effector hormone, angiotensin II (Ang II), have well-known endocrine properties which contribute to renal fibrosis.6.Billet S. Aguilar F. Baudry C. et al.Role of angiotensin II AT1 receptor activation in cardiovascular diseases.Kidney Int. 2008; 74: 1379-1384Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Previous studies showed that Ang II through type 1 (AT1) receptor activation stimulates LOX-1 expression.7.Chen J. Liu Y. Liu H. et al.Molecular dissection of angiotensin II-activated human LOX-1 promoter.Arterioscler Thromb Vasc Biol. 2006; 26: 1163-1168Crossref PubMed Scopus (38) Google Scholar In turn, LOX-1 activation upregulates AT1 receptor expression.8.Li D. Saldeen T. Romeo F. et al.Oxidized LDL upregulates angiotensin II type 1 receptor expression in cultured human coronary artery endothelial cells: the potential role of transcription factor NF-kappaB.Circulation. 2000; 102: 1970-1976Crossref PubMed Scopus (187) Google Scholar Activation of both AT1 and LOX-1 receptors induces a state of oxidative stress.4.Mehta J.L. Chen J. Hermonat P.L. et al.Lectin-like, oxidized low-density lipoprotein receptor-1 (LOX-1): a critical player in the development of atherosclerosis and related disorders.Cardiovasc Res. 2006; 69: 36-45Crossref PubMed Scopus (386) Google Scholar Recently, we used a mouse model of LOX-1 deficiency (hereafter called LOX-1 knockout mice) and showed that targeted deletion of LOX-1 reduces Ang II-induced hypertension and subsequent cardiac remodeling.9.Hu C. Dandapat A. Sun L. et al.Modulation of angiotensin II-mediated hypertension and cardiac remodeling by LOX-1 deletion.Hypertension. 2008; 52: 556-562Crossref PubMed Scopus (66) Google Scholar To gain further insight into the role of LOX-1 in hypertensive renal injury, we collected the kidneys of these mice and studied the effect of LOX-1 deletion on Ang II-mediated renal injury. Basal systolic blood pressure was similar in the wild-type and LOX-1 knockout mice and remained unchanged in all saline-treated mice. On the other hand, systolic blood pressure progressively increased during the Ang II infusion period, reaching a peak value on day 14 and remaining at plateau through day 28 in the wild-type mice (Figure 1). The rise in blood pressure was less in the LOX-1 knockout mice (P<0.01 vs wild-type mice), despite infusion with the same dose of Ang II. Typical histological photomicrographs of glomerulosclerosis, arterio-arteriolar sclerosis, and tubulointerstitial damage are shown in Figure 2 (left panel). Renal injury scores are shown in Figure 2 (middle panel). Glomerulosclerosis, arterio-arteriolar sclerosis, and tubulointerstitial damage were significantly increased after 28-day infusion of Ang II in the wild-type mice (P<0.01 vs saline-infused mice). The LOX-1 knockout mice, given Ang II infusion, exhibited less glomerulosclerosis, arterio-arteriolar sclerosis, and tubulointerstitial damage (P<0.01 vs wild-type mice). Sustained hypertension and resultant renal fibrosis is characterized by abundant accumulation of matrix proteins in the extracellular space.10.Nagae T. Mori K. Mukoyama M. et al.Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis.Kidney Int. 2008; 74: 70-80Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar We, therefore, determined the accumulation of collagen in multiple sections of kidneys from different animal groups. Results of Masson trichrome and Picro-sirius red staining were similar. In the wild-type mice, sustained hypertension following Ang II infusion resulted in a significant increase in collagen accumulation (Figure 2, right) (P<0.01 vs saline-infused mice). In contrast, the LOX-1 knockout mice showed less increase in collagen accumulation despite Ang II infusion (P<0.01 vs wild-type mice). As shown in Figure 3, the levels of plasma creatinine and BUN were increased in the Ang II-infused wild-type mice (P<0.01 vs saline-infused mice). However, LOX-1 knockout mice showed less increase in the plasma creatinine and BUN levels despite Ang II infusion (P<0.01 vs wild-type mice). Most of the cardiovascular actions of Ang II have been attributed to AT1 receptor activation.6.Billet S. Aguilar F. Baudry C. et al.Role of angiotensin II AT1 receptor activation in cardiovascular diseases.Kidney Int. 2008; 74: 1379-1384Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar We found that AT1 receptor mRNA expression increased in kidneys after infusion of Ang II in the wild-type mice (P<0.01 vs saline-infused mice). However, Ang II-infused LOX-1 KO mice showed less increase in AT1 receptor expression (P<0.01 vs wild-type mice) (Figure 4). The phosphorylation of MAPKs has been linked to the development of renal injury and fibrosis.10.Nagae T. Mori K. Mukoyama M. et al.Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis.Kidney Int. 2008; 74: 70-80Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar The protein levels of p38 and p44/42 MAPKs were unchanged in response to Ang II infusion, but their phosphorylation increased significantly following Ang II infusion (P<0.01 vs saline-infused mice). Importantly, LOX-1 knockout mice exhibited less phosphorylation of MAPKs despite Ang II infusion (P<0.01 vs wild-type mice) (Figure 5). CTGF is a fibrosis mediator.10.Nagae T. Mori K. Mukoyama M. et al.Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis.Kidney Int. 2008; 74: 70-80Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar As shown in Figure 5, the expression of CTGF increased after infusion of Ang II (P<0.01 vs saline-infused wild-type mice). However, LOX-1 knockout mice given Ang II infusion exhibited less increase in the expression of CTGF (P<0.01 vs wild-type mice) Our recent study showed that endothelium-dependent relaxation of aorta as well as eNOS expression are preserved in the LOX-1 knockout mice fed high cholesterol diet.11.Mehta J.L. Sanada N. Hu C.P. et al.Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet.Circ Res. 2007; 100: 1634-1642Crossref PubMed Scopus (374) Google Scholar In this study, we measured eNOS expression in kidney, and found that baseline eNOS expression in the LOX-1 knockout mice was higher than in the wild-type mice. eNOS expression decreased further after Ang II infusion in the wild-type mice (P<0.01); in contrast, LOX-1 knockout mice kidneys showed a smaller decrease in the expression of eNOS after Ang II infusion (P<0.01 vs wild-type mice, Figure 5). There are a host of studies showing inappropriate activation of the intrarenal renin–angiotensin system in the pathogenesis of hypertension and renal injury. Drug therapies directed at angiotensin-converting enzyme and AT1 receptor activation have certainly been shown to attenuate the disease process. However, there is still significant morbidity and mortality associated with hypertensive renal disease. Accordingly, it is necessary to understand other mechanisms responsible for renal injury in hypertension. Our study shows that renal injury caused by hypertension is markedly reduced with LOX-1 deletion. The reduction in renal injury is accompanied by a decrease in AT1 receptor expression and activation of MAPKs and an increase in eNOS expression. The rationale for this study comes from previous observations that oxidant stress induces LOX-1 expression.4.Mehta J.L. Chen J. Hermonat P.L. et al.Lectin-like, oxidized low-density lipoprotein receptor-1 (LOX-1): a critical player in the development of atherosclerosis and related disorders.Cardiovasc Res. 2006; 69: 36-45Crossref PubMed Scopus (386) Google Scholar Ang II through AT1 receptor activation induces LOX-1 expression at transcriptional level.7.Chen J. Liu Y. Liu H. et al.Molecular dissection of angiotensin II-activated human LOX-1 promoter.Arterioscler Thromb Vasc Biol. 2006; 26: 1163-1168Crossref PubMed Scopus (38) Google Scholar In turn, ox-LDL through LOX-1 upregulates the expression of AT1 receptors.8.Li D. Saldeen T. Romeo F. et al.Oxidized LDL upregulates angiotensin II type 1 receptor expression in cultured human coronary artery endothelial cells: the potential role of transcription factor NF-kappaB.Circulation. 2000; 102: 1970-1976Crossref PubMed Scopus (187) Google Scholar These observations have been the basis of the hypothesis of a cross talk between oxidant stress, LOX-1, and Ang II in the genesis of hypertension, atherosclerosis, myocardial ischemia, and renal injury. In a recent study,9.Hu C. Dandapat A. Sun L. et al.Modulation of angiotensin II-mediated hypertension and cardiac remodeling by LOX-1 deletion.Hypertension. 2008; 52: 556-562Crossref PubMed Scopus (66) Google Scholar we documented that Ang II-mediated hypertension is attenuated in the LOX-1 knockout mice; in contrast, norepinephrine-mediated hypertension, which does not involve the expression of LOX-1, is not affected by LOX-1 deletion. Other studies showed that LOX-1 deletion reduces ischemia-reperfusion injury in the wild-type mice12.Hu C. Chen J. Dandapat A. et al.LOX-1 abrogation reduces myocardial ischemia- reperfusion injury in mice.J Mol Cell Cardiol. 2008; 44: 76-83Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar as well as the development of atherosclerosis in the LDL receptor null mice.11.Mehta J.L. Sanada N. Hu C.P. et al.Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet.Circ Res. 2007; 100: 1634-1642Crossref PubMed Scopus (374) Google Scholar Now we show that LOX-1 deletion also reduces markers of renal injury in mice given Ang II. In keeping with the observation of increased AT1 receptor expression in the aortic and cardiac tissues in the wild-type mice given Ang II,9.Hu C. Dandapat A. Sun L. et al.Modulation of angiotensin II-mediated hypertension and cardiac remodeling by LOX-1 deletion.Hypertension. 2008; 52: 556-562Crossref PubMed Scopus (66) Google Scholar AT1 receptor expression in kidneys also increased in the wild-type mice given Ang II infusion. Importantly, LOX-1 knockout mice exhibited less increase in AT1 receptor expression despite Ang II infusion. These findings suggest that Ang II-AT1 receptor-LOX-1 loop is an important regulator of renal injury. Oxidant stress induces endothelial injury and causes downregulation of eNOS expression and activity. These processes have been implicated in hypertension-induced renal injury.3.Modlinger P.S. Wilcox C.S. Aslam S. Nitric oxide, oxidative stress, and progression of chronic renal failure.Semin Nephrol. 2004; 24: 354-365Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar Much of the effect of oxidant stress is mediated through LOX-1 expression/activation because its deletion restores eNOS expression and NO-dependent vasomotor activity.11.Mehta J.L. Sanada N. Hu C.P. et al.Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet.Circ Res. 2007; 100: 1634-1642Crossref PubMed Scopus (374) Google Scholar We now show that LOX-1 deletion attenuates the loss of eNOS expression following Ang II infusion. Ang II-mediated oxidant stress involves a host of intracellular signals, including activation of MAPKs.9.Hu C. Dandapat A. Sun L. et al.Modulation of angiotensin II-mediated hypertension and cardiac remodeling by LOX-1 deletion.Hypertension. 2008; 52: 556-562Crossref PubMed Scopus (66) Google Scholar,13.Chen J. Mehta J.L. Angiotensin II-mediated oxidative stress and procollagen-1 expression in cardiac fibroblasts: blockade by pravastatin and pioglitazone.Am J Physiol Heart Circ Physiol. 2006; 291: H1738-H1745Crossref PubMed Scopus (82) Google Scholar Although most studies show p38 MAPK activation, others have encountered enhanced p44/42 MAPK activity in Ang II-mediated oxidant stress.9.Hu C. Dandapat A. Sun L. et al.Modulation of angiotensin II-mediated hypertension and cardiac remodeling by LOX-1 deletion.Hypertension. 2008; 52: 556-562Crossref PubMed Scopus (66) Google Scholar,14.Xie Z. Singh M. Singh K. ERK1/2 and JNKs, but not p38 kinase, are involved in reactive oxygen species-mediated induction of osteopontin gene expression by angiotensin II and interleukin-1beta in adult rat cardiac fibroblasts.J Cell Physiol. 2004; 198: 399-407Crossref PubMed Scopus (72) Google Scholar An in vitro study showed that ox-LDL-mediated activation of LOX-1 is associated with p38 MAPK activation suggesting activation of pro-inflammatory pathways.15.Li D. Mehta J.L. Antisense to LOX-1 inhibits oxidized LDL-mediated upregulation of monocyte chemoattractant protein-1 and monocyte adhesion to human coronary artery endothelial cells.Circulation. 2000; 101: 2889-2895Crossref PubMed Scopus (374) Google Scholar p44/42 MAPK phosphorylation is also mediated by LOX-1 activation.9.Hu C. Dandapat A. Sun L. et al.Modulation of angiotensin II-mediated hypertension and cardiac remodeling by LOX-1 deletion.Hypertension. 2008; 52: 556-562Crossref PubMed Scopus (66) Google Scholar,12.Hu C. Chen J. Dandapat A. et al.LOX-1 abrogation reduces myocardial ischemia- reperfusion injury in mice.J Mol Cell Cardiol. 2008; 44: 76-83Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar This study shows activation of both p38 and p44/42 isoforms of MAPKs in the renal tissues of Ang II-infused mice. Interestingly, activation of both isoforms of MAPKs was reduced in the LOX-1 knockout mice despite infusion of Ang II. These data suggest a link between LOX-1 activation, MAPK activation, and renal injury in vivo. The extent of renal injury was quantified in this study by histological changes and measurement of plasma BUN and creatinine levels, as in earlier studies.16.Kobayashi N. Hara K. Tojo A. et al.Eplerenone shows renoprotective effect by reducing LOX-1–mediated adhesion molecule, PKCε-MAPK-p90RSK, and rho-kinase pathway.Hypertension. 2005; 45: 538-544Crossref PubMed Scopus (94) Google Scholar,17.Chiba Y. Ando K. Fujita T. The protective effects of taurine against renal damage by salt loading in Dahl salt-sensitive rats.J Hypertens. 2002; 20: 2269-2274Crossref PubMed Scopus (21) Google Scholar All three markers of hypertensive renal injury, glomerulosclerosis, arterio-arteriolar sclerosis and tubulointerstitial damage, were enhanced in the hypertensive wild-type mice, and LOX-1 deletion attenuated these histologic markers of renal injury. Notably, these alterations in renal structure resulted in a modest, but significant, reduction in plasma BUN and creatinine levels in LOX-1 knockout mice (vs wild-type mice). LOX-1 activation stimulates collagen formation.9.Hu C. Dandapat A. Sun L. et al.Modulation of angiotensin II-mediated hypertension and cardiac remodeling by LOX-1 deletion.Hypertension. 2008; 52: 556-562Crossref PubMed Scopus (66) Google Scholar, 18.Chen K. Mehta J.L. Li D. et al.Transforming growth factor beta receptor endoglin is expressed in cardiac fibroblasts and modulates profibrogenic actions of angiotensin II.Circ Res. 2004; 95: 1167-1173Crossref PubMed Scopus (135) Google Scholar, 19.Chen K. Chen J. Liu Y. et al.Adhesion molecule expression in fibroblasts: alteration in fibroblast biology after transfection with LOX-1 plasmids.Hypertension. 2005; 46: 622-627Crossref PubMed Scopus (36) Google Scholar, 20.Hu C. Dandapat A. Chen J. et al.LOX-1 deletion alters signals of myocardial remodeling immediately after ischemia-reperfusion.Cardiovasc Res. 2007; 76: 292-302Crossref PubMed Scopus (59) Google Scholar In this context, it is significant that renal fibrosis was reduced in the LOX-1 knockout mice despite Ang II infusion. The histological evidence was supported by CTGF expression, which was reduced in the basal state; its rise with the development of hypertension was also reduced in the LOX-1 knockout mice. Ando and Fujita21.Ando K. Fujita T. Role of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in the development of hypertensive organ damage.Clin Exp Nephrol. 2004; 8: 178-182Crossref PubMed Scopus (23) Google Scholar have emphasized the role of LOX-1 in rodents with chronic renal injury. Dominguez et al.22.Dominguez J.H. Mehta J.L. Li D. et al.Anti-LOX-1 therapy in rats with diabetes and dyslipidemia: ablation of renal vascular and epithelial manifestations.Am J Physiol Renal Physiol. 2008; 294: F110-F119Crossref PubMed Scopus (40) Google Scholar showed that obese rats, a model of metabolic syndrome in which hypertension is the dominant feature, show renal enlargement, intense oxidative stress, lipid accumulation, leukocyte infiltration, and renal dysfunction and fibrosis. These rats also show LOX-1 overexpression, and treatment with anti-LOX-1 antibody significantly limits or prevents the detrimental effects of obesity on renal function. In other studies by this group,23.Kelly K.J. Wu P. Patterson C.E. et al.LOX-1 and inflammation: a new mechanism for renal injury in obesity and diabetes.Am J Physiol Renal Physiol. 2008; 294: F1136-F1145Crossref PubMed Scopus (53) Google Scholar isolated proximal tubules from obese diabetic rats (NRK52E cells) were exposed to ox-LDL in vitro, and these cells changed their normally quiescent epithelial phenotype into a pro-inflammatory phenotype. These cells after exposure to ox-LDL exhibited suppression of anti-inflammatory transcription factor peroxisome proliferator-activated receptor-delta. In addition, ox-LDL impaired epithelial barrier function. Urine of obese diabetic rats contained more lipid peroxides and elevated levels of LOX-1. Levels of ICAM-1 and focal adhesion kinase, which participate in leukocyte migration and epithelial dedifferentiation, were also upregulated in the tubules of diabetic obese rats. All these alterations were prevented by an anti-LOX-1 antibody. Interestingly, the protective effects of taurine, eplerenone, and statins against renal injury have been attributed to LOX-1 suppression in hypertensive or diabetic rats.16.Kobayashi N. Hara K. Tojo A. et al.Eplerenone shows renoprotective effect by reducing LOX-1–mediated adhesion molecule, PKCε-MAPK-p90RSK, and rho-kinase pathway.Hypertension. 2005; 45: 538-544Crossref PubMed Scopus (94) Google Scholar, 17.Chiba Y. Ando K. Fujita T. The protective effects of taurine against renal damage by salt loading in Dahl salt-sensitive rats.J Hypertens. 2002; 20: 2269-2274Crossref PubMed Scopus (21) Google Scholar, 24.Wang L. Zhang L. Yu Y. et al.The protective effects of taurine against early renal injury in STZ-induced diabetic rats, correlated with inhibition of renal LOX-1-mediated ICAM-1 expression.Ren Fail. 2008; 30: 763-771Crossref PubMed Scopus (28) Google Scholar, 25.Zhou M.S. Schuman I.H. Jaimes E.A. et al.Renoprotection by statins is linked to a decrease in renal oxidative stress, TGF-beta, and fibronectin with concomitant increase in nitric oxide bioavailability.Am J Physiol Renal Physiol. 2008; 295: F53-F59Crossref PubMed Scopus (89) Google Scholar The present observations suggest that LOX-1 deletion modulates renal injury induced by Ang II infusion. However, whether the modulation of renal function is a direct effect of LOX-1 deletion on renal structures or a result of attenuation of hypertension cannot be discerned from this study Nonetheless, amelioration of renal structural changes and dysfunction by LOX-1 deletion strategy was significant. In this process, inhibition of AT1 receptor and MAPKs activity and overexpression of eNOS may have played a putative role. Further, it was obvious that Ang II-induced renal injury was not completely abolished by LOX-1 deletion, suggesting that there are other pathways by which Ang II induces renal injury. This study also does not rule out the possibility that the benefit of LOX-1 deletion in this model may be linked to concomitant alterations in other pathways. In keeping with this suggestion are our recent observations that LOX-1 inactivation leads to upregulation of immunoglobulins in cardiomyocytes challenged with Ang II.26.Kang B.Y. Hu C. Prayaga S. et al.LOX-1 dependent overexpression of immunoglobulins in cardiomyocytes in response to angiotensin II.Biochem Biophys Res Commun. 2009; 379: 395-399Crossref PubMed Scopus (25) Google Scholar C57BL/6 mice (also referred to as wild-type mice) were obtained from Jackson Laboratories (Bar Harbor, ME, USA). The homozygous LOX-1 knockout mice were developed as described recently,11.Mehta J.L. Sanada N. Hu C.P. et al.Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet.Circ Res. 2007; 100: 1634-1642Crossref PubMed Scopus (374) Google Scholar and backcrossed eight times with C57BL/6 strain to replace the genetic background. Male C57BL/6 and LOX-1 knockout mice weighing 22–26 g were utilized at 8–10 weeks of age. All animals received humane care in compliance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals published by the National Institutes of Health. These studies were approved by Institutional Animal Care and Usage Committee. An osmotic minipump (Alzet Model 2004) containing saline vehicle, Ang II (Sigma, St Louis, MO, USA) was implanted into the subcutaneous space of C57BL/6 and LOX-1 knockout mice anesthetized with pentobarbital sodium (80 mg/kg, i.p.). Ang II was delivered at an infusion rate of 50 ng/min for 28 days. The dosage of Ang II was chosen on the basis of published data.27.Weiss D. Kools J.J. Taylor W.R. Angiotensin II-induced hypertension accelerates the development of atherosclerosis in apoE-deficient mice.Circulation. 2001; 103: 448-454Crossref PubMed Scopus (301) Google Scholar,28.Cervenka L. Maly J. Karasova L. et al.Angiotensin II-induced hypertension in bradykinin B2 receptor knockout mice.Hypertension. 2001; 37: 967-973Crossref PubMed Google Scholar Systolic blood pressure was measured through a tail-cuff apparatus (AD Instruments, Bella Vista, NSW, Australia) in conscious mice 3 days before and then on days 0, 2, 5, 9, 14, 21, and 28 after implantation of a minipump. Systolic blood pressure values were derived from an average of five measurements per animal at each time point. Three preliminary training sessions were performed during 1 week before starting the experiment to ensure accurate measurements. In preliminary studies, the accuracy of tail cuff was established by comparing data obtained with biotelemetry system (Data Sciences, St Paul, MN, USA). Kidney sections were stained with hematoxylin and eosin, periodic acid-Schiff, and periodic acid-methenamine silver. Glomerulosclerosis, arterio-arteriolar sclerosis, and tubulointerstitial damage were scored as described.16.Kobayashi N. Hara K. Tojo A. et al.Eplerenone shows renoprotective effect by reducing LOX-1–mediated adhesion molecule, PKCε-MAPK-p90RSK, and rho-kinase pathway.Hypertension. 2005; 45: 538-544Crossref PubMed Scopus (94) Google Scholar The pathologist (JM) scored renal injury according to the method published in Figure 1a of reference 16 while unaware of source of kidney. Glomerulosclerosis was scored in 100 glomeruli in each section as G0 for a normal glomerulus; G1, mild sclerosis (<25%); G2, moderate segmental sclerosis (25–50%); G3, severe segmental sclerosis (50–75%); and G4, global sclerosis. Arterio-arteriolar sclerosis was scored in 20–30 arteries and arterioles in each section as A0, normal; A1, thickening of media; A2, focal hyalinosis with thickening of media; A3, medial thickening with global hyalinosis; A4, fibrinoid necrosis and/or cellular hyperplasia with narrowing of arteriolar lumen and/or thrombus formation. Tubulointerstitial damage was categorized in 25 areas per section randomly photographed as TI0, normal; TI1, periarterial fibrosis; TI2, peritubular fibrosis; TI3, hyaline casts with focal fibrosis; and TI4, focal fibrosis with cellular infiltration in a large area. The damage score was calculated in five mice in each group as (0 × number of S0+1 × number of S1+2 × number of S2+3 × number of S3+4 × number of S4)/(number of S0+S1+S2+S3+S4), where S represents glomerulosclerosis, arterio-arteriolar sclerosis, or tubulointerstitial damage. Renal fibrosis was assessed by analysis of collagen accumulation. Renal tissues were prepared and embedded in paraffin, cut into 5-μm thick sections, and stained with hematoxylin–eosin, Masson's trichrome and Picro-sirius red. Plasma creatinine and BUN were determined using a diagnostic kit from International Bio-Analytical Industries Inc. (Boca Raton, FL, USA). RT-PCR analysis for AT1 receptor was performed as described previously.20.Hu C. Dandapat A. Chen J. et al.LOX-1 deletion alters signals of myocardial remodeling immediately after ischemia-reperfusion.Cardiovasc Res. 2007; 76: 292-302Crossref PubMed Scopus (59) Google Scholar GAPDH was used as a standard control. The primers used are as follows. GAPDH: forward, 5′-AACTTTGGCATTGTGGAAGG-3′; reverse, 5′-ACACATTGGGGGTAGGAACA-3′. AT1 receptor: forward, 5′-TCATTTACTTTTATATTGTAA-3′; reverse 5′-TGAATTTCATAAGCCTTCTT-3′. Expression of p38 and p44/42 MAPK isoforms was assessed by western analysis using standard methodologies.13.Chen J. Mehta J.L. Angiotensin II-mediated oxidative stress and procollagen-1 expression in cardiac fibroblasts: blockade by pravastatin and pioglitazone.Am J Physiol Heart Circ Physiol. 2006; 291: H1738-H1745Crossref PubMed Scopus (82) Google Scholar All primary antibodies were purchased from Santa Cruz, CA, USA. The relative densities of protein bands were analyzed by Scan-gel-it.13.Chen J. Mehta J.L. Angiotensin II-mediated oxidative stress and procollagen-1 expression in cardiac fibroblasts: blockade by pravastatin and pioglitazone.Am J Physiol Heart Circ Physiol. 2006; 291: H1738-H1745Crossref PubMed Scopus (82) Google Scholar RT-PCR analysis for the expression of eNOS and CTGF was performed as described previously.20.Hu C. Dandapat A. Chen J. et al.LOX-1 deletion alters signals of myocardial remodeling immediately after ischemia-reperfusion.Cardiovasc Res. 2007; 76: 292-302Crossref PubMed Scopus (59) Google Scholar GAPDH was used as a standard control. The primers used are as follows. GAPDH: forward, 5′-GACCACAGTCCATGCCATCACT-3′; reverse, 5′-TCCACCACCCTGTTGCTGTAG-3′. eNOS: forward 5′-CCTTCCGCTACCAGCCAGA-3′; reverse, 5′-CAGAGATCTTCACTGCATTGGCTA-3; CTGF: forward, 5-GAGTGGGTGTGTGACGAGCCCAAGG-3′; reverse, 5′-ATGTCTCCGTACATCTTCCTGTAGT-3′. Data are presented as mean±s.e. The data regarding the expression of mRNA and protein and renal injury were analyzed by Kruskal–Wallis H-test. All other data were analyzed by a one-way ANOVA with a Newman–Student–Keul t-test. Four fold comparisons were performed and an adjusted value of P<0.05 was considered to be significant. All calculations were performed with SPSS version 12.0. All the authors declared no competing interests. This study was supported by funds from the Department of Veterans Affairs (JLM). This paper is dedicated to the memory of Prof. Thomas E. Andreoli, who suddenly passed away on 14 April 2009. He inspired and cultivated many clinician-scientists. We feel privileged to have worked under his guidance.
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