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Deletion of LOX-1 attenuates renal injury following angiotensin II infusion
original article
http://www.kidney-international.org & 2009 International Society of Nephrology
Deletion of LOX-1 attenuates renal injury following angiotensin II infusion Changping Hu1,2, Bum-Yong Kang1, Judit Megyesi1, Gur P. Kaushal1, Robert L. Safirstein1 and Jawahar L. Mehta1 1
Department of Medicine, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas, USA and 2Department of Pharmacology, School of Pharmaceutical Science, Central South University, Changsha, China
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 LOX1 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. Kidney International (2009) 76, 521–527; doi:10.1038/ki.2009.234; published online 24 June 2009 KEYWORDS: angiotensin; hypertension; LOX-1; oxidative stress; renal dysfunction
Glomerulosclerosis is thought to have a central pathogenetic role in the progression from chronic glomerulopathies to end-stage renal disease.1 It is characterized by relentless renal fibrosis. Stress states, such as sustained hypertension, result in glomerulosclerosis. Oxidant stress has been implicated in atherosclerosis2 and glomerulosclerosis.3 LOX-1 has recently been identified as an oxidized low-density lipoprotein (ox-LDL) receptor that is upregulated by redox state4 and is abundantly expressed in vascular endothelial cells and the renal tissues.5 LOX-1 is upregulated in the kidneys of saltloaded 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 The renin–angiotensin system and its effector hormone, angiotensin II (Ang II), have well-known endocrine properties which contribute to renal fibrosis.6 Previous studies showed that Ang II through type 1 (AT1) receptor activation stimulates LOX-1 expression.7 In turn, LOX-1 activation upregulates AT1 receptor expression.8 Activation of both AT1 and LOX-1 receptors induces a state of oxidative stress.4 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 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. RESULTS Blood pressure in response to Ang II
Correspondence: Jawahar L. Mehta, University of Arkansas for Medical Sciences, 4301 W Markham, Slot 532, Little Rock, Arkansas 72205, USA. E-mail: [email protected] Received 21 January 2009; revised 22 April 2009; accepted 5 May 2009; published online 24 June 2009 Kidney International (2009) 76, 521–527
Basal systolic blood pressure was similar in the wild-type and LOX-1 knockout mice and remained unchanged in all salinetreated 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 (Po0.01 vs wild-type mice), despite infusion with the same dose of Ang II. 521
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C Hu et al.: LOX-1, hypertension and renal injury
WT saline control (n=6)
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Figure 1 | Systolic blood pressure rise in response to angiotensin II (Ang II) infusion in wild-type (WT) and LOX-1 knockout (KO) mice. LOX-1 deletion attenuated blood pressure response to Ang II infusion. Data (in mean±s.e.) from 6–12 animals.
Renal morphology
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, arterioarteriolar sclerosis, and tubulointerstitial damage were significantly increased after 28-day infusion of Ang II in the wild-type mice (Po0.01 vs saline-infused mice). The LOX-1 knockout mice, given Ang II infusion, exhibited less glomerulosclerosis, arterio-arteriolar sclerosis, and tubulointerstitial damage (Po0.01 vs wild-type mice). Renal fibrosis and injury following sustained hypertension and effect of LOX-1 deletion
Sustained hypertension and resultant renal fibrosis is characterized by abundant accumulation of matrix proteins in the extracellular space.10 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) (Po0.01 vs saline-infused mice). In contrast, the LOX-1 knockout mice showed less increase in collagen accumulation despite Ang II infusion (Po0.01 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 (Po0.01 vs saline-infused mice). However, LOX-1 knockout 522
mice showed less increase in the plasma creatinine and BUN levels despite Ang II infusion (Po0.01 vs wild-type mice). Expression of AT1 receptor, MAPKs, CTGF, and eNOS induced by Ang II infusion, and effect of LOX-1 deletion
Most of the cardiovascular actions of Ang II have been attributed to AT1 receptor activation.6 We found that AT1 receptor mRNA expression increased in kidneys after infusion of Ang II in the wild-type mice (Po0.01 vs salineinfused mice). However, Ang II-infused LOX-1 KO mice showed less increase in AT1 receptor expression (Po0.01 vs wild-type mice) (Figure 4). The phosphorylation of MAPKs has been linked to the development of renal injury and fibrosis.10 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 (Po0.01 vs salineinfused mice). Importantly, LOX-1 knockout mice exhibited less phosphorylation of MAPKs despite Ang II infusion (Po0.01 vs wild-type mice) (Figure 5). CTGF is a fibrosis mediator.10 As shown in Figure 5, the expression of CTGF increased after infusion of Ang II (Po0.01 vs saline-infused wild-type mice). However, LOX-1 knockout mice given Ang II infusion exhibited less increase in the expression of CTGF (Po0.01 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 In this study, we measured eNOS expression in kidney, and Kidney International (2009) 76, 521–527
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C Hu et al.: LOX-1, hypertension and renal injury
*P< 0.01 vs wild-type saline +P<0.01 vs LOX-1 KO saline #P<0.01 vs wild-type Ang II
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Figure 2 | Renal morphology in mice given Ang II. The left panel shows typical histological photomicrographs of glomerulosclerosis, arterio-arteriolar sclerosis, and tubulointerstitial damage. Summary of renal injury scores is shown in the middle panel. LOX-1 knockout mice given Ang II infusion exhibited less glomerulosclerosis, arterio-arteriolar sclerosis, and tubulointerstitial damage (n ¼ 5). Staining for collagen in renal sections is shown in the right panel. Tissue sections were stained with Masson’s trichrome and Picrosirius red. LOX-1 knockout mice exhibited less increase in collagen accumulation despite Ang II infusion.
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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 (Po0.01); in contrast, LOX-1 knockout mice kidneys showed a smaller decrease in the expression of eNOS after Ang II infusion (Po0.01 vs wild-type mice, Figure 5).
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Figure 3 | Measurement of plasma creatinine and blood urea nitrogen (BUN). LOX-1 knockout mice given Ang II infusion showed less increase in the plasma levels of creatinine and BUN (n ¼ 6–12). Kidney International (2009) 76, 521–527
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 Ang II through AT1 receptor activation induces LOX-1 expression at transcriptional level.7 In turn, ox-LDL through LOX-1 upregulates the expression of AT1 receptors.8 These observations have been the basis of the hypothesis of a cross talk between oxidant stress, LOX-1, and Ang II in the genesis 523
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* P<0.01 vs Wild-type saline + P <0.01 vs LOX-1 KO saline # P <0.01 vs Wild-type Ang II 2
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Figure 4 | Expression of Angiotensin II type 1 receptor (AT1R). Ang II-infused LOX-1 knockout mice showed less increase in the expression of AT1R (n ¼ 5).
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of hypertension, atherosclerosis, myocardial ischemia, and renal injury. In a recent study,9 we documented that Ang IImediated 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 as well as the development of atherosclerosis in the LDL receptor null mice.11 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 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 Much of the effect of oxidant stress is mediated through LOX-1 expression/activation because its deletion
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Figure 5 | Expression of mitogen-activated protein kinases (MAPKs), connective-tissue growth factor (CTGF), and endothelial nitric oxide synthase (eNOS). Ang II-infused LOX-1 knockout mice exhibited less phosphorylation of MAPKs and less increase in the expression of CTGF. LOX-1 deletion was associated with a smaller decrease in the expression of eNOS despite Ang II infusion (n ¼ 5). 524
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C Hu et al.: LOX-1, hypertension and renal injury
restores eNOS expression and NO-dependent vasomotor activity.11 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.9,13 Although most studies show p38 MAPK activation, others have encountered enhanced p44/42 MAPK activity in Ang IImediated oxidant stress.9,14 An in vitro study showed that oxLDL-mediated activation of LOX-1 is associated with p38 MAPK activation suggesting activation of pro-inflammatory pathways.15 p44/42 MAPK phosphorylation is also mediated by LOX-1 activation.9,12 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.16,17 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.9,18–20 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 have emphasized the role of LOX-1 in rodents with chronic renal injury. Dominguez et al.22 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 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 Kidney International (2009) 76, 521–527
original article
protective effects of taurine, eplerenone, and statins against renal injury have been attributed to LOX-1 suppression in hypertensive or diabetic rats.16,17,24,25 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 MATERIALS AND METHODS Animals 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 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. Infusion of Ang II 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.27,28 Determination of blood pressure 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). Renal morphology and renal injury score Kidney sections were stained with hematoxylin and eosin, periodic acid-Schiff, and periodic acid-methenamine silver. Glomerulosclero525
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sis, arterio-arteriolar sclerosis, and tubulointerstitial damage were scored as described.16 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 (o25%); 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 þ 1 number of S1 þ 2 number of S2 þ 3 number of S3 þ 4 number of S4)/(number of S0 þ S1 þ S2 þ S3 þ S4), where S represents glomerulosclerosis, arterio-arteriolar sclerosis, or tubulointerstitial damage.
C Hu et al.: LOX-1, hypertension and renal injury
DISCLOSURE
All the authors declared no competing interests. ACKNOWLEDGMENTS
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. REFERENCES 1. 2. 3.
4.
5.
6.
Histological evaluation of renal fibrosis Renal fibrosis was assessed by analysis of collagen accumulation. Renal tissues were prepared and embedded in paraffin, cut into 5-mm thick sections, and stained with hematoxylin–eosin, Masson’s trichrome and Picro-sirius red.
7.
Measurement of plasma creatinine and BUN Plasma creatinine and BUN were determined using a diagnostic kit from International Bio-Analytical Industries Inc. (Boca Raton, FL, USA).
9.
8.
10.
Expression analysis RT-PCR analysis for AT1 receptor was performed as described previously.20 GAPDH was used as a standard control. The primers used are as follows. GAPDH: forward, 50 -AACTTTGG CATTGTGGAAGG-30 ; reverse, 50 -ACACATTGGGGGTAGGAACA30 . AT1 receptor: forward, 50 -TCATTTACTTTTATATTGTAA-30 ; reverse 50 -TGAATTTCATAAGCCTTCTT-30 . Expression of p38 and p44/42 MAPK isoforms was assessed by western analysis using standard methodologies.13 All primary antibodies were purchased from Santa Cruz, CA, USA. The relative densities of protein bands were analyzed by Scan-gel-it.13 RT-PCR analysis for the expression of eNOS and CTGF was performed as described previously.20 GAPDH was used as a standard control. The primers used are as follows. GAPDH: forward, 50 -GACCACAGTCCATGCCATCACT-30 ; reverse, 50 -TCCACCACCC TGTTGCTGTAG-30 . eNOS: forward 50 -CCTTCCGCTACCAGCC AGA-30 ; reverse, 50 -CAGAGATCTTCACTGCATTGGCTA-3; CTGF: forward, 5-GAGTGGGTGTGTGACGAGCCCAAGG-30 ; reverse, 50 -ATG TCTCCGTACATCTTCCTGTAGT-30 . Statistical analysis 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 Po0.05 was considered to be significant. All calculations were performed with SPSS version 12.0. 526
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http://www.kidney-international.org & 2009 International Society of Nephrology
Deletion of LOX-1 attenuates renal injury following angiotensin II infusion Changping Hu1,2, Bum-Yong Kang1, Judit Megyesi1, Gur P. Kaushal1, Robert L. Safirstein1 and Jawahar L. Mehta1 1
Department of Medicine, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas, USA and 2Department of Pharmacology, School of Pharmaceutical Science, Central South University, Changsha, China
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 LOX1 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. Kidney International (2009) 76, 521–527; doi:10.1038/ki.2009.234; published online 24 June 2009 KEYWORDS: angiotensin; hypertension; LOX-1; oxidative stress; renal dysfunction
Glomerulosclerosis is thought to have a central pathogenetic role in the progression from chronic glomerulopathies to end-stage renal disease.1 It is characterized by relentless renal fibrosis. Stress states, such as sustained hypertension, result in glomerulosclerosis. Oxidant stress has been implicated in atherosclerosis2 and glomerulosclerosis.3 LOX-1 has recently been identified as an oxidized low-density lipoprotein (ox-LDL) receptor that is upregulated by redox state4 and is abundantly expressed in vascular endothelial cells and the renal tissues.5 LOX-1 is upregulated in the kidneys of saltloaded 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 The renin–angiotensin system and its effector hormone, angiotensin II (Ang II), have well-known endocrine properties which contribute to renal fibrosis.6 Previous studies showed that Ang II through type 1 (AT1) receptor activation stimulates LOX-1 expression.7 In turn, LOX-1 activation upregulates AT1 receptor expression.8 Activation of both AT1 and LOX-1 receptors induces a state of oxidative stress.4 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 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. RESULTS Blood pressure in response to Ang II
Correspondence: Jawahar L. Mehta, University of Arkansas for Medical Sciences, 4301 W Markham, Slot 532, Little Rock, Arkansas 72205, USA. E-mail: [email protected] Received 21 January 2009; revised 22 April 2009; accepted 5 May 2009; published online 24 June 2009 Kidney International (2009) 76, 521–527
Basal systolic blood pressure was similar in the wild-type and LOX-1 knockout mice and remained unchanged in all salinetreated 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 (Po0.01 vs wild-type mice), despite infusion with the same dose of Ang II. 521
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WT saline control (n=6)
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Figure 1 | Systolic blood pressure rise in response to angiotensin II (Ang II) infusion in wild-type (WT) and LOX-1 knockout (KO) mice. LOX-1 deletion attenuated blood pressure response to Ang II infusion. Data (in mean±s.e.) from 6–12 animals.
Renal morphology
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, arterioarteriolar sclerosis, and tubulointerstitial damage were significantly increased after 28-day infusion of Ang II in the wild-type mice (Po0.01 vs saline-infused mice). The LOX-1 knockout mice, given Ang II infusion, exhibited less glomerulosclerosis, arterio-arteriolar sclerosis, and tubulointerstitial damage (Po0.01 vs wild-type mice). Renal fibrosis and injury following sustained hypertension and effect of LOX-1 deletion
Sustained hypertension and resultant renal fibrosis is characterized by abundant accumulation of matrix proteins in the extracellular space.10 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) (Po0.01 vs saline-infused mice). In contrast, the LOX-1 knockout mice showed less increase in collagen accumulation despite Ang II infusion (Po0.01 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 (Po0.01 vs saline-infused mice). However, LOX-1 knockout 522
mice showed less increase in the plasma creatinine and BUN levels despite Ang II infusion (Po0.01 vs wild-type mice). Expression of AT1 receptor, MAPKs, CTGF, and eNOS induced by Ang II infusion, and effect of LOX-1 deletion
Most of the cardiovascular actions of Ang II have been attributed to AT1 receptor activation.6 We found that AT1 receptor mRNA expression increased in kidneys after infusion of Ang II in the wild-type mice (Po0.01 vs salineinfused mice). However, Ang II-infused LOX-1 KO mice showed less increase in AT1 receptor expression (Po0.01 vs wild-type mice) (Figure 4). The phosphorylation of MAPKs has been linked to the development of renal injury and fibrosis.10 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 (Po0.01 vs salineinfused mice). Importantly, LOX-1 knockout mice exhibited less phosphorylation of MAPKs despite Ang II infusion (Po0.01 vs wild-type mice) (Figure 5). CTGF is a fibrosis mediator.10 As shown in Figure 5, the expression of CTGF increased after infusion of Ang II (Po0.01 vs saline-infused wild-type mice). However, LOX-1 knockout mice given Ang II infusion exhibited less increase in the expression of CTGF (Po0.01 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 In this study, we measured eNOS expression in kidney, and Kidney International (2009) 76, 521–527
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C Hu et al.: LOX-1, hypertension and renal injury
*P< 0.01 vs wild-type saline +P<0.01 vs LOX-1 KO saline #P<0.01 vs wild-type Ang II
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Figure 2 | Renal morphology in mice given Ang II. The left panel shows typical histological photomicrographs of glomerulosclerosis, arterio-arteriolar sclerosis, and tubulointerstitial damage. Summary of renal injury scores is shown in the middle panel. LOX-1 knockout mice given Ang II infusion exhibited less glomerulosclerosis, arterio-arteriolar sclerosis, and tubulointerstitial damage (n ¼ 5). Staining for collagen in renal sections is shown in the right panel. Tissue sections were stained with Masson’s trichrome and Picrosirius red. LOX-1 knockout mice exhibited less increase in collagen accumulation despite Ang II infusion.
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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 (Po0.01); in contrast, LOX-1 knockout mice kidneys showed a smaller decrease in the expression of eNOS after Ang II infusion (Po0.01 vs wild-type mice, Figure 5).
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Figure 3 | Measurement of plasma creatinine and blood urea nitrogen (BUN). LOX-1 knockout mice given Ang II infusion showed less increase in the plasma levels of creatinine and BUN (n ¼ 6–12). Kidney International (2009) 76, 521–527
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 Ang II through AT1 receptor activation induces LOX-1 expression at transcriptional level.7 In turn, ox-LDL through LOX-1 upregulates the expression of AT1 receptors.8 These observations have been the basis of the hypothesis of a cross talk between oxidant stress, LOX-1, and Ang II in the genesis 523
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* P<0.01 vs Wild-type saline + P <0.01 vs LOX-1 KO saline # P <0.01 vs Wild-type Ang II 2
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Figure 4 | Expression of Angiotensin II type 1 receptor (AT1R). Ang II-infused LOX-1 knockout mice showed less increase in the expression of AT1R (n ¼ 5).
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of hypertension, atherosclerosis, myocardial ischemia, and renal injury. In a recent study,9 we documented that Ang IImediated 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 as well as the development of atherosclerosis in the LDL receptor null mice.11 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 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 Much of the effect of oxidant stress is mediated through LOX-1 expression/activation because its deletion
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Figure 5 | Expression of mitogen-activated protein kinases (MAPKs), connective-tissue growth factor (CTGF), and endothelial nitric oxide synthase (eNOS). Ang II-infused LOX-1 knockout mice exhibited less phosphorylation of MAPKs and less increase in the expression of CTGF. LOX-1 deletion was associated with a smaller decrease in the expression of eNOS despite Ang II infusion (n ¼ 5). 524
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C Hu et al.: LOX-1, hypertension and renal injury
restores eNOS expression and NO-dependent vasomotor activity.11 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.9,13 Although most studies show p38 MAPK activation, others have encountered enhanced p44/42 MAPK activity in Ang IImediated oxidant stress.9,14 An in vitro study showed that oxLDL-mediated activation of LOX-1 is associated with p38 MAPK activation suggesting activation of pro-inflammatory pathways.15 p44/42 MAPK phosphorylation is also mediated by LOX-1 activation.9,12 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.16,17 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.9,18–20 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 have emphasized the role of LOX-1 in rodents with chronic renal injury. Dominguez et al.22 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 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 Kidney International (2009) 76, 521–527
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protective effects of taurine, eplerenone, and statins against renal injury have been attributed to LOX-1 suppression in hypertensive or diabetic rats.16,17,24,25 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 MATERIALS AND METHODS Animals 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 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. Infusion of Ang II 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.27,28 Determination of blood pressure 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). Renal morphology and renal injury score Kidney sections were stained with hematoxylin and eosin, periodic acid-Schiff, and periodic acid-methenamine silver. Glomerulosclero525
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sis, arterio-arteriolar sclerosis, and tubulointerstitial damage were scored as described.16 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 (o25%); 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 þ 1 number of S1 þ 2 number of S2 þ 3 number of S3 þ 4 number of S4)/(number of S0 þ S1 þ S2 þ S3 þ S4), where S represents glomerulosclerosis, arterio-arteriolar sclerosis, or tubulointerstitial damage.
C Hu et al.: LOX-1, hypertension and renal injury
DISCLOSURE
All the authors declared no competing interests. ACKNOWLEDGMENTS
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. REFERENCES 1. 2. 3.
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6.
Histological evaluation of renal fibrosis Renal fibrosis was assessed by analysis of collagen accumulation. Renal tissues were prepared and embedded in paraffin, cut into 5-mm thick sections, and stained with hematoxylin–eosin, Masson’s trichrome and Picro-sirius red.
7.
Measurement of plasma creatinine and BUN Plasma creatinine and BUN were determined using a diagnostic kit from International Bio-Analytical Industries Inc. (Boca Raton, FL, USA).
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8.
10.
Expression analysis RT-PCR analysis for AT1 receptor was performed as described previously.20 GAPDH was used as a standard control. The primers used are as follows. GAPDH: forward, 50 -AACTTTGG CATTGTGGAAGG-30 ; reverse, 50 -ACACATTGGGGGTAGGAACA30 . AT1 receptor: forward, 50 -TCATTTACTTTTATATTGTAA-30 ; reverse 50 -TGAATTTCATAAGCCTTCTT-30 . Expression of p38 and p44/42 MAPK isoforms was assessed by western analysis using standard methodologies.13 All primary antibodies were purchased from Santa Cruz, CA, USA. The relative densities of protein bands were analyzed by Scan-gel-it.13 RT-PCR analysis for the expression of eNOS and CTGF was performed as described previously.20 GAPDH was used as a standard control. The primers used are as follows. GAPDH: forward, 50 -GACCACAGTCCATGCCATCACT-30 ; reverse, 50 -TCCACCACCC TGTTGCTGTAG-30 . eNOS: forward 50 -CCTTCCGCTACCAGCC AGA-30 ; reverse, 50 -CAGAGATCTTCACTGCATTGGCTA-3; CTGF: forward, 5-GAGTGGGTGTGTGACGAGCCCAAGG-30 ; reverse, 50 -ATG TCTCCGTACATCTTCCTGTAGT-30 . Statistical analysis 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 Po0.05 was considered to be significant. All calculations were performed with SPSS version 12.0. 526
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