The Mechanism Of Renal Impairment Of Atherosclerotic Renal Artery Stenosis: An Experimental Study

BackgroundARAS is the most common cause for renal artery stenosis. As ARAS results in renal artery occlusion, significant reduction in the glomerular filtration rate (GFR) and kidney atrophy, ischemic nephropathy can be diagnosed. It is currently thought that the pathophysiology of severe renal artery stenosis and ischemic nephropathy is stenosis-induced renal ischemia, malignant hypertension secondary to continual activation of the blood pressure regulating systems (the renin-angiotension system and adrenal glands) and glomerular filtration dysfunction. Progressive renal artery stenosis is a major cause for end-stage ischemic nephropathy. Current indication for renal angioplasty is renal artery stenosis > 75%. Nevertheless, it is suggested that vascular stenting worsens renal function of patients with significant ARAS. Therefore, early diagnosis and treatment of ARAS is crucial. Nevertheless, since the mechanism of early ARAS remains unclear, no effective method to monitor and treat ARAS is available. It is demonstrated that ARAS is a manifestation of systematic atherosclerosis in the renal artery, and that atherosclerotic changes are essentially chronic progressive inflammatory reactions. The formation, rupture and progressive enlargement of atherosclerotic plaques are closely associated with chronic progressive inflammatory reactions. Clinical observations showed that the degree of mild to moderate ARAS does not parallel to the degree of renal damage. Clinical research has demonstrated that ARAS is not the main cause for renal parenchymal damage. Hence, it is postulated that chronic renal damage may result from other factors than renal artery stenosis in ARAS patients. These results suggest that atherosclerotic inflammatory reaction may also act on the renal parenchyma.Hence, we postulate that:①inflammatory reaction may be one of the important mechanisms for renal impairment progression in ARAS;②the severity of renal impairment ought to be consistent with that of renal artery atherosclerosis. Current methods to examine the renal artery cannot accurately reflect renal artery atherosclerosis and renal impairment. Nevertheless, the degree of ARAS can be inferred according to renal pathological changes and renal functional impairment, particularly tubular functional impairment. Regarding the timing for ARAS treatment, renal pathology and function should be stressed. In particular, dynamic detection of tubular function may help identify ARAS progression early;③ARAS-caused renal impairment is not always consistent with the severity of RAS; hence, the timing of vascular stenting cannot be determined according to the severity of renal artery stenosis.In the present study, ARAS models were established in ApoE-/- mice, and then the characteristics of renal impairment were dynamically observed during ARAS. The mechanisms of renal impairment in ARAS and the effect of rapamycin, an anti-inflammatory drug, on the progression of renal impairment were explored. Furthermore, in order to investigate the role of inflammation in ARAS-caused renal impairment and the timing for renal artery stent placement, renal arteriography, biopsy and tubular function testing were carried out in ARAS patients.Methods and results1. Establishment of ARAS models and observations of ARAS-caused renal impairment.The renal arteries and kidneys were harvested from C57BL/6J ApoE-knockout mice and wild-type mice of the same age. The renal arteries were embedded in paraffin, sectioned successively, and subjected to V.G staining. The sections were analyzed for renal artery stenosis using Image Pro-Plus image analysis system (Media Chernetics Inc. USA). Based on the severity of ARAS, the animals were divided into 3 groups (A: renal arterial luminal stenosis <50%; B: 50% -70%; C: >70%). In addition, group A was subdivided to A1 (without atherosclerotic plaque rupture) and A2 (with atherosclerotic plaque rupture) according to the status of atherosclerotic plaques. Kidney sections were subjected to conventional HE, PAS and Masson staining, and observed dynamically under electronmicroscopy and light microscopy for renal artery lesions and renal lesions.Results: 1. ARAS models were established successfully in ApoE-/- mice; 2. with the progression of renal artery atherosclerotic lesions, i.e., rupture and progressive enlargement of atherosclerotic plaque, tubular interstitial impairment occurred and aggravated, which resulted in tubular interstitial fibrosis and nephron destruction. 2. Mechanisms of renal impairment due to progression of renal artery atherosclerotic lesions in ApoE-/- mice.(1) ApoE-/- mice from groups A1 and A2 and wild-type mice of the same age were raised under the same conditions. In order to investigate the mechanism of renal impairment due to rupture of renal artery atherosclerotic plaques, NF-κB p65, ICAM-1 and P-sel expression was detected by Western blotting; IL-6 expression was detected by ELISA; IL-6 mRNA was detected by RT-PCR; macrophages were observed immunohistochemically; urine NAG was detected by direct enzyme-substrate coloration.(2) ApoE-/- mice were used in the experimental group, and wild-type mice were included in the control group. In order to investigate the effect of progressive lesion enlargement after renal artery atherosclerotic plaque rupture on renal inflammatory impairment and tubular interstitial fibrosis in ApoE-/-mice,α-SMA, ICAM-1, P-sel, NF-κBp65 expression and macrophages (F4/80) in the kidney tissue were detected immunohistochemically; IL-6 and TF expression was detection by ELISA; urine NAG was detected by direct enzyme-substrate coloration; blood creatinine was determined using an automatic biochemistry analyzer.(3) In order to investigate the role of mononuclear macrophages in ARAS-caused renal impairment of ApoE-/- mice, kidney tissue of groups A1, A2, B and of the control group were observed under laser confocal scanning microscopy for changes in the distribution of proinflammatory mononuclear macrophages;α-SMA was detected immunohistochemically; the expression of Col I and Col III was observed under polarizing microscopy. Results:①NF-κB p65 dominated ARAS progression, which played important roles in tubular-interstitial cell phenotypic transformation, tubular interstitialα-SMA expression upregulation, extensive necrosis of tubular epithelial cells and interstitial fibrosis, and inflammatory cell infiltration, as well as interstitial inflammatory impairment.②with rupture and enlargement of renal artery atherosclerotic plaques, pro-inflammatory, activated macrophages accumulated at the site of plaque ruptures and in tubular interstitium.③Aggravation of inflammatory reactions was the major cause for rupture and progressive enlargement of renal artery atherosclerotic plaques and subsequent renal artery stenosis, and was also one of the key factors causing progressive tubular interstitial impairment and nephron destruction. 3. Effect of rapamycin on the progression of ARAS-caused renal impairment in ApoE-/- mice.(1) In vivo study: 24 male mice were used in the study:①33-week-old ApoE-/- mice;②medication group: 33-week-old ApoE-/-mice, which were intragastrically administered 100ug/kg rapamycin daily for 8 to 41 weeks;③41-week-old ApoE-/-mice;④control group: 41-week-old wild-type mice (n=6 for each group). The severity of renal artery stenosis, M1-type macrophage infiltration in the renal artery and kidney, NF-κBp65 and IL-6mRNA expression, and the severity of renal fibrosis were investigated in each group.(2) In vitro cell culture: In order to investigate the effect of rapamycin on the proinflammatory properties of mononuclear macrophages, mouse peritoneal macrophages were co-stimulated with rapamycin and LPS, and the changes in NF-κBp65 and MHCII expression and IL-6mRNA transcription were detected in each group. Results:①rapamycin significantly reduced M1-type macrophage infiltration in renal artery atherosclerotic plaques and renal interstitium, downregulated NF-κBp65 expression by the renal artery and renal tissue, the transcription of IL-6 (a central regulator of inflammatory reaction), and the expression of renalα-SMA and types I, III collagen;②rapamycin inhibited the activation by LPS of MHCII and NF-κBp65 expression by mouse peritoneal macrophages in a dose dependent manner. These findings support inflammatory reaction as an important pathological mechanism of ARAS caused renal impairment.4. In patients on high risk of ARAS, tubular interstitial inflammatory reaction was assessed by determining urine NAG/Ucr and the urine macrophage detection rate and performing renal biopsy, if necessary. ARAS patients were screened through color Doppler of the renal artery or/and nephrogram, and were treated by renal artery stenting after definite diagnosis by arteriography. Changes in urine NAG/Ucr and the urine macrophage detection rate were observed before and after stent placement and in patients who did not undergo stenting.Results: 1. Inflammatory reaction was found to be an important mechanism for ARAS caused renal impairment, which affected tubular interstitium first; 2. Urine NAG/UCr and the urine macrophage detection rate reflected tubular interstitial impairment, thus helping monitor ARAS progression and assess the therapeutic effect of stenting; 3. The severity of ARAS stenosis was not an indicator for treatment, but active ARAS lesions should be treated.Conclusions1. ARAR models were established successfully in C57BL/6J ApoE-/- mice. Active ARAS lesions in ApoE-/- mice, i.e., rupture and progressive enlargement of atherosclerotic plaques, are an important factor promoting and aggravating renal lesions, and tubular interstitium is the first to be affected.2. Aggravation of inflammatory reaction acts as an important mechanism of active ARAS lesions, i.e., rupture and progressive enlargement of renal artery atherosclerotic plaques, and ARAS-caused renal impairment is mediated by this mechanism. Early control of renal artery inflammatory reaction is beneficial for delaying or suppressing the occurrence of tubular interstitial lesions and protecting renal function.3. Both in vivo and in vitro experiments indicated that rapamycin①reduces the accumulation of macrophages in the renal artery and renal tissue of ApoE-/- mice and suppresses the proinflammatory action of macrophages;②downregulates the expression of inflammatory factors;③reduces renal mesenchymal cells. Therefore, rapamycin suppresses renal artery atherosclerosis and tubular interstitial fibrosis, and protects renal function. These results support inflammatory reaction as an important pathogenetic mechanism of ARAS.4. Clinical studies preliminarily indicate that:①active ARAS lesions should be treated;②urine testing and renal biopsy may indicate active renal artery lesions, thus helping guide the treatment;③a preliminary strategy to treat ARAS is to combine anti-inflammatory therapy with early renal artery stenting.