| BACKGROUNDHypertension is one of the main causes of chronic kidney disease(CKD).Renal damage secondary to hypertension presents as benign or malignant hypertensive nephrosclerosis(MHN).MHN is usually the result of hypertensive emergency,which is a severe clinical syndrome presented with rapid rise of blood pressure(>180/120 mmHg)accompanied by acute target organ injuries including retina,brain,kidney and microangiopathy.Despite the rapid progress of antihypertensive drugs,the incidence and prevalence of MHN remains unchanged.Therefore,it is still of significance to study the clinicopathologic features,pathogenesis and prognosis of MHN.The role of renin-angiotensin system(RAS),especially the local renal RAS,in the pathogenesis of MHN is not clear.Megalin,an endocytotic protein expressed in the brush border of renal proximal tubules,is proved to be involved in the reabsorption of components such as angiotensinogen(Agt),renin and Angiotensin ?(Ang ?).Cilastatin,inhibitor of dehydropeptidase I,was proved to ameliorate nephrotoxicity mediated by megalin through competitive binding drugs.Since hypotension is one of the adverse reactions of cilastatin,we presumed that it might take part in the regulation of RAS components reabsorption in kidney.This study aimed to describe the clinicopathologic characteristics of MHN,and identify the risk factors of long-term prognosis based on a large MHN cohort.An Ang ?-induced hypertension mouse is used to simulate the RAS activation state of MHT patients.These mice were treated with cilastatin to study its impact on local RAS and blood pressure.OBJECTIVE1.To observe the clinicopathological characteristics of malignant hypertensive nephrosclerosis(MHN),especially the activation of renin-angiotensin system(RAS),and analyze the risk factors of long-term prognosis.2.Try to establish a mouse model of Ang ?-induced hypertension and to observe whether cilastatin can interfere with the expression of megalin and local RAS in the kidney.METHODS1.Clinical data collectionThe patients diagnosed as MHN by renal biopsy from January 1,2003 to May 31,2019 at Peking Union Medical College Hospital were enrolled.The clinical,pathological and follow-up data were carefully reviewed.The primary end-point was defined as renal replacement therapy for over 3 months,kidney transplantation,or death.Clinical data include demographic information,medical history,clinical manifestations,laboratory examinations and treatment,etc were extracted.Histological slides were scanned using Aperio digital section scanning and image analysis system.Glomerular sclerosis index(GSI),tubular atrophy ratio(TA)and interstitial fibrosis ratio(IF)were semi-quantitatively evaluated.The expression of renin,ACE2,megalin and DKK3 in renal tissue of MHN was evaluated by immunohistochemical staining.Benign hypertensive nephrosclerosis(BHN)and glomerular minimal lesion(GML)were used as controls.The correlation between renin,ACE2,megalin and DKK3 expression and clinical manifestation and prognosis were analyzed.2.Establishment of Ang ?-induced hypertension mouse modelAng ? was continuously released at a speed of 400 ng/(kg·min)by micro-osmotic pump implanted subcutaneously in C57 mice for 4 weeks.The general condition and blood pressure of mice were monitored.After 4 weeks,the blood and urine were collected for biochemical test.The mice were then sacrificed.The kidney tissues were stained with megalin,DKK3 andACE2 immunohistochemistry evaluation.3.Statistical analysisKolmogorov Smirnov test was used for normality test.The continuous variable with normal distribution is represented by mean ± standard deviation;the non-normal distribution variable is represented by median(25 quantiles,75 quantiles);the categorical variable is represented by constituent ratio.Student's t test and Wilcoxon rank test were used to analyze the difference of continuous variables,while chi square test and Fisher exact test were used to analyze the sample composition ratio.Pearson correlation analysis was used for continuous variables of normal distribution,and Spearman correlation analysis was used for non-normal distribution and rank variables.In the survival analysis,the Kaplan Meier survival curve was used to analyze the cumulative survival rate of kidney,and the Cox proportional risk model was used to analyze the multiple factors regression.Bilateral test p<0.05 was defined as statistically significant difference.RESULTS1.Clinicopathologic characteristics and prognosis of MHN1.1 Clinicopathologic characteristicsIn this study,124 MHN patients were included,110 were male(Male:Female 7.8:1).Their average age was 34.1±8.2 years old,and the female patients were younger(28.9±.8 vs 34.7±8.1,p=0.011).The mean estimated glomerular filtration rate(eGFR)at discharge was 25.18 ml/min·1.73 m2.93.5%of the patients had reached CKD stage 3 or worse.Most of them had metabolic abnormalities such as dyslipidemia(78.9%),overweight or obesity(76.8%),hyperuricemia(73.0%).More than half of the patients had abnormalities in serum calcium and phosphorus levels and 80%of the patients had elevated parathyroid hormone(PTH).Glomerular wrinkling and sclerosis due to ischemia were observed.The median glomerular sclerosis index(GSI)was 1.49(1.30,1.70).Glomerular sclerosis was more significant in women(p<0.001).PASM and Masson staining showed that the ratio of TA and IF was 65%(50%,80%).Serum creatinine correlated with GSI(r=0.227,p=0.002),TA(r=0.473,p<0.001)and IF(r=0.482,p<0.001)significantly.The eGFR at discharge was significantly correlated with age(r=-0.196,p=0.029),systolic blood pressure(r=-0.196,p=0.029),diastolic blood pressure(r=-323,p<0.001),hemoglobin(r=0.576,p<0.001),serum uric acid(R=-0.286,p=0.001),GSI(r=-0.270,p=0.002),TA(r=-0.574,p<0.001),IF(r=-0.606,p<0.001).1.2 RAS activation in MHN patientsMore than 80%of patients presented abnormal activation of RAS,including elevated plasma renin activity(PRA),Ang ? and aldosterone(ALD).Immunohistochemical staining showed that the expression of megalin(average optical density,AOD 0.0019±0.0007)in MHN patients was significantly lower than that in benign hypertensive nephrosclerosis(BHN)(0.0050±0.0022,p<0.001)and glomerular mild lesion(GML)(0.0082±0.0033,P<0.001).The expression of ACE2 in MHN was also significantly lower than in BHN(0.0029± 0.0014 vs 0.0043±0.0016,p=0.039).1.3Prognosis and risk factorsThe cumulative renal survival rates in the first,third,fifth and tenth year were 91%,76%,64%and 33%,respectively.Multivariate Cox regression analysis showed that eGFR(RR 0.92[0.86,0.98],p=0.009),24 hour urinary protein(RR 2.04[1.33,3.11],p<0.001),and blood pressure?140/90 mmHg(RR 6.92[1.59,30.23],p=0.010)were independent risk factors for poor renal prognosis.2.Renin-angiotensin system in MHN2.1 Ang ?-induced hypertension animal model.One week after subcutaneous implantation of Ang ? micro-osmotic pump,the blood pressure in the Ang ? group was significantly higher than that in the sham operation group,and keeping this difference until the end of the experiment(123.6±22.3 vs 102.5±8.6 mmHg,p=0.006).At the end of the experiment,the average urinary Ang?/Cr ratio was raised(230.8±42.5 vs 179.1±43.7 pg/mg,p=0.010).The weight of Ang ?group was significantly heavier(26.76±1.23 vs 25.11±1.63 g,p=0.011)in the end.2.2 Effect of cilastatin on Ang ?-induced hypertension mouse modelNo significant difference in blood pressure or weight was observed between mice treated with and without cilastatin.There was no significant difference in urine protein,creatinine and electrolyte,neither.HE staining showed proliferation of the juxtaglomerular apparatus(JGA)in mice(p=0.001)with Ang ? pump,and cilastatin could further aggravate the difference(p=0.003).Immunohistochemistry staining of megalin showed that Ang ? could reduce the expression of megalin in the proximal tubule(p=0.003),while cilastatin brought a further reduce(p=0.027).Cilastatin also decesased renin excretion in Ang ?-induced hypertension mice.CONCLUSIONIn this study:1.MHN is more common in young men,with severe renal damage,abnormal activation of RAS,multiple metabolic diseases and electrolyte disorders.The onset age of female patients is younger,and the pathological injury is more prominent.2.The cumulative renal survival rates in the first,third,fifth and tenth years were 91%,76%,64%and 33%,respectively.The lower eGFR,24-hour urinary protein,and BP over 140/90 mmHg were independent risk factors for poor renal prognosis.3.Ang ? can induce the proliferation of juxtaglomerular apparatus(JGA)and decrease the expression of megalin and ACE2,Cilastatin further increases JGA proliferation and reduces the expression of megalin.Cilatatin increases excretion of renin in Ang ?-induced hypertension mice,but shows no significant effect on blood pressure. |
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