Research On The Biomarkers Of Kidney Injury And The Protective Role Of Ang-(1-7) To Podocyte Injury In Preeclampsia

PART ⅠClinical research on renal injury in preeclampsia1、The risk factors of preeclampsiaObjective:Explore the risk factors of preeclampsia. Test the serum and urine biochemical indicators of normal pregnant women and patients with pregnancy-induced hypertension and preeclampsia.Methods:In this cross-sectional study,75patients were enrolled from the Department of Gynaecology and Obstetrics, The Fifth People’s Hospital of Shanghai, Fudan University, from May2011to March2012. The study included three groups, they are preeclampsia, gestational hypertension and normal pregnant women and each group has25patients. Detection of prenatal and postnatal blood pressure, kidney function and liver function, the level of urine albumin and urine creatinine. We also observe the levels of lipids, hsCRP, immune parameters and coagulation function. SPSS16.0was used for data entry and statistical analysis.Results:1、Compared with normal pregnant group, the preeclampsia group has obviously differences in gestational age at childbirth, weight, BMI, more multipara cases,cesarean section,edema than all the normal pregnancy group. The preeclampsia group also has higher systolic and diastolic blood pressure5min and2h after the birth, and has higher BUN, Scr, UA, alanine aminotransferase level;and the eGFR Resultss from the MDRD formula and CKD-EPI formula descented, as well as the total protein, albumin, globulin and A/G(P<0.05). IgQ C4were also significant changed (P <0.05).24-hour urinary protein excretion, urinary albumin, urine ACR rised, the difference was statistically significant (P<0.001). Compared with the gestational hypertension group, the preeclampsia group has obviously differences in gestational weeks,total protein,albumin,24-hour urinary protein excretion, urinary albumin, urine ACR.blood lipids, coagulation, and other immune indexes both changes, but the difference was not statistically significant.2、Compared with the normal pregnancy group, the study showed that the preeclampsia group has a high proportion of low birth weight and premature children, including weight, length significant difference (P<0.001), the preeclampsia group has lower Apgar score than the normal pregnancy group, but the difference was not statistically significant.3、The two classification of multivariate logistic regression analysis shows that the maternal age<20years or>33years old, family history of hypertension, edema,higher alanine transaminase, low albumin are independent risk factors to hypertensive disorders in preeclampsia. Conclusion: Compared with normal pregnant women, Scr, BUN,UA, urine ACR and age, BMI, family history of hypertension, edema, serum alanine aminotransferase, serum albumin, and IgQ C4changes in preeclampsia The probability of high incidence of premature and low birth weight was increased markedly in patients with preeclampsia. Age, family history of hypertension, edema,low albumin and elevated serum alanine aminotransferase were the main risk factors for preeclampsia. 2、Research on biomarkers of kidney injury in preeclampsiaObjective:Find the sensitive biomarkers preeclampsia renal injury by testing the serum and urine biomarkers about renal injury in the normal pregnant women and patients with gestational hypertension and preeclampsiaMethods:In this cross-sectional study,75patients were enrolled from the Department of Gynaecology and Obstetrics, The Fifth People’s Hospital of Shanghai, Fudan University, from May2011to March2012. The study included three groups, they are preeclampsia, gestational hypertension and normal pregnant women and each group has25patients. Detect the kidney function, the level of serum cystatin C, RBP, NGAL and IL-18, we also tested the level of urine KIM-1, RBP and of NGAL, IL-18. SPSS16.0was used for data entry and statistical analysis.Results:1、Compared with the normal pregnancy group, the level of serum Cystatin C, NGAL and urine-RBP, urine-NGAL, urine-IL-18, urine-KIM-1were significantly increased in preeclampsia (P<0.05).2、Compared with the normal pregnancy group, the study found that the levels of serum RBP and IL-18were reduced in preeclampsia, but the difference was not statistically significant (P>0.05).3、Serum and urine RBP、urine NGAL were positive correlated to Scr, serum IL-18had a significantly negative correlation with Scr and urine-ACR, Serum cystatin C、 urine-IL-18、serum and urine NGAL had a significantly positive correlation with urine-ACR, but the correlations were not closely (r<0.5).4、Urine-RBP and urine-ACR had significant positive correlation (P<0.01),Serum cystatin C and NGAL, urine-KIM1and urine IL-18had a significantly positive correlation with serum creatinine (P<0.05), and the correlations were closely (r>0.5).5、Serum Cystatin C, serum RBP and urinary ACR has no significant correlation (P> 0.05).6、The cutoff value with the highest Youden’s index (correct diagnosis index) was defined as the optimal predictors of preeclampsia:serum Cystatin C<0.935mg/l, urine-RBP<0.600mg/l, urine NGAL<39.889ng/ml, urine KIM-1<1.6432ng/ml. The sensitivity and specificity was80%/64%;80%/78%,70%/90%,80%/80%.7、Combining serum Cystatin C、urine-RBP、urine-NGAL and urine-KIM1, the sensitivity and specificity were96%/49.92%(serum Cystatin C and urine-RBP),94%/57.60%(serum Cystatin C and urine-NGAL),96%/51.20%(serum Cystatin C and urine-KIM1),94%/70.2%(Urine-RBP and urine-NGAL),96%/62.40%(Urine-RBP and urine-KIM1),94%/72%(Urine-NGAL and urine-KIM1);92%/88%(serum Cystatin C+urine-RBP+urine-KIM-1);94%/80%(serum Cystatin C+urine-NGAL+urine-KIM-1),90%/72%(血Cystatin C+urine-RBP+urine-NGAL),94%/94%(urine-RBP+urine-NGAL+urine-KIM-1),100%/98.20%(serum Cystatin C+urine-NGAL+urine-RBP+urine-KIM-1).Conclusion:The level of renal injury biomarkers serum Cystatin C, NGAL, urine-RBP, NGAL, IL-18and KIM-1are significantly increased in preeclampsia. The level of serum Cystatin C and NGAL, urine-NGAL and urine-IL-18are closely related to SCr, urine-RBP and urine-ACR is closely related in preeclampsia. The renal injury biomarkers serum Cystatin C, urine-RBP, urine KIM-1have the highest sensitivity about80%, KIM-1has the highest specificity up to80%. Combining two biomarkers, urine-NGAL and urine-KIM1has the most sensitivity and specificity to diagnose preeclampsia renal injury. Combining three biomarkers, urine-RBP, urine-NGAL and urine-KIM-1has the most sensitivity and specificity. When combined the four biomarkers,the sensitivity is100%and the specificity is98.20%to diagnose preeclampsia renal injury. PART II Experimental study on renal injury in preeclampsiaObjective’Study whether podocyte apoptosis is involved in preeclampsia, explore the possible mechanism of podocyte injury and reveal a new mechanism for preeclampsia to provide a theoretical basis for clinical treatment of this disease.Methods:Podocyte were either kept untreated or treated with the serum of normal pregnancies, gestational hypertension and preeclampsia cases for24hour, then collected and observed the morphology changes, trypan blue and Annexin-V/PI were used to observe cell apoptosis. CCK8was used to observe cell activity and Western-blot and RealtimePCR were used to measure the expression of podocin, WT1and Mas.Results:1. Podocyte structure changes:Intervened by various pregnancy women serum stimulation for24h, the podocyte treated by preeclampsia serum were smaller than other groups, arranged disorderly, body atrophied and secondary structure disappeared, and a part of the cell apoptosis.2. Cell activity detectted by trypan blue:Compared with the normal podocyte group and the group intervened by normal pregnancy women serum, the cell activity of podocyte intervened by preeclampsia serum stained by trypan blue descented, the difference was statistically significant (P<0.001). Compared with the group intervened by normal pregnancy women serum and gestational hypertension women serum, the, cell viability of preeclampsia descented (P<0.01).3. Cell activity detectted by CCK8:Read the OD at450nm with a microplate reader. Compared with the normal podocyte group, the cell activity of podocyte intervened by preeclampsia and gestational hypertension descented, the difference was statistically significant (P<0.01). Compared with the groups intervened by normal pregnancy women serum and gestational hypertension women serum, the, cell viability of preeclampsia descented, but the difference was not statistically significant. 4. Apoptosis detectted by Annexin V-FITC/PI double staining:The use of flow cytometry or fluorescence microscopy detected the occurrence of apoptosis. Flow cytometry analysis showed that podocyte intervened by preeclampsia serum had the highest apoptosis proportion. Compared with the normal podocyte, intervened by normal and gestational hypertension women serum, the number of apoptosis podocyte in preeclampsia group was increased, the difference was statistically significant (P <0.001), the number of apoptosis podocyte interfered with preeclampsia serum was most observed under fluorescence microscope,5. Realtime and Western Blot Resultss:Compared with the normal podocyte group, the expression of podocin, Mas mRNA of preeclampsia group was significantly decreased, the difference was statistically significant (P<0.05). Compared with the groups intervened by normal pregnancy and gestational hypertension women serum,the expression podocin, Mas mRNA was also decreased, but the difference was not statistically significant. Compared with the normal podocyte and normal pregnancy women serum intervened group, the expression of podocin, WT1and Mas protein of preeclampsia group was significantly decreased, the difference was statistically significant (P<0.01). Compared with the group intervened by gestational hypertension women serum, the expression podocin, WT1and Mas protein was also decreased, but the difference was not statistically significant.Conclusion:Podocyte injury is involved in renal injury of the pathogenesis of preeclampsia. Podocyte treated with the serum of preeclampsia can cause an increase of podocyte apoptosis. Mas receptor expression in podocyte decreased, which may play an important role on kidney injury in patients with preeclampsia. PART ⅢResearch on the protective role of Ang-(1-7) to podocyte injury in preeclampsiaObjective:Research on the protective role of Ang-(1-7) to podocyte injury in preeclampsia in order to provide a new theoretical basis for clinical treatment to preeclampsia.Methods:Podocyte were cultured by preeclampsia serum prior to treated with angiotensin-(1-7)(Ang-(1-7)(10-7、10-6,10-5mol/l) for24h, then observed cell apoptosis and cell activity by trypan blue, Annexin-V/PI, CCK8detection, detected podocyte injury use the Real-time PCR and Western-Blot.Results:1. Cell activity detectted by trypan blue:Compared with the podocyte intervened by preeclampsia serum group, the cell activity of podocyte of the groups intervened by preeclampsia serum and Ang-(1-7)10-6was increased, the difference was statistically significant (P<0.01). the groups of Ang-(1-7)10"5treated and Ang-(1-7)10"7treated group cell viability was also increased, but the difference was not statistically significant.2. Cell activity detectted by CCK8:Read the OD at450nm with a microplate reader. Compared with the podocyte intervened by preeclampsia serum group, the cell activity of podocyte of the groups intervened by preeclampsia serum and Ang-(1-7)10-5and Ang-(1-7)10-6was increased, the difference was statistically significant (P <0.05). the group interfered with preeclampsia serum and Ang-(1-7)10"7cell viability was also increased, but the difference was no statistically significant.3. Apoptosis detectted by Annexin V-FITC/PI double staining:The use of flow cytometry or fluorescence microscopy detected the occurrence of apoptosis. Flow cytometry analysis showed that podocyte intervened by preeclampsia serum and Ang-(1-7)10-5and Ang-(1-7)10-6group cell apoptosis proportion was decreased (P <0.05). The number of apoptosis podocyte intervened by preeclampsia serum and Ang-(1-7)10-7was reduced, but with no significant difference. Observed under fluorescence microscope, there was the least number of apoptosis podocyte interfered with preeclampsia serum and Ang-(1-7)10-6.4. Realtime and Western Blot Resultss:Compared with the normal podocyte group, the expression of podocin,WTl and Mas was significantly increased of the groups of podocyte intervened by the serum of preeclampsia and Ang-(1-7)10-6(P<0.05).Conclusion:Exogenous administration of Ang-(1-7) can improve the apoptosis of podocyte, and Ang (1-7)10-6is the most optimal concentration of podocyte protection.