| Background and ObjectiveCongenital hydronephrosis is a common urinary malformation, accounting for 1%-2% of newborns, is an important cause of renal failure in infants and children. In the pediatric population, obstruction at the ureteropelvic junction (UPJ) is a common cause of hydronephrosis and the obstruction is mostly partial and congenital. Decreased urine concentration ability is one of the functional changes of hydronephrotic kidney during the early stage. It is still entirely unclear about molecular biological mechanism of decreased urine concentration and dilution ability in the hydronephrotic kidney. After Agre and his colleagues found the existence of aquaporins (AQP) in the 1990s, the research on the renal concentration function mechanism at the molecular level had made a significant progress.At least thirteen different AQPs have been described in humans, with AQP1, AQP2, AQP3 and AQP4 shown to occur in the mammalian kidney. AQPs, membrane-inserted water channel proteins, have been identified to play a highly important role in the reabsorption of water from the renal tubular fluid and are the major basis to maintain the body's water balance. It has been reported that AQP1 is present in the proximal tubule and in the descending thin limb but is absent in highly water-impermeable segments of ascending thin limb, thick ascending limb, and distal tubule. Moreover, it is absent from the collecting duct, making it clear that AQP1 does not participate in the regulation by vasopressin of renal water excretion. AQP2 is confined to the apical regions of the principal cells of the distal tubule and collecting tubule and is the principal target for short-term regulation of collecting duct permeability by arginine vasopressin (AVP). Basolateral permeability of these cells is mediated by AQP3 and AQP4. AQP3 is localized to the basolateral plasma membranes of the connecting tubule cells and collecting duct principal cells in cortex and outer medulla. AQP3 appears to be regulated by vasopressin and aldosterone. AQP4 is most abundant in the inner medulla, with a gradual decrease in expression towards the cortex. AQP4 has also been reported to be expressed in the basolateral plasma membrane of proximal tubule S3 segments.Animal studies showed that changes of urine concentration and dilution function in the hydronephrotic kidney were related to abnormal expression of aquaporins. Recent studies in unilateral ureter obstruction (UUO) and bilateral ureter obstruction (BUO) rats'model have identified the role of aquaporins in both short-term and long-term regulation of body water balance and have elucidated their critical roles in multiple water balance disorders. The changes in the abundance of renal aquaporins may play an important role for the urinary concentrating defect in obstructed kidney. At present, very few information were reported on the expression of AQPs in human hydronephrotic kidney. The study on it will be helpful to reveal the mechanism of renal function changes in children with hydronephrotic kidney.The relationship between expression of AQP1-4 mRNA and protein in the hydronephrotic kidney and ultrasonographic appearance, renal excretory function, histopathological changes and glomerular filtration rate (GFR) of hydronephrotic kidney is unclear. However, these are important issues for the clinicians to understand. Therefore, the purposes of this study were to examine expression of AQP1-4 mRNA and protein in hydronephrotic kidney in children due to congenital UPJO and correlate to different degree of hydronephrosis graded by ultrasound and intravenous pyelography (IVP); to investigate the expression of AQP1-4 mRNA and protein with the different degree of pathology of congenital hydronephrotic kidneys in children due to UPJO; to investigate the relationship between AQP1-4 protein expression, renal parenchyma thickness and GFR in hydronephrotic kidney in children due to UPJO.Methods1. Renal tissue samples were harvested from 22 kidneys (7 left and 15 right kidneys) in 15 boys and 7 girls (58.9±54.3 month-old) who underwent Anderson-Hynes pyeloplasty. Renal control samples were obtained from 8 patients (5 boys and 3 girls, averaged 58.0±37.7 month-old) undergoing nephrectomy for nephroblastoma, from the normal kidney tissue which was confirmed histologically.2. Grading of hydronephrotic kidneys by ultrasound. According to the grading system of the Society of Fetal Urology (SFU) which is based on the long-axis sonographic appearance of the renal parenchyma and pelvicalyceal system, all hydronephrotic kidney belonged to gradeⅢ(8 cases), andⅣ(14 cases).3. All hydronephrotic kidney tissue samples were classified into two groups according to the development time of renal pelvis and calyces as well as image appearance of the hydronephrotic kidneys:(1) detected hydronephrotic kidney:10 cases, kidney is visualized in IVP within 60 minutes after giving contrast intravenously; (2) undetected hydronephrotic kidney:12 cases, kidney is undetected in IVP at time of over 60 minutes after giving contrast intravenously.4. Immunohistochemistry, RT-PCR and Western Blot were used to determine the immunolocalization of AQP1-4, the expression of AQP1-4 mRNA and protein in the hydronephrotic kidney and normal kidney.5. Statistical analysis:The Statistical Program for Social Sciences, version 13.0 (SPSS, Chicago,Ill), was used for statistical analysis. Results are expressed as mean±SE. P<0.05 was considered significant. One-way analysis of variances (ANOVA) was used to compare the AQP1-4 levels between groups.Results1. Immunohistochemistry showed that positive immunoreactivity of AQP1 was found in plasma membranes of proximal tubule, thin descending limb of Henle, and descending vasa recta; AQP2 in the apical membrane of collecting duct principal cells; AQP3 and AQP4 in the basolateral membrane of the same cells. In kidneys with severe hydronephrosis there was reduction in the protein abundance of all 4 AQP isoforms which was more pronounced compared with the protein abundance seen in kidneys with mild hydronephrosis and control kidney.2. RT-PCR showed that expression of AQP1-4 mRNA was significantly reduced in severe hydronephrotic kidney group graded by ultrasound compared to those in mild hydronephrotic kidney group graded by ultrasound and normal controls (AQP1: 0.237±0.154 Vs.0.624±0.084 Vs.0.858±0.122; AQP2:0.283±0.124 Vs.0.583±0.112 Vs.0.976±0.134; AQP3:0.460±0.146 Vs.0.760±0.066 Vs.1.006±0.084; AQP4: 0.196±0.124 Vs.0.439±0.076 Vs.0.739±0.201; P<0.01; respectively). RT-PCR showed that expression of AQP1-4 mRNA was significantly reduced in severe hydronephrotic kidney group graded by IVP compared to those in mild hydronephrotic kidney group graded by IVP and normal controls (AQP1: 0.194±0.118 Vs.0.598±0.092 Vs.0.858±0.122; AQP2:0.247±0.089 Vs.0.566±0.105 Vs.0.976±0.134; AQP3:0.426±0.126 Vs.0.74±0.074 Vs.1.006±0.084; AQP4: 0.171±0.115 Vs.0.420±0.081 Vs.0.739±0.201; P<0.01; respectively).3. Western Blot showed that expression of AQP1-4 protein was significantly reduced in severe hydronephrotic kidney group graded by ultrasound compared to those in mild hydronephrotic kidney group graded by ultrasound and normal controls (AQP1:0.349±0.096 Vs.0.705±0.059 Vs.0.895±0.040; AQP2:0.242±0.113 Vs. 0.609±0.037 Vs.0.868±0.084; AQP3:0.195±0.133 Vs.0.575±0.050 Vs.0.806±0.076; AQP4:0.241±0.067 Vs.0.598±0.074 Vs.0.809±0.079; P<0.01; respectively). Western Blot showed that expression of AQPl-4 protein was significantly reduced in severe hydronephrotic kidney group graded by IVP compared to those in mild hydronephrotic kidney group graded by IVP and normal controls (AQP1: 0.343±0.099 Vs.0.692±0.062 Vs.0.895±0.040; AQP2:0.217±0.126 Vs.0.567±0.069 Vs.0.868±0.084; AQP3:0.17±0.127 Vs.0.543±0.082 Vs.0.806±0.076; AQP4: 0.225±0.063 Vs.0.537±0.078 Vs.0.809±0.079; P<0.01; respectively).Methods1. Renal tissue samples were harvested from 26 kidneys (11 left and 15 right kidneys) in 17 boys and 9 girls (55.9±49.5 month-old) who underwent Anderson- Hynes pyeloplasty. Renal control samples were obtained from 10 patients (6 boys and 4 girls, averaged 63.7±36.8 month-old) undergoing nephrectomy for nephroblastoma, from the normal kidney tissue which was confirmed histologically.2. All hydronephrotic kidney tissue samples were classified into two groups according to histopathologic changes. Mild hydronephrotic kidney group (n=12; including gradesⅡ-Ⅲ) and severe hydronephrotic kidney group (n=14, gradesⅣ-Ⅴ).3. Immunohistochemistry, RT-PCR and Western Blot were used to determine the immunolocalization of AQP1-4, the expression of AQP1-4 mRNA and protein in the hydronephrotic kidney and normal kidney.4. Statistical analysis:The Statistical Program for Social Sciences, version 13.0 (SPSS, Chicago,111), was used for statistical analysis. Results are expressed as mean±SE. P<0.05 was considered significant. One-way analysis of variances (ANOVA) was used to compare the AQP1-4 levels between groups.Results1. Immunohistochemistry showed the results were consistent with those in the Part One.2. RT-PCR showed that expression of AQP1-4 mRNA was significantly reduced in severe hydronephrotic kidney group graded by kidney histopathologic changes compared to those in mild hydronephrotic kidney group graded by kidney histopathologic changes and normal controls (AQP1:0.154±0.12 Vs.0.618±0.10 Vs. 0.858±0.12; AQP2:0.229±0.11 Vs.0.535±0.12 Vs.0.946±0.14; AQP3:0.402±0.15 Vs.0.724±0.08 Vs.0.978±0.10; AQP4:0.169±0.11 Vs.0.423±0.08 Vs.0.756±0.18;P <0.01; respectively).3. Western Blot showed that expression of AQP1-4 protein was significantly reduced in severe hydronephrotic kidney group graded by kidney histopathologic changes compared to those in mild hydronephrotic kidney group graded by kidney histopathologic changes and normal controls (AQP1:0.335±0.096 Vs.0.697±0.057 Vs.0.878±0.051; AQP2:0.195±0.133 Vs.0.569±0.069 Vs.0.816±0.072; AQP3: 0.229±0.104 Vs.0.553±0.089 Vs.0.797±0.081; AQP4:0.241±0.067 Vs.0.528±0.084 Vs.0.833±0.104; P<0.01; respectively).Methods1. Renal tissue samples were harvested from 10 kidneys (10 left) in 6 boys and 4 girls (62.3±18.3 month-old) who underwent Anderson-Hynes pyeloplasty. Renal control samples were obtained from 6 patients (4 boys and 2 girls, averaged 62.7±17.1 month-old) undergoing nephrectomy for nephroblastoma, from the normal kidney tissue which was confirmed histologically.2. Hydronephrotic renal parenchyma thickness measured by ultrasound preoperatively and was verified at operation. Renal glomerular filtration rate (GFR) was assessed by using 99mTc-labeled diethylene triamine pentaacetic acid (99mTc-DTPA) scintigraphy preoperatively.3. Western Blot was used to determine the expression of AQP1-4 protein in the hydronephrotic kidney and normal kidney.4. Statistical Analysis:All statistical analyses were made by SPSS 13.0 for windows software. All values were presented as means±SE. Correlation analysis, unpaired and paired Student t tests were used for the comparisons. Bivariate analysis was presented as Pearson correlation coefficient (r). P values less than 0.05 were considered to be statistically significant.Results1. Western Blot showed that expression of AQP1-4 protein was markedly reduced in hydronephrotic kidney group compared to those in normal controls (AQP1: 0.40±0.14 vs.0.74±0.21; AQP2:0.48±0.18 vs.0.84±0.19; AQP3:0.47±0.21 vs. 0.82±0.20; AQP4:0.55±0.22 vs.0.82±0.14; P<0.05; respectively).2. The mean renal parenchyma thickness of hydronephrotic kidney was 1.8-8.5mm, with a mean of 4.59±2.25mm measured preoperative. Obstructed kidney GFR before surgery was 24.01-56.65ml/min, with a mean of 39.93±11.52 ml/min. Contralateral kidney GFR before surgery was 84.08-118.09ml/min, with a mean of 105.36±20.38 ml/min. GFR was markedly reduced in obstructed kidney compared to contralateral kidney (P<0.05).3. Expression of AQP1-4 protein was down-regulated with the decrease of renal parenchyma thickness, a significant correlation was found between the levels of AQP1,2,3 and 4 protein and hydronephrotic renal parenchyma thickness (r=0.754, 0.724,0.726,0.716, respectively; P<0.05). Expression of AQP1,2,3 and 4 protein was correlated with preoperative renal GFR (r=0.690,0.705,0.691,0.750, respectively; P<0.05). Renal parenchyma thickness was correlated with renal GFR (r=0.728, P<0.05).ConclusionsThrough all three-part study, we get the following conclusions.1. Expression of AQP1-4 mRNA and protein was reduced in the hydronephrotic kidney, with the progress of hydronephrosis severity, their expression get lower and lower which indicated that the expression of AQP1-4 mRNA and protein abundance is reduced in proportion with the degree of hydronephrosis graded by ultrasound in human congenital hydronephrotic kidney as well as changes of AQP1-4 expression may be an important factor that involved in the earlier renal morphological and functional change in hydronephrotic kidney.2. The significant decrease in AQP1-4 expression in IVP undetected hydronephrotic kidney indicated that the decreased expressions of AQP1-4 are related to the degree of hydronephrosis, and may be involved in the mechanism of losing renal concentration and dilution function during the development of hydronephrotic kidney.3. Expression of AQP1-4 is down-regulated in pediatric hydronephrotic kidney at the mRNA and protein level dependent on the degree of hydronephrosis. These changes in AQP1-4 expression may play a role in the pathogenesis of congenital hydronephrotic kidney.4. In hydronephrotic kidney, expression of AQP1-4 protein was correlated well with atrophy of kidney and renal GFR. It is helpful to explain why the thinner renal parenchyma thickness, the weaker renal function of the hydronephrotic kidney.
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