The Study On Mechanism Of Downregulation Of Aquaporin2and Na-K-2Cl Cotransporter2in Obstructed Kidney In Rat And Follow Up The Clinical Features Of Children With Hydronephrosis

BackgroundPediatric hydronephrosis is a common disorder encountered by physicians in their daily practice. Nowadays, fetal hydronephrosis is more frequently detected due to popular used antenatal ultrasonography. It is well known that one of the most common causes of congenital hydronephrosis is ureteropelvic junction obstruction (UPJO) which has been previously characterized in the literature.Its incidence is around1/1000～1/1500. Ureteral obstruction significantly affect the renal blood flow(RBF), glomerular filtration rate(GFR) and renal tubular function. And those cause the reabsorption and excretion dysfunction of H+, Na+,K+. The release after obstruction results in hypotension urine.As a transmembrane protein, Aquaporin-2(AQP2) is also the most important aquaporin in kidney. Na-K-2Cl Cotransporter2(NKCC2),which specifically express in kidney, is one of the most significant moleculars which regulate the reabsorption of water and salt. It has been shown that, the expression level of AQP2and Na-K-2CL decreased after ureteral obstruction. Renin-angiotensin system plays an important role in the pathophysiological processes of renal function change after ureteral obstruction. However, the regulation mechanism is unclear. To investigate the regulation of angiotensin Ⅱ on the expression of AQP2,NKCC2, and renal function,we administrated ANG II AT1receptor inhibitor on BUO rats, to see its effect on the expression of AQP2, NKCC2and renal function in part one.In2008, the outbreaks of melamine related urinary stone (MRUS) occurred in China. Actually, hydornephrosis is one of the main complications in infants with MRUS, approximately reached1/3of cases. Clinically, hydronephrosis induced by urinary melamine stone (HNMUS) need to be distinguished with the hydronephrosis due to congenital ureteropelvic junction obstruction (HNUPJO) considering the different treatment and follow up strategy. However, the differential diagnosis is not always easy, especially in cases with mild degree of hydronephrosis and that induced by a small or occult MRUS. Although early diagnosis of patients with hydronephrosis is not difficult due to the advances in ultrasonography during the last two decades, disagreement exists over the different diagnostic tests to define obstruction accurately or predict which kidney will benefit from surgical intervention. Furthermore, the indications for and timing of surgery or initial non-operative approach for severe hydronephrosis are still debated. Therefore, we studied the different clinical features and outcome between HNMUS and HNUPJO in part two, to provide more evidence for the diagnosis and treatment of these two diseases.ObjectivePart1(1)To investigate the regulation of angiotensin II on the expression of Aquaporin2(AQP2) and renal function after bilateral ureteral obstruction (BUO);(2)To investigate the regulation of angiotensin II on the dysregulation of Na-K-2C1Cotransporter2(NKCC2) and renal function in response to bilateral ureteral obstruction (BUO);(3) To investigate the early time course of dysregulation of AQP2and renal function after2-,6-,12-,24hours unilateral ureteral obstruction (UUO).Part2To study the difference of clinical features and follow-up reasults in HNMUS with that due to HNUPJO in infants at admission and the24-month follow up.Materials and MethodsPart1Protocal1:Tweenty-four Munich-Wistar rats were randomly divided into three groups (BUO, n=8; CAN n=8; Sham n=8). The BUO (and BUO+CAN) model was built by bilateral ureteral ligation, then the osmotic minipumps contained isotonic saline (n=8) or candesartan (n=8) were implanted subcutaneously. Age-and time-matched sham-operated controls (n=8) were prepared and observed in parallel. The rats were monitored for another48h after the24h BUO was released. The blood samples were collected and kidneys were harvested to examine the effects of angiotensin Ⅱ receptor antagonist candesartan on the dysregulation of AQP2by semi quantitative immunoblottling.Protocal2:Thirty Munich-Wistar rats were randomly divided into three groups (BUO, n=10; CAN n=10; Sham n=10). The BUO (and BUO+CAN) model was built by bilateral ureteral ligation, then the osmotic minipumps contained isotonic saline (n=10) or candesartan (n=10) were implanted subcutaneously. Age-and time-matched sham-operated controls (n=10) were prepared and observed in parallel. The rats were monitored for another48h after the24h BUO was released. The blood samples were collected and kidneys were harvested to examine the effects of angiotensin Ⅱ receptor antagonist candesartan on the dysregulation of NKCC2by semi quantitative immunoblottling.Protocal3:Twenty eight Munich-Wistar rats underwent UUO afterwards were followed2h (UUO-2, n=7),6h (UUO-6, n=7),12h (UUO-12, n=7),24h (UUO-24, n=7). Age-and time-matched sham-operated controls (n=7) were prepared and observed in parallel for each UUO group. The blood samples were collected and obstructed kidneys were harvested to examine the dysregulation of AQP2by semiquantitative immunoblottling.Part2Forty six infants (18.5±10.0months)(34boys and12girls) with HNMUS and85infants (20.4±9.3months)(68boys and17girls) with HNUPJO were included. Diagnostic criteria include the history of feeding melamine-contaminated infant formula; having one or more clinical manifestations such as unexplained crying and/or vomiting; macroscopic or microscopic hematuria (urinary red blood cell morphology shows normal morphology of red blood cells), acute obstructive renal failure:oliguria or anuria; parathyroid hormone test (normal); ultrasound B exam indicated urinary system stone imaging. The hydronephrosis diagnosis is mainly depending on the ultrasonography, which was graded according to the Society for Fetal Urology (SFU) guidelines. Radiographic, functional, and laboratory data of urinary system were recorded. All HNMUS and30HNUPJO infants with mild pelvic dilation similar to those of HNMUS were followed up for24months. Urinary ultrasonography, renal function and urinalysis were evaluated at admission and follow up. The clinical features of two groups were compared.ResultsPart1Protocall: Release of BUO resulted in a marked polyuria(BUO vs. Sham:92±7vs.25±3μl·min-1·kg-1, n=8; P<0.05) and a reduced urine osmolality(BUO vs. Sham:636±55vs.1853±163mosmol/kgH2O, n=8; P<0.05), which persisted throughout the experimental period. Administration of candesartan partly prevented this increase in postobstructive urine production (55±5vs.92±7ul·min-1·kg-1, n=8; P<0.05) and decrease in urine osmolality (783±47vs.636±55mosmol/kgH2O, n=8; P<0.05). BUO induced a significantly increase in plasma osmolality (336±10vs.303±7mosmol/kgH2O, n=8; P<0.05) and plasma aldosterone concentration (4.1±0.2vs.1.4±0.1nmol·L-1, n=8; P<0.05) in BUO vs. in sham-operated control rats. Candesartan partly attenuated the increase of plasma aldosterone (2.8±0.5vs.4.1±0.2nmol·L-1, n=8; P<0.05). BUO resulted in a significantly decreased expression of AQP2compared with control, and candesartan prevented the reduction of AQP2(PO.05).Protocal2: Release of BUO resulted in a marked polyuria (BUO vs. Sham:99±6vs.28±4ul·min-1·kg-1, n=10; P<0.05), increase of urine Na (BUO vs. Sham:7.0±0.7vs.4.0±0.4μmol·min-l·kg-l, n=10; PO.05) and a reduced urine osmolality (BUO vs. Sham:647±57vs.1879±157mosmol/kgH2O, n=10; PO.05), which persisted throughout the experimental period. BUO induced a significantly increase in plasma osmolality (BUO vs. Sham:340±8vs.305+9mosmol/kgH2O, n=10; PO.05) and plasma aldosterone concentration (BUO vs. Sham:4.5±0.2vs.1.4±0.1nmol·L-1, n=10; P<0.05) in BUO vs. in sham-operated control rats. Administration of candesartan partly prevented this increase in postobstructive urine production (BUO+CAN vs. BUO:60±7vs.99±6ul·min-1·kg-1, n=10; P<0.05), increase of urine Na (BUO+CAN vs. BUO:4.9±0.4vs.7.0±0.7μmol·min-1·kg-1, n=10; P<0.05) and decrease in urine osmolality (BUO+CAN vs. BUO:806±61vs.647±57mosmol/kgH2O, n=10; P<0.05). Candesartan attenuated partly the increase of plasma osmolality and aldosterone [(BUO+CAN vs. BUO:(325±7vs.340±8mosmol/kgH2O) and (2.9±0.4vs.4.5±0.2nmol·L-1), n=10; P<0.05]. BUO resulted in a significantly decreased expression of NKCC2compared with control, and candesartan prevented the reduction of NKCC2(P<0.05).Protocal3:In rats subjected to UUO for varying durations between2and24h, osmolality and electrolyte concentrations in plasma were determined. Comparing with sham group, two-hour UUO resulted in a significant increase in the level of plasma creatinine.(39.6±5.8vs.10.5μmol/L), urea (9.2±0.4vs.6.9±0.3mmol/L) serum potassium (5.3±0.2vs.4.3±0.1mmol/L). Rats subjected to6,12, and24h of obstruction had decreased plasma sodium levels(135.6±1.3vs.137.4±0.8,126.3±1.3vs.136.5±1.2,135.2±1.0vs.139.7±1.5mmol/L, respectively) compared with sham group. Plasma osmolality was higher after24h UUO compared with sham group (347.6±1.7vs.304.3±1.3mosmol/kgH2O). Semiquantitative immunoblotting was performed to determine the time frame of the AQP2regulation in a UUO model. It showed that, AQP2protein abundance did not change in kidneys from2-and6-h UUO rats, but after12and24h UUO significant down regulation was observed (69%and22%, respectively), compared with sham group.Part2There was no significant difference in male-to-female ratio of HNMUS vs. HNUPJO (2.8:1vs.4.0:1); however, male preponderance in each group is obvious. Hydronephrosis was unilateral in35(76%) HNMUS and77(91%) HNUPJO patients. More bilateral hydronephrosis were found in HNMUS than in HNUPJO (24%,11/46vs.9%,8/85, P<0.05). HNMUS show more symptomatic (abdominal mass, dysuria, renal colic (unexplained crying, oliguria or anuria, macroscopic or microscopic haematuria and hypertension) than those of HNUPJO (67%vs.41%, p<0.05). Five cases of acute renal failure were observed in HNMUS and none in HNUPJO. More than2kinds of symptoms were found simultaneously in4of HNMUS and in11of HNUPJO. According to the SFU guidelines of hydronephrosis graduation, there were27HNMUS cases and30HNUPJO of a mild degree (SFU grade0,1),19HNMUS cases and44HNUPJO cases of a moderate degree (SFU grade2,3) and0HNMUS cases and11HNUPJO of a severe degree (SFU grade4). Mean degree of hydronephrosis of HNMUS was significantly lighter than HNUPJO (p<0.05). The PH value of urine was significantly lower in HNMUS children compared with HNUPJO (p<0.05), whereas the SUA was significantly higher in HNMUS children compared with HNUPJO (p<0.05). There was no significant difference of the BUN and Scr between HNMUS and HNUPJO (p>0.05).Conservative short-term treatments were used in all HNMUS patients immediately after diagnosis. Hemodialysis was performed in5HNMUS with severe renal failure, and all cases gradually recovered after1to4times of hemodialysis treatments. Extracorporeal shock wave lithotripsy was performed in1HNMUS cases. Primary pyeloplasty was performed in55cases with HNUPJO, whereas follow up was carried out in the remaining30cases HNUPJO, in which the degree of hydronephrosis is similar to those of HNMUS at discharging. During the follow up,5HNUPJO patients underwent pyeloplasty due to progressive hydronephrosis and/or reduction in renal function. At24-month of follow-up, no mortality was observed, and98%(45/46) HNMUS and83%(25/30) HNUPJO showed asymptomatic. Ultrasonogram follow-up showed that hydronephrosis of HNMUS decreased rapidly during the first6months and then more gradually with time. Hydronephrosis resolved spontaneously in43(93%) HNMUS and in none of HNUPJO, remained stable in2(4%) HNMUS and24(80%) HNUPJO. The dilation degree of hydronephrosis decreased significantly in HNMUS compared to those of HNUPJO (6.5%,3/46vs.97%,29/30, P<0.05). The renal function of all the patients were recovered to normal, and the urinary output of whom returned to normal levels.ConclusionsPart1(1)Angiotensin Ⅱ receptor antagonist prevents dysregulation of AQP2in response to BUO. Angiotensin Ⅱ is involved in the pathophusilogical changes in renal function after release of BUO;(2)Angiotensin Ⅱ receptor antagonist prevents dysregulation of NKCC2in response to BUO which is likely to contribute to the associated urinary concentrating defect. Angiotensin Ⅱ is involved in the pathophusilogical changes in renal function after release of BUO;(3)AQP2protein abundance did not change in obstructed kidneys from2-and6h UUO rats, but significant downregulate after12-and24h UUO. The damage of renal function observed is earlier than the downregulation of AQP2protein.Part2Significant difference of clinical feature existed between HNMUS and HNUPJO. The incidence of hydronephrosis in MRUS was28%. Comparing to HNUPJO group, the incidence of bilateral hydronephrosis in HNMUS was higher. The sex ratio of the two groups was similar. More male infants suffered from hydronephrosis. Comaring to HNUPJO group, the infants in HNMUS group had more severe clinical symptoms, and were more easily to suffer acute renal functional failure. However, the hydronephrosis degree in HNMUS was lower and the abdominal mass was not obvious. Ultrasound was the optimal method to differentiate the two kinds of hydronephrosis. It was especially useful for detcting the melamine stone in infants. The primary treatment for HNMUS was the conservative medical management. Alkalization of urine and the close follow-up were safe and effective for most of the HNMUS infants. The hydronephrosis in HNMUS infants would disappear with the excretion of the stone. However, the hydronephrosis in HNUPJO group wouldn’t disappear automatically. In the24-month follow-up, the structure and function of the urinary system had no significant change, and no uninary system tumor occurred in HNMUS infants. Whereas, the hydroneprosis in HNUPJO group increased, and the morphology of kidney changed.