| Background and ObjectiveObstructive nephropathy(ON), is due to the urinary tract obstruction caused by some diseases. It mainly defined as urine output was obstructed and large quantity of urine gathered in the renal pelvis or ureter, then the pressure reverse conducted to the kidney, caused increased pressure in renal pelvis and tubules, and thus resulted in renal parenchyma injury and renal dysfunction. ON is a common cause of acute or chronic renal failure, and also the main reason for recurrent urinary tract infection. In children, ON is a common cause of end-stage renal disease(ESRD), accounting for21% of chronic kidney disease(CKD) and for 16% of children listed for transplantation. In the UK, obstructive kidney disease is the third most common etiology for CKD in children, accounting for 15% of the cases. In the United States,renal replacement therapy as a result of acquired obstruction accounts for 1.4% of prevalent patients.The major etiologies of ON vary with patient age. Anatomic abnormalities are commonly seen in children while acquired causes are more frequent in adults.Pelvic-ureteral junction obstruction(PUJO) is the most common anatomic cause of hydronephrosis detected by ultrasound in utero with an incidence of 1%-2%. Other causes of obstruction in children include ureterovesical junction obstruction, posterior urethral valves, urethral atresia or stricture and neuropathic bladder. Prostatic obstruction, tumors, nephrolithiasis, ureteral strictures and retroperitoneal fibrosis are the primary causes of ON in adults.PUJO is the most common cause of congenital hydronephrosis(CHN), while the obstruction is usually incomplete. Indeed, congenital obstructive nephropathy is a common cause of renal failure in infants and children. Approximately 50% of all cases of urinary tract dilatation have no evidence of a physical obstruction and no abnormality in the structural components of the pelvic or ureteral wall.The cause of these conditions is thought to be abnormalities in the smooth muscle of the urinary outflow tract(renal pelvis, ureters, or bladder) or impaired peristalsis, but the underlying pathogenic mechanisms that contribute to impaired ureter peristalsis are unknown.The pathophysiology of CHN is not well clearified entil now. Although there have reports about animal models with CHN from fetus, it usually caused by gene mutation or knockoff. So, the process of the hydronephrosis development in these animal models is very different form the nature development of CHN in human fetus.The renal function of newborn rats is not well developed, and its maturity is very the same as fetus kidney of the third trimester of pregnancy. Therefore, newborn rats are usually used as the animal model with ureter obstruction for imitating human CHN to explore the pathophysiology of human CHN. However, most of the reports are concerning about the renal protein expression changes after ureter obstruction, the effects of ureter obstruction on the renal pelvic pressure and the peristaltic rhythms of renal pelvis and ureter are less reported.It is well-known that the early changes of obstructive nephropathy is enlargement of renal pelvis and calices, and expansin of collecting duct. Clinical and experimental studies showed that the dysfunction of renal urine concerntration ability after ureter obstruction is associated with the down regulation of renal aquaporin 2(AQP2). AQP2 is one of the members of aquaporins family, and maily expressed in the apical plasma membrane and vesicles of principal cells, controlling the water reabsorption ablity of collecting duct. It was reported that even though releasing obstruction by surgery in early stage, the renal urine concerntration ability could not recover completely. And this is resulted from the renal AQP2 could not recover timely after releasing of obstruciton.Although many studies focus on how to protect the renal concentration ability with ureter obstruction or after releasing of obstruction, but it is still not found suitable method for preventing the down regulation of renal AQP2 expression in infants with obstructive nephropathy. Antidiuretic hormone(ADH) could regulate the expression and distribution of renal AQP2 under physiological state, but it has no obvious effect on the diuresis after releasing of obstruction, this maybe due to that the down regulation of renal ADH V2 receptors and increased of prostaglandin E2(PGE2)synthesis could antagonize the effect of ADH, and the down regulation of renal AQP2 expression. The angiotensin receptor blockers could prevent the down regulation of renal AQP2 expression caused by obstruction, but the application of such drugs may increase the risk of kidney damage caused by obstruction, limiting its clinical application.In recent years, Erythropoietin(EPO) could protect the renal function in obstructed kidney has attracted the attention of many researchers. Nakazawa et al reported that EPO could protect the renal function by antiapoptosis but not hematopoietic function. Kitamura et al found that use of EPO and its derivatives(carbamyl EPO) could prevent apoptosis, alleviate renal interstitial fibrosis, reduce smooth muscle actin expression. Souza et al found that EPO could inhibit NF-kappa B, reduce inflammation in acute renal failure animal models, and antagonize its inhibitional effect on AQP2 expression, avoid down regulation of AQP2. Gong et al also found that EPO could prevent the down regulation of AQP2 m RNA and protein in ischemia-reperfusion kidney injury animal model.However, whether EPO could prevent the down regulation of renal AQP2 expression caused by partial unilateral ureter obstruction(PUUO), and promote the recovery of renal AQP2 expression, renal function and water and salt handling function in bilateral ureter obstruction-released(BUO-R) still were not reported. So far, EPO has been used for the the treatment of children with hypoxic ischemia encephalopathy in clinical practice, however, whether EPO could protect the renal function of CHN children still needs further research.Rat hydronephrosis model caused by UO has been used for the study of down regulation of AQP2 expression due to obstruction, production of inflammatory mediators, renal tubular cell apoptosis and renal interstitial fibrosis.PUUO model is more similar to pathophysiological changes in CHN children caused by partial ureteral obstruction, however, the degree of partial ureteral obstruction is not easy to control and quantize in rat model. While complete ureteral obstruction model is easy to control and quantize the degree of obstruction, and often used to verify the renal pathophysiology caused by obstruction, but complete obstruction of CHN children in clinical practice are rare.Therefore, partial ureteral obstruction rat model and complete ureteral obstruction model were employed in this study to verify the degree of ureteral obstruction in newborn rats and explore the mechanisms of pathophysiological changes caused by obstruction in young rats. So, the main purpose of this study include(1) establish the newborn rat PUUO model, and examine the quality of PUUO model built by embedding the ureter into a psoas muscle tunnel and test the upper urinary tract pressure through renal puncture, and investigate the effect of urete obstruction on renal pelvic pressure and peristaltic rhythms;(2) establish PUUO young rat model, and given EPO intervention, to explore the effect of EPO on the expression of renal AQP2 in PUUO kidney;(3) establish BUO-R young rat model,and given EPO intervention, to discuss the effect of EPO on BUO-R rat kidney AQP2expression;(4) establish BUO-R young rat model, and given EPO intervention, to explore the effect of EPO on renal function, water and salt handling ability and its mechanisms.This research mainly focus on the establishment of newborn PUUO rat model,and verify the quality control of PUUO model bulit by embedding the ureter into a psoas muscle tunnel method, and investigate the effect of partial ureter obstruction on renal pelvis pressure and peristaltic function and how to promote the recovery of renal function and functional protein expression after ureter obstruction, in order to explore the mechanism and treatment of CHN.Part One The establishment of PUUO model in newborn rats and the effects of PUUO on renal pelvic peristalsis and pressureMaterials and Methods1. Thirty 2d-old newborn SD rats, weight(8.07±0.39)g, equally divided into PUUO group, CUUO group and Sham group randomly, n=10. After the newborn rat was anesthetized, open the abdomen and expose the left ureter. In PUUO group rats,the left ureter was imbedded in the psoas below the renal vein level 0.3cm; in CUUO group rats, the left ureter was ligatured also below the renal vein level 0.3cm; while in the Sham group rats, only blunt separation of the left ureter, but not imbedded or ligatured.2. One week after operation, weighted rats body weight again. Then, open the abdominal cavity after they were anesthetized, and exposed the left kidney, measuring the left kidney size. After the intravenous indwelling needle was connected to the piezometric tube and then the latter was connected to the urodynamics. The starting perfusion speed was set to 5 ml/h, measuring the pelvic pressure while puncture into the pelvis throug the outer of kidney. After the puncture needle was put into the renal pelvis and pelvic pressure was steady, records 3min of pressure curve, then adjust the perfusion speed respectively to 10ml/h, 20ml/h, 30ml/h, 40ml/h, 50ml/h, 60ml/h and80ml/h, and each perfusion speed records 3min of pressure curve. Record and compare the parameters of pelvic pressure, frequency and amplitude of pelvic peristalsis at each perfusion speed.3. Statistical analysis: The Statistical Program for Social Sciences, version 10.0(SPSS 10.0), is used for statistical analysis. Results of measurement data are expressed as mean ± SD. Independent-samples T test is used for comparison between two grouops, and One-way analysis of variances(ANOVA) is used for comparison among three or more groups. Results of enumeration data are expressed as ratio,chi-square test is used for the comparison among groups. The significant level was set as α=0.05, if the P value was less than 0.05, then consider the differences are statistically significant.Results1. Kidney appearance and size comparison: In CUUO group rats, the left kidney was pink, more lighter and the largest among the three groups, while with severe hydronephrosis, which was visible outside the renal hilum; In PUUO group rats, the left kidney was grayred, slightly ligher and larger than that of Sham group, and still had hydronephrosis but was not visible; The left kidney of Sham group rats was reddish brown, more heavier and the smallest among the three groups, had no hydronephrosis, and the pelvis was not visible outside the renal hilum.2. The comparison renal pelvic pressure parameters: In Sham group rats, the renal pelvic pressure increased gradually with the increase of perfusion speed, but increased slowly; The frequency of peristaltic wave increased rapidly with the increase of perfusion speed, while it reached the highest level(up to 0.0875) at the speed of 20ml/h, and then droped with the the increase of perfusion speed. The amplitude of peristaltic wave was similar to the frequency, at the speed of 30ml/h,reached the highest level(up to 36 cm H2O) and then droped with the the increase of perfusion speed. When the perfusion speed was up to 20ml/h, minor wave of pelvic peristalsis began to appear after the main wave, and the amplitude reduced with the increase of perfusion speed. The intervals between the peak of minor waves and the peak of main waves were relatively constant(all were 4s). In PUUO group rats, the renal pelvic pressure also increased gradually with the increase of perfusion speed,but all higher than those of Sham group, and increased rapidly; The frequency of peristaltic wave was higher than that of Sham group, and it was relatively constant under the perfusion speed of 40ml/h, but when perfusion speed increased again, the frequency began to decline. The amplitude of peristaltic wave increased quickly and then began to decline with the increase of perfusion speed, and more rapidly than Sham group. When the perfusion speed was up to 20 ml/h, minor wave also began to appear after the main wave, and the amplitude reduced with the increase of perfusion speed, while they were all lower than those of Sham group. The intervals of peak between minor waves and main waves were variate greatly(from 3.9s to 6.1s, with an average of 5.1s). The basic pelvic pressure of CUUO group rats was 12 cm H2 O, and rised gradually at the perfusion rate of 5ml/h, no plateau was appeared, until the pelvic pressure reached 73 cm H2O, when the perfusion liquid retrograded from the side of puncture needle. The renal pelvic pressure slightly decreased and then balanced. no regular renal pelvic peristaltic waves was observed throughout the whole perfusion process.Part Two Experimental study on the effects of EPO on the expression of AQP2 in kidney of young rats with PUUOMaterials and Methods1. Twenty-four 6-7 weeks old SD rats, weight(160±8)g, were randomly divided into 3 groups(PUUO group, PUUO+EPO group and Sham group, all group n=8).The PUUO model was built by embedding the upper quarter of the left ureter into psoas muscle. EPO(500U/kg) was given to PUUO+EPO rats at 1h after surgery, and then repeated 1, 3, 5 days after surgery by intraperitoneal injection and the same volume of normal saline was simultaneously given to PUUO group rats. The Sham group was prepared in parallel by laparotomy and free dissection of the left ureter, but not embedded.2. One week after surgery, all group rats were given the renal magnetic resonance imaging examine, then the left kidneys of rats were harvested to examine the effect of EPO on the expression of AQP2 in inner medulla by immunohistochemistry, real-time PCR and semi quantitative immunoblottling.3. Statistical analysis: The Statistical Program for Social Sciences, version 10.0(SPSS 10.0), is used for statistical analysis. Results of measurement data are expressed as mean ± SD. Independent-samples T test is used for comparison between two grouops, and One-way analysis of variances(ANOVA) is used for comparison among three or more groups. Results of enumeration data are expressed as ratio,chi-square test is used for the comparison among groups. The significant level was set as α=0.05, if the P value was less than 0.05, then consider the differences are statistically significant.Results1. All of the rats were performed MRI to confirm the degree of hydronephrosis.The MRI results showed that in PUUO group and PUUO+EPO group rars, the left kidney all had mild hydronephrosis 7 days after surgery.2. Immunohistochemistry shows that the AQP2 positive staining mainly located in the principle cell of collecting duct in the inner medulla. The positive staining intensity in Sham group is significantly stronger than that in PUUO+EPO and PUUO groups, whereas, it is the weakest in PUUO group. Also, the diameter of the collecting duct, and the thickness of the collecting duct wall in PUUO+EPO and PUUO groups are larger and thinner than those in Sham group.3. Real-time PCR shows that the level of AQP2 m RNA in Sham group is the highest, while the level of that in PUUO+EPO is medium, and in PUUO group is the least. The ΔCt value of three groups are:-7.6±0.18 vs.-9.7±0.54 vs.-10.8±0.31,P<0.05). When calculated by the formula 2-ΔΔCt, it shows that, the level of AQP2 m RNA in Sham group was 2.1 folds of PUUO+EPO group, 9.2 folds of PUUO group,while the level of AQP2 m RNA in PUUO+EPO group was 4.3 folds of PUUO group.4. The semi quantitative immunoblottling results shows that the relative quantity of AQP2 in PUUO group is lower than that of PUUO+EPO and Sham group. The relative level of AQP2 in PUUO group, PUUO+EPO group and Sham group are:0.29±0.016, 0.64±0.106 and 0.84±0.134, P<0.05.Part Three Experimental study on the effects of EPO on the expression of AQP2 in BUO-R kidney of young ratsMaterials and Methods1. Thirty-two young SD rats, weight(163 ± 6)g, were equally divided into 4groups randomly(BUO group, BUO-R group, BUO-R+EPO group and Sham group;n=8). The BUO model was built by bilateral ureteral ligation. The obstruction was released after 24 h in BUO-R group rats and BUO-R+EPO group rats. EPO(500U/kg)was given to BUO-R+EPO rats at 2h after released of BUO, and then repeated 6h,12 h, 24 h and 36 h thereafter and the same volume of normal saline was simultaneously given to BUO-R rats. The Sham group was prepared in parallel by laparotomy and free dissection of bilateral ureters but not ligated.2. Both side kidneys were harvested 48h(72h for Sham group) after released of BUO to examine the effect of EPO on the expression of AQP2 in inner medulla by immunohistochemistry, real-time PCR and semi quantitative immunoblottling.3. Statistical analysis: The Statistical Program for Social Sciences, version 10.0(SPSS 10.0), is used for statistical analysis. Results of measurement data are expressed as mean ± SD. Independent-samples T test is used for comparison between two grouops, and One-way analysis of variances(ANOVA) is used for comparison among three or more groups. Results of enumeration data are expressed as ratio,chi-square test is used for the comparison among groups. The significant level was set as α=0.05, if the P value was less than 0.05, then consider the differences are statistically significant.Results1. Immunohistochemistry shows that the AQP2 positive cells are stained as claybank, and mainly expressed in the apical membrane and cytoplasm of principle cell in the inner medulla collecting duct. The positive staining intensity of AQP2 in the collecting duct of BUO group rats was significantly down-regulated than that in Sham group, whereas, it was slightly weaker in BUO-R and BUO-R+EPO groups than Sham group. Also, the positive staining intensity of AQP2 in BUO-R group was slightly weaker than that in BUO-R+EPO group. Moreover, the diameter and the thickness of the collecting duct wall in BUO, BUO-R and BUO-R+EPO groups are larger and thinner than those in Sham group.2. Real-time PCR shows that the level of AQP2 m RNA in Sham group was the highest, and BUO-R+EPO group takes the second place, while and BUO-R group takes the third place, and in BUO group was the lowest. The ΔCt value of the four groups are:-3.7±0.18 vs.-1.2±0.17 vs.-0.3±0.11 vs. 0.9±0.23, P<0.05. When calculated by the formula 2-ΔΔCt, it shows that the level of AQP2 m RNA in Sham group was 24.3 folds of BUO group, 10.6 folds of BUO-R group and 5.7 folds of BUO-R+EPO group respectively.3. The semi quantitative immunoblottling results shows that the relative quantity of AQP2 in PUUO group is lower than that of PUUO+EPO and Sham group. The relative level of AQP2 in Sham group is the highest, BUO-R+EPO group takes the second place and BUO-R group takes the third place, while the BUO group was also the lowest: 0.97±0.022, 0.68±0.025, 0.54±0.022 and 0.43±0.118, P<0.05.Part Four Experimental study on mechanisms of the protective effects of EPO on renal function in BUO-R young ratsMaterials and MethodsThirty-two young SD rats, weight(165±7)g, were equally divided into 4 groups randomly(BUO group, BUO-R group, BUO-R+EPO group and Sham group; n=8).The BUO model was built by bilateral ureteral ligation. The obstruction was released after 24 h in BUO-R group rats and BUO-R+EPO group rats. EPO(500U/kg) was given to BUO-R+EPO rats immediately after releasing of BUO, and then repeated 2d,4d and 6d thereafter and the same volume of normal saline was simultaneously given to BUO-R rats. The Sham group was prepared in parallel by laparotomy and free dissection of bilateral ureters but not ligated.2. The urine samples were collected by metabolic cage 24 h before death and then blood samples were collected from inferior vena cava. Both side kidneys were harvested 7d after released of BUO to examine the effect of EPO on the expression of TNF-α and IL-6 by immunohistochemistry and real-time PCR.Results1. The osmotic pressure of BUO-R+EPO group was higher than that of BUO-R group, while lower than that of Sham group((1164±93 vs. 715±76 vs. 1835±98)mosm/kg, P<0.05). The urine output in BUO-R+EPO group was higher than that in Sham group, while lower than in BUO-R group((41±5 vs. 24±3 vs. 66±7) μl/min kg,P<0.05). The concentrations of urinary K+, Na+ and CREA in BUO-R+EPO group were lower than those in Sham group, but higher than those in BUO-R group((132.5±4.95mmol/L, 131.3±3.06mmol/L, 3875±343μmol/L) vs.(152.3±5.21mmol/L,168.5±3.54mmol/L, 4698±744μmol/L) vs.(105.1±6.32mmol/L, 98.3±3.25mmol/L,2509±556μmol/L), P<0.05). The concentration of hematal K+ in BUO group was higher than that in Sham group, BUO-R group and BUO-R+EPO group((11.5±0.71 vs. 6.9±0.16, 7.9±0.66 and 7.5±0.31) mmol/L, P<0.05); While the hematal Na+concentration in BUO group lower than that in Sham group, BUO-R group and BUO-R+EPO group((123.1±6.03 vs. 137.8±5.37, 146.7±4.53 and 142.9±6.51)mmol/L, P<0.05). The hematal concentrations of Urea and CREA in BUO group(58.4±4.63mmol/L and 277.3±5.06μmol/L) were higher than those in other three groups; While they were both higher in BUO-R+EPO group(6.2±1.34 mmol/L and25.7±0.58 μmol/L) than those in Sham group, but lower than those in BUO-R group(7.7±0.27 mmol/L and 32.6±0.83 μmol/L); P<0.05.2. Immunohistochemistry showed that TNF-α and IL-6 were mainly expressed in renal tubules and collecting ducts, while IL-6 also expressed in glomerulus. The expressions of TNF-α and IL-6 were strongest in BUO group and weakest in Sham group, while they wre stronger in BUO-R group than in BUO-R+EPO group.3. Real-time PCR showed that the level of TNF-α m RNA in BUO group was 2.5folds of BUO-R group, 6.1 folds of BUO-R+EPO group and 11.3 folds of Sham group respectively(The ΔCt value of 4 groups were: 1.8±0.12 vs. 3.1±0.15 vs.4.4±0.18 vs. 5.3±0.22, P<0.05). The level of IL-6 m RNA in BUO group was 4.9folds of BUO-R group, 7.5 folds of BUO-R+EPO group and 13.0 folds of Sham group respectively(The ΔCt value of 4 groups were:-1.8±0.14 vs. 0.5±0.11 vs.1.1±0.13 vs. 1.9±0.12, P<0.05).Conclusion1. The method by embedding a small segment of the ureter into the psoas muscles can successfully establish PUUO model in newborn rat, and degree of hydronephrosis is relative uniform, so the quality control is good;2. PUUO has a significant impact on renal pelvic peristalsis function. The changes of peristaltic frequency, amplitude and pelvic pressure have certain regularities to follow, which may provide some thoughts to explore the mechanisms of pelvic peristalsis after PUUO.3. EPO could partially prevent the downrelation of AQP2 in young PUUO rats kidney.4. EPO could promote the recovery of AQP2 in young BUO-R rats kidney.5. EPO could promote the recovery of renal function and normalize the altered renal water and salt handling in young BUO-R rats by inhibiting inflammatory reaction. |
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