Dysregulation Of Renal Aquaporins, Sodium Transporters And Acid-base Transporters In Rats With Urinary Tract Obstruction

The major function of kidney is to regulate body water and sodium balance. This process is achieved and intimately regulated by a number of cellular and molecular processes, including reabsorption of water and sodium by renal tubules through water channels (aquaporins, AQPs) and sodium (co)transporters. Another major function of kidney is to maintain systemic acid-base homeostasis, which is involved H secretion and HCO 3 reabsorption along the whole renal tubules. There are also some special channels or (co)transporters in charge of that courses. All of these functions are under control of hormones and nerves by means of intracellular signaling pathways.AQPs are a family of membrane proteins sharing a similar structure with six membrane spanning domains in the lipid bilayer. Those proteins function as water channels and mediate facilitated movement of water across the biological membranes. Out of 12 mammalian aquaporin isoforms, at least 7 (AQPl, 2, 3, 4, 6, 7and 8) are known to present in kidney at distinct sites along the nephron and collecting ducts, and play key roles for reabsorption of water in kidney.AQPl is extremely abundant in proximal tubule (PT) and descending thin limb where it plays a pivotal role for constitutive water reabsorption. Whereas AQPl is absent in water impermeable segments of ascending thin limb and thick ascending limb (TAL) and distal convoluted tubule (DCT). AQP1 deficient animals were polyuric and unable to concentrate their urine in response to thirsting. Consistent with other animal experiment results demonstrates that AQPl plays an essential role for urinary concentration.AQP2 is exclusively expressed in apical plasma membrane and intracellular vesicles of principal cells of connecting tubule and collecting duct, and is the water channel predominately regulated by the antidiuretic hormone, vasopressin. Watertransport across apical membrane is mediated by AQP2. Recent studies have elucidated important roles of AQP2 in multiple water balance disorders.AQP3 and AQP4 are both located in the basolateral membrane of collecting duct principal cells and represent exit pathways for water reabsorbed apically via AQP2. The importance of AQP3 and AQP4 was demonstrated in AQP3 and AQP4 deficient mice. They are essential to the volume and concentration of final urine.Recently several major renal sodium transporter proteins have been identified, which are involved in physiological processes of transepithelial sodium and water movement alone the nephron. A series of studies have identified that dysregulated sodium transporters play an important role in animal models with various disorders of sodium and water balance.In the proximal tubule, two-third of filtered load of sodium is reabsorbed. NHE3 (type 3 Na+/H+ exchanger) provides the main route for sodium transport across apical plasma membrane of this segment. Na+,K+-2C1" cotransporters (BSC-1) are expressed in the apical membranes of thick ascending limb of Henle's loop, and mediate sodium entry. This part of nephron plays a key role in the regulation of renal water excretion where BSC-1 mediates an increased osmolality in renal medulla through the countercurrent multiplier mechanism and dilutes the tubule fluid. NaCl cotransporter (TSC) is located in the distal convoluted tubules and responsible for a large fraction of net sodium and chloride reabsorption under the control of mineralcorticoid aldosterone. Na,K- ATPase is heavily expressed in the basolateral membranes of renal tubule cells and widely considered to be the driving force for active salt and water movement across electrolyte-transporting epithelial tissue.Kidney maintains systemic acid-base homeostasis by two processes: 1) reabsorption of filtered HCO J , which occurs fundamentally in the proximal convoluted tubule; 2) excretion of fixed acid through the titration of urinary buffers and the excretion of ammonium, which takes place primarily in the distal nephron. Both are involved several acid-base transporters presented along the nephron and collecting ducts.The proximal tubule of the mammalian kidney reabsorbs more than 80% of the filtered bicarbonate by the apically expressed NHE3 in conjunction with the basolaterally expressed electrogenic NaV HCOacotransporter (NBCl). The study on NHE3 null mice shows that HCO 3 reabsorbtion in PT decreased up to -60%. Whereas the Mutations in gene encoded NBCl cause permanent isolated proximal renal tubular acidosis. NHE3 and NBCl plays major role in HCOireabsorption in PT.In the thick ascending limb of Henle's loop, 10-15% of the filtered HCO 3 is reabsorbed by combination of apical NHE3 and basolateral anion exchanger type 2, AE2(1). Moreover, the electroneutral, NBCnl, which is localized in the basolateral plasma membrane of TAL cells in the outer medulla, may also participate in transporting HCOJ into cells and maintain intracellular pH levels. NBCnl may also play a role in NH| reabsorption, medullary accumulation, and urinary excretion of NH?The collecting duct (CD) is a final place to regulate the acid-base balance. H+-ATPase, which exhibit in the apical plasma membrane and apical vesicles of type A cell, is in charge of H+ excretion. Patients with secretory-defect distal renal tubular acidosis demonstrated that H+-ATPase immunoreactivity was almost absent in all 11 patients. Studies reveal that pendrin expressed in type B cell may play a key role in HCO 3 secretion in CD.Urinary tract obstruction is a common and serious disorder both in children and adults, which can impair renal function. After release of obstruction, there occurs not only decreased ability to concentrate urine, but also defect in urinary acidification. The purpose of the present study is to establish the expression of aquaporins, sodium transporters and acid-base transporters undergone regulation, e.g. in condition with 24h bilateral obstruction of ureters (BUO), and after release of BUO, and with metabolic acidosis. The method for checking those proteins is semiquantitative immunoblotting. This thesis consists of 3 parts. Part one The Regulation of AQPs in Rats with Bilateral Ureteral ObstructionMaterials and methods:1. Experiment animals: Male Munich-Wistar rats initially weight 250 g were maintained on a standard rodent diet with free access to water and kept in individual metabolic cages.2. Rats were randomly allocated to the protocol indicates below. Age- and time-matched, sham operated controls were in parallel with each experiment group. Protocol 1:1) BUO for 24h (BUO, n=7): kidneys were removed for semiquantitative immunoblotting.2) Sham operated rats (Sham, n=7): kidneys were removed for semiquantitative immunoblotting.Protocol 2:1) Release of BUO (BUO-R, n=l 1): BUO for 24h, followed by release and animals were observed the next 4 days. Kidneys were removed for semiquantitative immunoblotting.2) Sham operated rats (Sham, n=13).3. Membrane fraction for immunoblotting.Kidney was dissected into cortex and outer stripe of outer medulla (OSOM+C), inner stripe of outer medulla (ISOM) and inner medulla (IM). The homogenate was used for making Gel samples.4. Semiquantitative immunoblotting was used for checking transporter proteins by compared experimental group with sham group.5. Primary antibodies: Antibodies to AQP1, AQP2, AQP3, AQP4 respectively. Secondary antibody: P448.6. Statistical analysis: The excel software was used to analyze results. Comparisons between experiment group and sham control group were made by unpaired t test. P<0.05 was considered significant.Results1. The kidneys were bigger and heavier in BUO rats than in sham rats after 24h BUO.2. BUO 24h increased the plasma osmolality, potassium concentration, plasma creatinine and urea (P<0.01 respectively); But the sodium concentration decreased (P<0.01). After release of BUO, the osmolality, potassium, creatinine and urea were still higher (P<0.05). The sodium concentration returned to normal.3. Following the release of BUO, there was hypopolyuria. BUO resulted in a highly significant increase in urine output and parallel reduction in urine osmolality in BUO-R.4. BUO and release of BUO was associated with down-regulation of AQPl.The expression of AQP1 in 24h BUO was 46.6% of sham level (P<0.01), and still was 67.1% (PO.01) 4 days after release of BUO in PT. It was decreased to 55.5% (PO.01) in descending thin limb of Henle's loop in BUO-R.5. AQP2 was decreased to 68.1% (PO.01) in BUO 24h, and remained down-regulated 4 days after release of BUO (52.6%, PO.01).6. The expressions of AQP3 and AQP4 were also reduced to 76.3% (PO.01) and 58.1% (PO.01) of sham levels respectively in response to BUO 24h.Conclusion1. BUO 24h in rats can impair renal function, therefore damage systemic electrolyte and fluid balance.2. Release of BUO is followed by significant polyuria and impairment of urinary concentrating capacity.3. BUO and release of BUO is associated with down-regulation of AQP1 in proximal tubules, which suggests impairment of water reabsorption in that segment. The decreased level of AQP1 in the descending thin limbs might affect countercurrent multiplication.4. The down-regulation of AQP2 in CD may contribute to the polyuria and impairment of urinary concentration.5. The decreased abundance of AQP3 and AQP4 may be also the reasons of reduced water re-absorption in CD.The observed down-regulation of AQPs may provide a molecular explanation for the functional defects in urinary concentrating capacity associated with obstructive nephropathy.Part twoThe Regulation of Na+ Transporters in Rats with Bilateral UreteralObstructionMaterials and methods:Almost same to part one.Primary antibodies: Antibodies to NHE3, Na-K-ATPase, BSC-1 and TSC respectively. Secondary antibody: P448 or P447 Results1. Release of BUO was associated with altered renal sodium and water handling. The filtered load of sodium ((Fl^a) and urinary sodium excretion (UNa*Uvoi) were significant reduced (P<0.01 respectively). Net reabsorption of sodium (NetReab. ofNa) and free water reabsorption (TCH2O) were markedly decreased (P<0.01 respectively). Decreased creatinine clearance (CCr) indicated GFR reduced (P<0.01).2. Consistent with polyuria after release of BUO, water drinking was increased, but the food intake was dramatically decreased (P<0.01 respectively).3. The expression of NHE3 in BUO 24h was significantly down regulated in PT (26.3%, P<0.01) and TAL (27.1%, P<0.01). After release of BUO, the levels of NHE3 were still lower in those two segments (52.8% and 32.4% respectively, P<0.01).4. BUO and release of BUO were associated with dramatically down-regulation of BSC-1 in thick ascending limbs (2.8% and 26.2% respectively, P<0.01).5. Na,K-ATPase was significantly decreased in all the sections of kidney in response to BUO and release of BUO.6. Expression of TSC was also reduced in DCT in BUO (82.4%, PO.01) and BUO (77.6%, PO.01). Conclusion1. BUO results in impairment of tubular function in sodium and water handling. Na+ reabsorption is reduced, and secretion is also decreased.2. Urinary reduced Na+ excretion partly due to decreased GFR and food intake.3. The down-regulation of NHE3 and Na,K-ATPase in proximal tubule may contribute to reduced Na+ reabsorption in that segment.4. The decreased abundance of BSC-1, NHE3 and Na,K-ATPase reduces NaCl reabsorption in TAL, therefore impairs countercurrent multiplication, which results in the defect in concentrating urine in collecting ducts.5. Down-regulation of TSC can reduce NaCl reabsorption in DCT.6. At last, the lower level of Na,K-ATPase in collecting ducts may also the reason of reduced Na+ reabsorption.Part threeThe Urinary Acidification Defect and Regulation of Acid-base Transporters in Response to Bilateral Ureteral Obstruction Materials and methods:1. The other methods are same to part one. The protocol is different.2. Protocol Protocol 1:1) BUO for 24h (BUO, n=7): kidneys were removed for semiquantitative immunoblotting.2) Sham (n=7): kidneys were removed for semiquantitative immunoblotting. Protocol 2:1) Release of BUO (BUO-R, n=l 1): BUO for 24h, followed by release and animals were observed for the next 4 days.2) Release of BUO+NH4C1 (BUO-R-A, n=8): BUO for 24h, followed by release and animals were observed for the next 4 days. NH4CI was given by gavage in last two days.3) Sham (Sham, n=7)4) Sham+NH4C1 (Sham-A, n=7) NH4C1 was given by gavage in last two days.3. Primary antibodies: Antibodies to NHE3, NBC1, NBCnl, H-ATPase, BSC-1 and Pendrin respectively. Secondary antibody: P448 or P447.4. Statistical analysis: The excell and SPSS software were used to analyze results. P<0.05 was considered significant.Results1. BUO 24h resulted in acidosis in rats. The plasma pH and HCO3 were much lower than that in controls. The plasma pH reversed to normal in BUO-R, but experimental metabolic acidosis was made in BUO-R-A with NH4CI gavage in 2 days. Same acid treatment did not change plasma pH in Sham-A rats.2. BUO reduced the ability of urinary acidification. After NH4CI treatment, the BUO-R-A rats had very lower plasma pH, but the urine pH was higher compared with Sham-A group.3. The expression of NHE3 was reduced in PT and TAL in response to BUO, BUO-R, but increased to Sham level under the metabolic acidosis.4. The abundance of NBC 1 was reduced in BUO, reversed to normal in BUO-R. It was not change after NH4CI gavage.5. BUO and BUO-R were associated with downregulation of BSC-1. It reached to the normal level in BUO-R-A after acid treatment.6. The expression of NBCnl was down regulated in BUO, BUO-R. It increased to sham level in BUO-R-A.7. BUO did not affect the abundance of H+-ATPase. It increased in ISOM in BUO-R-A.8. There were markedly increased levels of NHE3, NBC, NBCnl and BSC-1 in Sham-A compared to BUO-R-A, indicating compensating ability was reduced in BUO-R rats.9. Pendrin was reduced in all of experiment groups. Conclusion1. With the semiquantitative immunoblotting method, we are the first ones to examine the acid-base transporters regulations in response to BUO, release of BUO, and under the condition of acidosis.2. BUO 24h in rats results in systemic acidosis. The obstruction of urinary tracts can impair renal tubular capacity of urinary acidification.3. The down regulation of NHE3 and NBC1 in PT contributes to reduced reabsorption of HCO j*.4. The decreased expression of BSC-1, NHE3 and NBCnl in TAL can reduce NH 4 reabsorption, accumulation in medulla, and even affect H+ secretion in CD.5. BUO reduces the renal compensating abilities in HCO 3 reabsorption and H+ secretion.6. The activity of H+-ATPase may be inhibited in BUO. The mechanism is not clear.7. Reduced expression of pendrin is compensating regulation.