OBJECTIVETo investigate the expression and function of PKD1 and PKD2 in different kidney tissues and cell lines.

METHODSImmunoprecipitation, Western blotting, In situ hybridization and immunohistochemical staining methods were used to observe the expression of PKD1 mRNA and PKD2 mRNA and their protein abundance in different kidney tissues and cell lines.

RESULTSCoordinate expressions of PKD1 and PKD2 were found in all kidney tissues and cell lines. Distribution of PKD1 mRNA and PKD2 mRNA and their protein polycystin-1 and polycystin-2 in normal human adult kidney tissue were mainly expressed in the medullary collecting ducts and distal tubules. Positive staining was also found in the majority of cyst-lining epithelial cells of PKD1 cystic kidney tissue, PKD1 cyst-lining epithelia cell line and LLC-PK1. The expression level of them in cystic epithelia of ADPKD kidney tissue was much higher than that in adult renal tubules (P < 0.01).

CONCLUSIONSSimilar expression pattern of PKD1 and PKD2 and their different tissue distribution in different kidney tissues show that the molecular mutuality of PC-1 and PC-2 might be the base of their functional correlation. Polycystins might play an important role in the maintenance of tubular architecture.

Adult ; Animals ; Cell Line ; Gene Expression ; Humans ; Kidney ; metabolism ; Kidney Tubules, Collecting ; metabolism ; Kidney Tubules, Distal ; metabolism ; Kidney Tubules, Proximal ; cytology ; Polycystic Kidney, Autosomal Dominant ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Swine ; TRPP Cation Channels ; metabolism

An improved approach for determination of Na(+)-K(+)-ATPase activity in single proximal renal tubule of rat reported by Doucet and his colleagues has been suggested. The single proximal renal tubules were isolated by hand under stereomicroscope from collagenase II-treated renal cortical tissue in rats. The length of every single renal tubule segment obtained was measured. The single proximal renal tubules were treated with a hypoosmotic solution and with freeze-thaw successively, before incubation with [gamma-(32)P]-ATP. (32)Pi hydrolyzed from [gamma-(32)P]ATP by Na(+)-K(+)-ATPase in the single proximal renal tubules was assayed by liquid scintillation counting. The activity of Na(+)-K(+)-ATPase in the single proximal renal tubules was calculated by applying a modified formula. There was no significant difference in the measurement result of Na(+)-K(+)-ATPase activity between the method of Doucet et al. and the improved one, but the latter has advantage of less time, less reagents and being easy to operate.
Adenosine Triphosphate ; metabolism ; Animals ; Kidney Tubules, Proximal ; enzymology ; Male ; Microscopy ; Rats ; Rats, Wistar ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Time Factors

The present study was designed to investigate the effect of administration of adrenomedullin (ADM) into subfornical organ (SFO) on renal tubular Na(+),K(+)-ATPase activity in rats. Rats under anesthesia were injected with ADM 0.1 mL (20 ng/mL) via an implanted cannula into SFO (n=6). Plasma ADM and serum endogenous digitalis-like factor (EDLF) levels were assayed with radioimmunoassay, and urine samples were collected via a canoula intubated in bladder. Urinary sodium concentration was assayed with flame spectrophotometry. Single proximal renal tubule segments were obtained by hand under stereomicroscope and its Na(+),K(+)-ATPase activity was measured by liquid scintillation counting. In addition, single proximal renal tubule segments from normal rats (n=6) were incubated with serum from animals administered with ADM into SFO, and then the Na(+),K(+)-ATPase activity was determined. The results showed that both urinary volume and sodium excretion amounted to the peak value at 30 min after ADM administration, and sustained a significant high level at 60 min (P<0.01). At 30 min after ADM administration, there was a significant increase in serum EDLF and a decrease in Na(+),K(+)-ATPase activity of proximal tubule (P<0.01, respectively), but not in plasma ADM level. Na(+),K(+)-ATPase activity was decreased significantly in single proximal renal tubule segments from normal rats incubated with serum from rats administered with ADM into SFO (P<0.01). These results suggest that the diuretic and natriuretic responses following administration of ADM into SFO are associated with the inhibition of renal tubule Na(+),K(+)-ATPase activity. The inhibition of renal tubule Na(+),K(+)-ATPase activity is related to the increase in the serum level of EDLF.
Adrenomedullin ; pharmacology ; Animals ; Kidney Tubules, Proximal ; drug effects ; enzymology ; Rats ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Subfornical Organ

HCO3(-) reabsorption in the renal tubules plays a critically important role in maintaining the global acid-base balance. Loss of HCO3(-) causes metabolic acidosis. Proximal renal tubule is the major site for HCO3(-) reabsorption, accounting for more than 80% of total HCO3(-) reabsorption along the nephron. Over the past more than half centuries, tremendous progresses have been made on understanding the molecular mechanisms underlying the HCO3(-) reabsorption in proximal tubules. The transepithelial movement of HCO3(-) involves the coordinated operation of machineries on both the apical and the basolateral membranes of the epithelial cells. On the apical domain, Na(+)-H(+) exchanger NHE3 and the vacuolar H(+)-ATPase are two major pathways mediating the apical uptake of HCO3(-)-related species. Taken together, NHE3 and H(+)-ATPase are responsible for about 80% of HCO3(-) reabsorption in the proximal tubule. The remaining 20% is likely mediated by pathways yet to be characterized. On the basolateral membrane, NBCe1 represents the only major known pathway mediating the extrusion of HCO3(-) coupled with Na(+) into the interstitial space. In the present article, we provide a historical view about the studies on the mechanisms of HCO3(-) reabsorption since 1940s. Moreover, we summarize the latest progresses emerging over the past decade in the physiological as well as pathological roles of acid-base transporters underlying the HCO3(-) reabsorption in proximal tubules.
Acidosis ; physiopathology ; Animals ; Bicarbonates ; metabolism ; Humans ; Kidney Tubules, Proximal ; physiopathology ; Sodium-Hydrogen Exchangers ; physiology ; Vacuolar Proton-Translocating ATPases ; physiology

Animals ; Drug Carriers ; Drug Delivery Systems ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; Maleates ; metabolism ; Molecular Weight ; Oligodeoxyribonucleotides, Antisense ; metabolism ; Polyvinyls ; metabolism ; Prodrugs ; metabolism ; Proteins ; metabolism

OBJECTIVETo investigate renal sodium handling of ouabain-hypertensive rats and the role of renal sodium handling in pathogenesis of hypertension using endogenous trace lithium as a marker of proximal sodium reabsorption.

METHODSForty male Sprague-Dawley (SD) rats weighting 180-220 g were equally divided into normal control group and ouabain group randomly. Rats were infused with normal saline 1 ml/ (kg x d) or ouabain 27.8 microg/ (kg-d) intraperitoneally once a day respectively. Systolic blood pressure (SBP) and body weight were recorded weekly. Rats were sacrificed after 2 and 6 weeks respectively. Blood and 24 hour urine sample were collected to measure the serum and urinary concentration of sodium, potassium, trace lithium, and creatinine. Endogenous creatinine clearance rate (Ccr), fractional excretions of sodium (FE(Na)), and fractional excretions of lithium (FE(Li)), the ratios of urinary lithium to sodium (U(Li)/U(Na)), the ratios of urinary potassium to sodium (U(K)/U(Na)), and fractional reabsorption of sodium in the postproximal tubules (FDR(Na)) were also calculated. All were studied on their normal diet and ate salt freely.

RESULTSBlood pressure had no significant difference in these two groups after 2 weeks (P > 0.05); After 4 weeks, however, blood pressure was significantly higher in ouabain group than in control group (P < 0.01). Body weight of rats had no significant difference during the experiment period (P > 0.05). Ccr and FE(Na) were similar in these 2 groups (P > 0.05). FE(Li), U(Li)/U(Na), U(K)/U(Na), and FDR(Na) of ouabain group were significantly lower than control group after 2 and 6 weeks (P < 0.05, P < 0.01, P < 0.001 respectively).

CONCLUSIONThe reabsorption of sodium increases in the proximal tubule in ouabain-hypertensive rats, and such increase occurs before the development of hypertension. Therefore, increase of proximal reabsorption of sodium may be involved in the pathogenesis of ouabain-induced hypertension.

Animals ; Hypertension ; chemically induced ; metabolism ; Kidney Tubules, Proximal ; metabolism ; Lithium ; metabolism ; Male ; Natriuresis ; Ouabain ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sodium ; metabolism

OBJECTIVETo investigate the changes in renal sodium transport during development of hypertension in ouabain-hypertensive rats (OHR) and further elucidate the role of ouabain in the pathogenesis of hypertension.

METHODSEighty male SD rats weighing 80-100 g were randomized equally into normal control and ouabain groups and treated with intraperitoneal injection of normal saline (1 ml/kg) and ouabain (27.8 microg/kg) once daily, respectively. Systolic blood pressure (SBP) and body weight of the rats were recorded weekly. One week before sacrifice scheduled at weeks 2, 4, 6 and 8, respectively, the rats were individually housed in metabolic cages to determine food consumption twice. Blood and 24-hour urine samples were collected to measure serum and urine concentration of sodium, trace lithium and creatinine. Endogenous creatinine clearance rate (Ccr), fractional excretions of sodium (FENa), fractional excretions of lithium (FELi) and fractional reabsorption of sodium in the distal tubules (FDRNa) were calculated.

RESULTSThe body weight and food intake between ouabain groups and control groups were comparable during the experiment (P>0.05). Blood pressure was also comparable in the two groups after 2 weeks (P>0.05). At week 4, however, blood pressure of ouabain group was significantly higher than that of the control group (P<0.001) and increased in a dose-dependent manner. The SBP in ouabain group appeared to reach a plateau at week 7. Ccr and plasma sodium (PNa) were similar in the 2 groups during the experiment (P>0.05). FELi was significantly lower at weeks 2, 4 and 6 in ouabain group than in the control group (P<0.01), and FELi decrement in ouabain group was accompanied by reduced sodium excretion. FENa was significantly lower at week 4 in ouabain group than in the control group (P<0.05), but this difference was not significant in weeks 2 and 6 (P>0.05). At weeks 2, 4 and 6, ouabain group showed significantly lower FDRNa than the control group (P<0.05), suggesting the compensation of the distal nephron segments. After 8 weeks, FENa, FELi and FDRNa were similar between the two groups (P>0.05).

CONCLUSIONSOuabain can increase renal proximal tubule reabsorption of sodium and consequently decrease renal sodium excretion in OHR, which can contribute to alteration of the pressure-natriuresis relationship in OHR, and play an important role in the development and maintenance of hypertension of OHR.

Animals ; Blood Pressure ; Creatinine ; blood ; Hypertension ; chemically induced ; metabolism ; physiopathology ; Ion Transport ; Kidney Tubules, Proximal ; metabolism ; Male ; Ouabain ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sodium ; blood ; metabolism

AIMTo study the endogenous mechanism for the inhibition of aquaporin-1 expression in rat renal proximal tubule epithelial cells in response to acetazolamide.

METHODSPrimary cultured rat renal proximal tubule epithelia cells were divided into two groups: one was subjected to 1 x 10(-5) mol.L-1 acetazolamide, the other served as normal control. When grown to sub-confluency, the cells were disintegrated to perform isoelectrofocusing electrophoresis in order to find the differential proteins induced by the acetazolamide treatment. The differential proteins were defined by peptide mass fingerprinting technology.

RESULTSTwo differential proteins were found in the cell disintegrant. The pI 3.8 protein was reduced after treatment, which showed 21.4% similarity with the brush border membrane myosin from rat brain and testis, and 27% with glycogen phosphorylase; The pI 5.5 protein was increased on the contrary, with 20% similarity to phosphatidylinositol transfer protein alpha isoform.

CONCLUSIONAcetazolamide inhibited AQP1 expression probably by affecting the expression of pI 3.8 and pI 5.5 proteins.

Acetazolamide ; pharmacology ; Animals ; Aquaporin 1 ; Aquaporins ; antagonists & inhibitors ; metabolism ; Diuretics ; pharmacology ; Epithelial Cells ; metabolism ; Isoelectric Focusing ; Kidney Tubules, Proximal ; cytology ; metabolism ; Male ; Peptide Mapping ; Rats ; Rats, Sprague-Dawley

We investigated the protective effects of electromagnetic field (EMF) on the survival of the human renal proximal tubular cell line, HK-2, using an in vitro hypoxia/reoxygenation (H/R) injury model. The survival rate of cells cultured under H/R condition declined significantly, while the intracellular reactive oxygen species (ROS) levels markedly increased. The 10 Hz/1 mT EMF exposure reversed the H/R induced reduction in cell survival and induction of intracellular ROS. Our results suggest that 10 Hz/1 mT EMF exposure could inhibit H/R-induced cell death of HK-2 via suppression of intracellular ROS production and that this treatment might be clinically useful for the amelioration of renal ischemia/reperfusion injury.
Cell Hypoxia ; Cell Line ; Electromagnetic Fields ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; radiation effects ; Reactive Oxygen Species ; metabolism ; Reperfusion Injury ; prevention & control

OBJECTIVETo observe the effect of bone morphogenetic protein-7 (BMP-7) on aristolchic acid induced renal tubular epithelial cell trans-differentiation to look for new therapeutic approach for aristolchic acid nephropathy (AAN).

METHODSIn vitro cultured human proximal renal tubular epithelial cell line HK-2 cells were treated with different concentrations of BMP-7 (75 ng/mL, 150 ng/mL and 300 ng/mL) after trans-differentiation of the cells was induced by AA (10 microg/mL). Levels of alpha-SMA mRNA and protein expressions were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively.

RESULTSBMP-7 reversed the AA inducing alpha-SMA expressions in HK-2 cells in a dose-dependent manner.

CONCLUSIONBMP-7 can inhibit the trans-differentiation of human renal tubular epithelial cell induced by AA, thereby might be a new potential drug for AAN prevention and treatment.

Actins ; metabolism ; Aristolochic Acids ; adverse effects ; Bone Morphogenetic Protein 7 ; pharmacology ; Cell Line ; Cell Transdifferentiation ; drug effects ; Epithelial Cells ; cytology ; Humans ; Kidney Tubules, Proximal ; cytology