The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.
Animals ; Cell Line ; Immune Sera/genetics/immunology/*metabolism ; Male ; Mice ; Protozoan Proteins/genetics/*metabolism ; Rabbits ; Recombinant Proteins/immunology ; Sheep ; Sodium-Hydrogen Antiporter/genetics/immunology/*metabolism ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/parasitology/prevention & control

PURPOSE: Diabetic nephropathy (DN) is a prevalent chronic microvascular complication of diabetes mellitus involving disturbances in electrolytes and the acid-base balance caused by a disorder of glucose metabolism. NHE1 is a Na+/H+ exchanger responsible for keeping intracellular pH (pHi) balance and cell growth. Our study aimed to investigate roles of NHE1 in high glucose (HG)-induced apoptosis in renal tubular epithelial cells. MATERIALS AND METHODS: Renal epithelial tubular cell line HK-2 was cultured in medium containing 5 mM or 30 mM glucose. Then, cell apoptosis, oxidative stress, NHE1 expression, and pHi were evaluated. NHE1 siRNA and inhibitor were used to evaluate its role in cell apoptosis. RESULTS: HG significantly increased cell apoptosis and the production of reactive oxygen species (ROS) and 8-OHdG (p<0.05). Meanwhile, we found that HG induced the expression of NHE1 and increased the pHi from 7.0 to 7.6 after 48 h of incubation. However, inhibiting NHE1 using its specific siRNA or antagonist DMA markedly reduced cell apoptosis stimulated by HG. In addition, suppressing cellular oxidative stress using antioxidants, such as glutathione and N-acetyl cysteine, significantly reduced the production of ROS, accompanied by a decrease in NHE1. We also found that activated cyclic GMP-Dependent Protein Kinase Type I (PKG) signaling promoted the production of ROS, which contributed to the regulation of NHE1 functions. CONCLUSION: Our study indicated that HG activates PKG signaling and elevates the production of ROS, which was responsible for the induction of NHE1 expression and dysfunction, as well as subsequent cell apoptosis, in renal tubular epithelial cells.
Antioxidants/metabolism ; Apoptosis/*drug effects ; Cation Transport Proteins/*metabolism ; Cell Cycle/drug effects ; Cell Line ; Dose-Response Relationship, Drug ; Epithelial Cells/*cytology/drug effects/*metabolism ; Glucose/*pharmacology ; Glutathione/metabolism ; Humans ; Kidney Tubules/*cytology ; Oxidative Stress/*drug effects ; Reactive Oxygen Species/metabolism ; Signal Transduction/drug effects ; Sodium-Hydrogen Antiporter/*metabolism

Metabolic acidosis is a cause of renal disease progression, and alkali therapy ameliorates its progression. However, there are few reports on the role of renal acid-base transporters during alkali therapy. We evaluated the effect of sodium bicarbonate therapy and the role of acid-base transporters on renal disease progression in rats with a remnant kidney. Sprague-Dawley rats consumed dietary sodium bicarbonate (NaHCO3) or sodium chloride (NaCl) with 20% casein after a 5/6 nephrectomy. After being provided with a casein diet, the NaHCO3-treated group had higher levels of serum bicarbonate than the control group. At week 4, the glomerular filtration rate in the NaHCO3 group was higher than that in the NaCl group, and the difference became prominent at week 10. The glomerulosclerosis and tubulointerstitial damage indices in the NaHCO3 group were less severe compared with controls at week 4 and 10. The expression of the Na/H exchanger (NHE) was decreased, and apical reactivity was decreased in the NaHCO3 group, compared with the NaCl group. Endothelin-1 levels in the kidney were also decreased in the NaHCO3 group. Dietary sodium bicarbonate has the effects of ameliorating renal disease progression, which may be related to the altered expression of NHE in the remaining kidney.
Acidosis/*drug therapy ; Alkalies/*therapeutic use ; Animals ; Caseins/administration & dosage ; Disease Progression ; Glomerular Filtration Rate/drug effects ; Glomerulosclerosis, Focal Segmental/drug therapy ; Kidney/injuries ; Male ; Nephrectomy ; Nephritis, Interstitial/drug therapy ; Rats ; Rats, Sprague-Dawley ; Renal Insufficiency/*drug therapy ; Sodium Bicarbonate/*therapeutic use ; Sodium Chloride/administration & dosage ; Sodium-Hydrogen Antiporter/*antagonists & inhibitors

The role of the kidney in combating metabolic acidosis has been a subject of considerable interest for many years. The present study was aimed to determine whether there is an altered regulation of renal acid base transporters in acute and chronic acid loading. Male Sprague-Dawley rats were used. Metabolic acidosis was induced by administration of NH4Cl for 2 days (acute) and for 7days (chronic). The serum and urinary pH and bicarbonate were measured. The protein expression of renal acid base transporters [type 3 Na+/H+ exchanger (NHE3), type 1 Na+/HCO3- cotransporter (NBC1), Na-K+ ATPase, H(+)-ATPase, anion exchanger-1 (AE-1)] was measured by semiquantitative immunoblotting. Serum bicarbonate and pH were decreased in acute acid loading rats compared with controls. Accordingly, urinary pH decreased. The protein expression of NHE3, H(+)-ATPase, AE-1 and NBC1 was not changed. In chronic acid loading rats, serum bicarbonate and pH were not changed, while urinary pH was decreased compared with controls. The protein expression of NHE3, H(+)-ATPase was increased in the renal cortex of chronic acid loading rats. These results suggest that unaltered expression of acid transporters combined with acute acid loading may contribute to the development of acidosis. The subsequent increased expression of NHE3, H(+)-ATPase in the kidney may play a role in promoting acid excretion in the later stage of acid loading, which counteract the development of metabolic acidosis.
Acidosis ; Adenosine Triphosphatases ; Ammonium Chloride ; Animals ; Humans ; Hydrogen-Ion Concentration ; Immunoblotting ; Kidney ; Male ; Proton-Translocating ATPases ; Quaternary Ammonium Compounds ; Rats ; Rats, Sprague-Dawley ; Sodium-Hydrogen Antiporter

Thiazide is known to decrease urinary calcium excretion. We hypothesized that thiazide shows different hypocalciuric effects depending on the stimuli causing hypercalciuria. The hypocalciuric effect of hydrochlorothiazide (HCTZ) and the expression of transient receptor potential vanilloid 5 (TRPV5), calbindin-D(28K), and several sodium transporters were assessed in hypercalciuric rats induced by high calcium diet and vitamin D3. Urine calcium excretion and the expression of transporters were measured from 4 groups of Sprague-Dawley rats; control, HCTZ, high calcium-vitamin D, and high calcium-vitamin D with HCTZ groups. HCTZ decreased urinary calcium excretion by 51.4% in the HCTZ group and only 15% in the high calcium-vitamin D with HCTZ group. TRPV5 protein abundance was not changed by HCTZ in the high calcium-vitamin D with HCTZ group compared to the high calcium-vitamin D group. Protein abundance of NHE3, SGLT1, and NKCC2 decreased in the hypercalciuric rats, and only SGLT1 protein abundance was increased by HCTZ in the hypercalciuric rats. The hypocalciuric effect of HCTZ is attenuated in high calcium and vitamin D-induced hypercalciuric rats. This attenuation seems to have resulted from the lack of HCTZ's effect on protein abundance of TRPV5 in severe hypercalciuric condition induced by high calcium and vitamin D.
Animals ; Calcium/therapeutic use/urine ; Calcium Channels/genetics/metabolism ; Cholecalciferol/*toxicity ; Hydrochlorothiazide/*therapeutic use ; Hypercalciuria/chemically induced/*drug therapy ; Rats ; Rats, Sprague-Dawley ; Sodium Chloride Symporter Inhibitors/*therapeutic use ; Sodium-Glucose Transporter 1/genetics/metabolism ; Sodium-Hydrogen Antiporter/genetics/metabolism ; Sodium-Potassium-Chloride Symporters/genetics/metabolism ; TRPV Cation Channels/genetics/metabolism

TRPV5 is believed to play an important role in the regulation of urinary calcium excretion. We assessed the effects of hydrochlorothiazide (HCTZ) on the expression of TRPV5, calbindin-D28K, and several sodium transporters in hypercalciuric rats. Sprague- Dawley rats were divided into 4 groups; control, HCTZ, high salt, and high salt with HCTZ group in experiment 1; control, HCTZ, high calcium (Ca), and high Ca with HCTZ group in experiment 2. To quantitate the expression of TRPV5, calbindin- D28K, and sodium transporters, western blotting was performed. In both experiments, HCTZ significantly decreased urinary calcium excretion. TRPV5 protein abundance decreased in all hypercalciuric rats, and restored by HCTZ in both high salt with HCTZ and high Ca with HCTZ group. Calbindin-D28K protein abundance increased in the high salt and high salt with HCTZ groups, but did not differ among groups in experiment 2. Protein abundance of NHE3 and NKCC2 decreased in all hypercalciuric rats, and were restored by HCTZ in only high Ca-induced hypercalciuric rats. In summary, protein abundance of TRPV5, NHE3, and NKCC2 decreased in all hypercalciuric rats. The hypocalciuric effect of HCTZ is associated with increased protein abundance of TRPV5 in high salt or calcium diet-induced hypercalciuric rats.
Animals ; Biological Transport ; Calcium/urine ; Calcium Channels/chemistry ; Calcium-Binding Protein, Vitamin D-Dependent/*biosynthesis ; Hydrochlorothiazide/pharmacology ; Hypercalciuria/*therapy ; Male ; Models, Biological ; Rats ; Rats, Sprague-Dawley ; Sodium/*metabolism ; Sodium-Hydrogen Antiporter/chemistry ; Sodium-Potassium-Chloride Symporters/metabolism ; TRPV Cation Channels/*biosynthesis/chemistry ; Thiazides/*pharmacology

The serum phosphorus level is maintained through a complex interplay between intestinal absorption, exchange intracellular and bone storage pools, and renal tubular reabsorption. The kidney plays a major role in regulation of phosphorus homeostasis by renal tubular reabsorption. Type IIa and type IIc Na+/Pi transporters are important renal Na+-dependent inorganic phosphate (Pi) transporters, which are expressed in the brush border membrane of proximal tubular cells. Both are regulated by dietary Pi intake, vitamin D, fibroblast growth factor 23 (FGF23) and parathyroid hormone. The expression of type IIa Na+/Pi transporter result from hypophosphatemia quickly. However, type IIc appears to act more slowly. Physiological and pathophysiological alteration in renal Pi reabsorption are related to altered brush-border membrane expression/content of the type II Na+/Pi cotransporter. Many studies of genetic and acquired renal phosphate wasting disorders have led to the identification of novel genes. Two novel Pi regulating genes, PHEX and FGF23, play a role in the pathophysiology of genetic and acquired renal phosphate wasting disorders and studies are underway to define their mechanism on renal Pi regulation. In recent studies, sodium-hydrogen exchanger regulatory factor 1 (NHERF1) is reported as another new regulator for Pi reabsorption mechanism.
Fibroblast Growth Factors ; Homeostasis ; Hypophosphatemia ; Intestinal Absorption ; Kidney ; Membranes ; Microvilli ; Parathyroid Hormone ; Phosphoproteins ; Phosphorus ; Sodium-Hydrogen Antiporter ; Sodium-Phosphate Cotransporter Proteins ; Vitamin D

Hemodynamic factors play an important role in the development and/or progression of diabetic nephropathy. We hypothesized that renal sodium transporter dysregulation might contribute to the hemodynamic alterations in diabetic nephropathy. Otsuka Long Evans Tokushima Fatty (OLETF) rats were used as an animal model for type 2 diabetes. Long Evans Tokushima (LETO) rats were used as controls. Renal sodium transporter regulation was investigated by semiquantitative immunoblotting and immunohistochemistry of the kidneys of 40-week-old animals. The mean serum glucose level in OLETF rats was increased to 235+/-25 mg/dL at 25 weeks, and the hyperglycemia continued up to the end of 40 weeks. Urine protein/ creatinine ratios were 10 times higher in OLETF rats than in LETO rats. At 40th week, the abundance of the epithelial sodium channel (ENaC) beta-subunit was increased in OLETF rats, but the abundance of the ENaC gamma-subunit was decreased. No significant differences were observed in the ENaC alpha-subunit or other major sodium transporters. Immunohistochemistry for the ENaC beta-subunit showed increased immunoreactivity in OLETF rats, whereas the ENaC gamma-subunit showed reduced immunoreactivity in these rats. In OLETF rats, ENaC beta-subunit upregulation and ENaC gamma-subunit downregulation after the development of diabetic nephropathy may reflect an abnormal sodium balance.
Animals ; Blood Glucose/analysis ; Diabetes Mellitus, Type 2/*metabolism ; *Disease Models, Animal ; Epithelial Sodium Channel/*analysis ; Hypertension/complications ; Immunoblotting ; Immunohistochemistry ; Kidney/*metabolism ; Male ; Rats ; Sodium/*metabolism ; Sodium-Hydrogen Antiporter/genetics ; Sodium-Potassium-Chloride Symporters/genetics

The present study was done to determine whether endogenous nitric oxide (NO) plays a role in the regulation of sodium transporters in the kidney. Male Sprague-Dawley rats were treated with NG-nitro-L-arginine methyl ester (L-NAME, 100 mg/L drinking water) for 4 weeks. Control rats were supplied with tap water without drugs. Expression of Na, K-ATPase, type 3 Na/H exchanger (NHE3), Na/K/2Cl cotransporter (BSC1), and thiazide-sensitive Na/Cl cotransporter (TSC) proteins was determined in the kidney by Western blot analysis. Catalytic activity of Na,K-ATPase was also determined. The treatment with L-NAME significantly and steadily increased the systemic blood pressure. Total and fractional excretion of urinary sodium decreased significantly, while creatinine clearance remained unaltered. Neither plasma renin activity nor aldosterone concentration was significantly altered. The alpha1 subunit expression and the catalytic activity of Na, K-ATPase were increased in the kidney. The expression of NHE3, BSC1 and TSC was also increased significantly. These results suggest that endogenously-derived NO exerts a tonic inhibitory effect on the expression of sodium transporters, including Na, K-ATPase, NHE3, BSC1, and TSC, in the kidney.
Animals ; Blotting, Western ; Carrier Proteins/*biosynthesis ; Enzyme Inhibitors/pharmacology ; Kidney/drug effects/metabolism ; Male ; NG-Nitroarginine Methyl Ester/*pharmacology ; Na(+)-K(+)-Exchanging ATPase/biosynthesis ; Nitric Oxide Synthase/*antagonists & inhibitors/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Drug/biosynthesis ; Sodium/*metabolism ; Sodium Chloride Symporters/biosynthesis ; Sodium-Hydrogen Antiporter/biosynthesis ; Sodium-Potassium-Chloride Symporters/biosynthesis

PURPOSE: The present study was aimed to determine whether there exist an altered regulation of tubular transporters and nitric oxide system in the kidneys in maleic acid-nduced metabolic acidosis. METHODS: Male Sprague-awley rats were treated with maleic acid (2 mmol/kg, every 24 hours, intraperitoneally) for 2 days. Control rats were injected with saline. At 24 hours following the second injection, rats were killed by decapitation. Plasma HCO3-and anion gap were measured. The protein expression of type 3 Na+/H+ exchanger (NHE3), type 1 Na+:HCO3- cotransporter (NBC1), and aquaporin (AQP)-1 in the cortex of the kidneys was determined by Western blot analysis. In addition, the expression of isoforms of nitric oxide synthase (NOS) was determined. Contents of nitric oxide metabolites (nitrite/ nitrate, NOx) were also measured in urine by colorimetric assay. RESULTS: Plasma concentrations of HCO3- were significantly decreased following the treatment of maleic acid, while plasma anion gap was did not differ between the experimental and the control groups. In the experimental group, the protein expression of NHE3 was significantly increased in the cortex of the kidney although the expression of NBC1 was not altered significantly. The expression of inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) was significantly increased in the cortex of the kidney. Accordingly, urine NOx contents were increased in the experimental group. In contrast, the expression of AQP1 was not altered. CONCLUSION: These results indicated that upregulation of NHE3 and nitric oxide system may play a role in regulation of acid-ase balance.
Acid-Base Equilibrium ; Acidosis* ; Animals ; Blotting, Western ; Decapitation ; Humans ; Kidney* ; Male ; Neurons ; Nitric Oxide Synthase ; Nitric Oxide* ; Plasma ; Protein Isoforms ; Rats* ; Sodium-Bicarbonate Symporters ; Sodium-Hydrogen Antiporter ; Up-Regulation