Structural repurposing of SGLT2 inhibitor empagliflozin for strengthening anti-heart failure activity with lower glycosuria

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been reapproved for heart failure (HF) therapy in patients with and without diabetes. However, the initial glucose-lowering indication of SGLT2i has impeded their uses in cardiovascular clinical practice. A challenge of SGLT2i then becomes how to separate their anti-HF activity from glucose-lowering side-effect. To address this issue, we conducted structural repurposing of EMPA, a representative SGLT2 inhibitor, to strengthen anti-HF activity and reduce the SGLT2-inhibitory activity according to structural basis of inhibition of SGLT2. Compared to EMPA, the optimal derivative JX01, which was produced by methylation of C2-OH of the glucose ring, exhibited weaker SGLT2-inhibitory activity (IC50 > 100 nmol/L), and lower glycosuria and glucose-lowering side-effect, better NHE1-inhibitory activity and cardioprotective effect in HF mice. Furthermore, JX01 showed good safety profiles in respect of single-dose/repeat-dose toxicity and hERG activity, and good pharmacokinetic properties in both mouse and rat species. Collectively, the present study provided a paradigm of drug repurposing to discover novel anti-HF drugs, and indirectly demonstrated that SGLT2-independent molecular mechanisms play an important role in cardioprotective effects of SGLT2 inhibitors.

Role of sodium hydrogen exchanger isoform 1 in tumor microenvironment / 国际肿瘤学杂志

The reversed pH gradient across the cell membrane on account of intracellular alkalinization and extracellular acidosis is an importart characteristic of tumor microenvironment.Sodium hydrogen exchanger isoform 1 (NHE1) is ubiquitously expressed at the plasma membrane of many types of cells,which plays a critical role in intracellular pH and cell volume homeostasis.NHE1 plays an important role in the regulation of tumor microenvironment and involves in tumor migration and invasion,which may be a potential new target in anti-cancer therapy.

Amiloride slows down calpain-mediated ABCA1 degradation through in-hibition of hypoxia-induced NHE1 expression / 中国病理生理杂志

AIM:To examine the effects of hypoxia on sodium-hydrogen exchange 1 (NHE1) expression, in-tracellular Ca2+concentration ( [ Ca2+] i ) and calpain activity, and to explore the effect of amiloride on adenosine triphos-phate-binding cassette transporter A1 (ABCA1) degradation and its calpain-related mechanism.METHODS:RAW264.7 cells were exposed to hypoxia for 0 h, 12 h, 24 h and 48 h.The cell viability was measured by MTT assay and the expres-sion of NHE1 at mRNA and protein levels was detected by real-time PCR and Western blot.[ Ca2+] i was analyzed by flow cytometry.Calpain activity was assessed by the method of Suc-LLVY-aminoluciferin.Furthermore, the protein levels of ABCA1 in the RAW264.7 cells exposed to hypoxia for 24 h were determined after 6 h or 12 h treatment with NHE1 inhibi-tor amiloride in the presence of cycloheximide.ABCA1 protein levels and calpain activity were detected after 12 h incuba-tion with calpain inhibitor ALLN or intracellular calcium-chelating agent BAPTA.RESULTS: Hypoxia inhibited the cell viability in a time-dependent manner.Hypoxia up-regulated the mRNA and protein expression of NHE1, and increased [ Ca2+] i and calpain activity.Hypoxia increased the degradation of ABCA1 and amiloride slowed down the ABCA1 degra-dation.ALLN or BAPTA increased ABCA1 protein level and decreased calpain activity.CONCLUSION:NHE1 inhibitor amiloride attenuates the calpain-mediated degradation of ABCA1, indicating that hypoxia-induced NHE1 might, at least in part, participate in the ABCA1 degradation.

Expression of sodium-hydrogen exchanger 1 in gastric carcinoma and paraneoplastic tissue and its clinical significance / 中国医师进修杂志

Objective To determine the expression of sodium-hydrogen exchanger (NHE) 1 in gastric carcinoma tissue,and investigate the relationship between NHE1 expression and clinicopathological characteristics.Methods The expression levels of NHE1 mRNA and protein were detected in both gastric carcinoma tissue (48 cases) and paraneoplastic tissue (40 cases) by reverse transcription polymerase chain reaction (RT-PCR) and Western blot.The relationship between the expression levels of NHE1 mRNA and protein and the clinicopathological characteristics was analyzed.Results The relative expression levels of NHE1 mRNA and protein in gastric carcinoma tissue were 0.791 ± 0.286 and 1.475 ± 0.142,in paraneoplastic tissue were 0.352 ± 0.069 and 0.329 ± 0.048,and there were statistical differences (P ＜ 0.01).The expression level of NHE1 mRNA was positively correlated with NHE 1 protein in the gastric carcinoma tissue (r =0.375,P ＜ 0.05).The expression levels of NHE1 mRNA and protein were associated with the depth of invasion,lymph node metastasis and TNM staging (P ＜ 0.05).However,the expression levels of NHE1 mRNA and protein were no associated with age,gender and tumor differentiation (P ＞ 0.05).Conclusion The expression levels of NHE1 mRNA and protein are significantly up-regulated in gastric carcinoma tissue,which may be involved in the development of gastric carcinoma.

Altered Regulation of Renal Acid Base Transporters in Response to Ammonium Chloride Loading in Rats

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.

Kidney and Phosphate Metabolism

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.

Altered Renal Sodium Transporter Expression in an Animal Model of Type 2 Diabetes Mellitus

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.

Altered Regulation of NHE3, NBC1 and Nitric Oxide System in the Kidney of Rats with Maleic Acid-nduced Metabolic Acidosis / 대한신장학회잡지

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.

Effects of hammerhead ribozyme on NHE-1 activity and proliferation in pulmonary artery smooth muscle cells of rats in vitro / 第三军医大学学报

Objective To explore the effects of hammerhead ribozyme on the expression and activity of NHE 1 and pHi in pulmonary artery smooth muscle cells (PASMCs) of rats and its role in PASMCs proliferation in vitro . Methods According to the secondary structure of NHE 1 mRNA in rats, NHE 1 specific hammerhead ribozyme was designed with the assistance of computer. The recombinant vector of retroviral plasmid pLXSN and hammerhead ribozyme, PRZ, was transfected into the cultured PASMCs. G418 resistant cell clones were screened with 100 ?g/ml G418. Then, the expression of NHE 1 mRNA was detected by RT PCR, intracellular pH(pHi) value and recovery rate of pHi after intracellular acid loading were measured by fluorescent probe BCECF. 22 Na uptake and 3H TdR incorporation were determined, respectively. Results Compared with the cells transfected with pLXSN and non transfected cells, NHE 1 mRNA, pHi value, pHi recovery rate, 22 Na uptake and 3H TdR incorporation decreased significantly in cells transfected with recombinant vector PRZ. No significance was found between the pLXSN transfected group and non transfected group. Conclusion Hammerhead ribozyme can cleave the target NHE 1 mRNA specifically, reduce the expression of NHE 1 mRNA, induce intracellular acidosis and consequently suppress the proliferation of PASMCs.

Change of sodium-hydrogen exchanger m RNA expression in lung of patients with pulmonary hypertension and its clinical significance / 第二军医大学学报

Objective:To examine the sodium-hydrogen exchanger(NHE-1) m RNA expression in lung of patients with congenital heart disease,and to explain the cause of pulmonary vascular remodeling.Methods:Twenty-two patients with congenital heart disease were divided into 2 groups:non-pulmonary-hypertension group(s PAP