Objective: To clarify the molecular basis of patients with Bartter syndrome type I and explore the therapeutic effect of trafficking-defective variations by chemical chaperone 4-Phenylbutyric acid(4-PBA). Methods: The clinical characteristics, laboratory findings and genetic data of 3 patients diagnosed with Bartter syndrome type I who were admitted to Department of Nephrology, Children's Hospital of Nanjing Medical University from 2017 to 2018 were retrospectively analyzed. Wild type and variant SLC12A1 gene constructs were transiently overexpressed in HEK293 cells. Western blotting was used to detect the expression levels of Na⁺-K⁺-2Cl^-cotransporter(NKCC2) protein. Immunofluorescent staining was applied to investigate the subcellular localization of NKCC2 protein. In addition, the effect of the chemical chaperone 4-PBA on the expression and localization of the SLC12A1 gene variants was investigated. Unpaired t test was used for statistical analysis of 4-PBA treatment. Results: All the 3 patients (2 males and 1 female), aged 3.0, 4.0 and 1.2 years, respectively. All patients had antenatal onset with polyhydramnios and were born prematurely. After birth, all patients presented with hypochlorine alkalosis accompanied by hypokalemia and hyponatremia. Sequencing analysis revealed that the 3 patients were homozygotes or compound heterozygotes for variants in the SLC12A1 gene. In HEK293 cells, the surface expression of NKCC2 in 3 variants (p.L463S, p.L479V, p.507-510del) are all lower than in wild type (0.718±0.039, 0.287±0.081, 0.025±0.156 vs. 1.001±0.028, t=5.92, 8.35, 30.49, all P<0.01). Moreover, the total protein expression of p.L479V and p.507-510del group were all lower than that in wild type group (0.630±0.032, 0.043±0.003 vs. 1.000±0.111, t=3.21, 8.65, all P<0.05). 4-PBA treatment increased the mature protein expression level of the p.L463S and p. L479V group in 4-PBA treatment group are all higher than the untreated group (0.459±0.018 vs. 1.123±0.024, 0.053±0.012 vs. 1.256±0.037, t=2.75, 18.35, all P<0.05). Cytoplasmic retention of the L479V and 507-510del variants were observed by immunofluorescent staining. 4-PBA treatment could rescue a number of NKCC2 L479V variants to the membrane. Conclusions: The 3 SLC12A1 variants cause expression or subcellular localization defects of the protein. The findings that plasma membrane expression and activity can be rescued by 4PBA might help to develop novel therapeutic strategy for Bartter syndrome type Ⅰ.
Bartter Syndrome/genetics* ; Child, Preschool ; Female ; HEK293 Cells ; Homozygote ; Humans ; Infant ; Male ; Pregnancy ; Retrospective Studies ; Solute Carrier Family 12, Member 1/genetics*

BACKGROUNDLittle information is available regarding the effect of angiotensin II (Ang II) on the bumetanide-sensitive sodium-potassium-2 chloride cotransporter (NKCC2), the thiazide-sensitive sodium-chloride cotransporter (NCC), and the Cl- channel (CLC)-K2 at both mRNA and protein expression level in Ang II-induced hypertensive rats. This study was conducted to investigate the influence of Ang II with chronic subpressor infusion on nephron-specific gene expression of NKCC2, NCC and CLC-K2.

METHODSSprague Dawleys rats were treated subcutaneously with either Ang II (100 ng.kg-1.min-1) or vehicle for 14 days. Expression of NKCC2, NCC and CLC-K2 mRNA in kidneys was determined by real time polymerase chain reaction (PCR). Western blotting analysis was used to measure NKCC2 and NCC protein expression.

RESULTSAng II significantly increased blood pressure and up-regulated NKCC2 mRNA and protein expression in the kidney. Expression of CLC-K2 mRNA in the kidney increased 1.6 fold (P < 0.05). There were no changes in NCC mRNA or protein expression in AngII-treated rats versus control.

CONCLUSIONSChronic subpressor Ang II infusion can significantly alter NKCC2 and CLC-K2 mRNA expression in the kidney, and protein abundance of NKCC2 in kidney is positively regulated by Ang II. These effects may contribute to enhanced renal Na+ and Cl- reabsorption in response to Ang II.

Angiotensin II ; pharmacology ; Animals ; Blood Pressure ; drug effects ; Gene Expression Regulation ; drug effects ; Hypertension ; chemically induced ; metabolism ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Sodium-Potassium-Chloride Symporters ; genetics ; Solute Carrier Family 12, Member 1