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Introduction: Recent studies suggest that endocytosis of ROMK channels is important for regulation of K+ secretion in cortical collecting ducts. In this study the effect of V364D mutation is examined on the membrane turnover and stability of ROMK2 channel when expressing in Xenopus laevis oocytes. Materials and Methods: In this experimental study, oocytes were isolated by standard protocols using collagenase (Type 1A). Mutations of the cytoplasmic termini of ROMK2 were constructed using the quikchange approach for site-directed mutagensis. Xenopus oocytes were injected with cRNA encoding ROMK2 or V364D mutant three days prior to treatment with BFA solution (time 0). Brefeldin A (BFA) was added to the OR3 medium (+BFA) at concentrations of 5-25 μM (inhibit insertion of new proteins into the cell membrane) or ethanol as BFA vehicle (-BFA). Two-electrode voltage clamp (TEVC) was used to measure oocyte ROMK-dependent currents and membrane potential. Data was analysed using Student’s t-tests or ANOVA as appropriate. Results: Incubation of oocytes expressing ROMK2 channels in both 5µM and 25 µM BFA caused a reduction in the normalized steady state currents. The effect of BFA was dose dependent. In oocytes expressing the V364D mutant, there was no decay in current at any time point during incubation with BFA at either 5 M or 25 M. The fractional current for ROMK2 at 48h following treatment of oocytes with BFA was 0.24  0.05 (n=16) which was significantly different to V364D mutant (1.17  0.09). Conclusion: These results show that the V364D mutation increases the general stability of ROMK and renderes the protein resistant to endocytosis, consistent with the idea that there is an interaction between the C-terminal of ROMK2 and components of the endocytotic pathway. A functional PDZ domain (the S-E-V) plays a key role in determining stability of ROMK.

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Background: In this study, the effects of S362A and S362D mutations on the membrane turnover and the stability of ROMK2 channel when expressing in Xenopus laevis oocytes are examined . Methods and Materials:In this experimental study, oocytes were isolated by standard protocols using collagens (Type 1A). Mutations of the cytoplasmic termini of ROMK2 were constructed using the quick-change approach for site-directed mutagenesis. Xenopus oocytes were injected with cRNA encoding ROMK2 or S362A or S362D mutant three days prior to treatment with BFA solution (time 0). Brefeldin A (BFA) was added to the OR3 medium (+BFA) at concentrations of 25µM (inhibit insertion of new proteins into the cell membrane) or ethanol as BFA vehicle (-BFA). Two-electrode voltage clamp (TEVC) was used to measure oocyte ROMK-dependent currents and membrane potential. Results: In oocytes was expressing ROMK2 and/or the S362A mutant, there was significant reduce in current and membrane voltage of K. The fractional currents for ROMK2 and S362D mutant demonstrated a slight difference 48h following treatment of oocytes with BFA, 0.160.05(n=18) and 0.110.05(n=18) respectively. This was however, significantly different from the fractional current of S362A mutant which stood at 0.960.05(n=24). Conclusion: Mutant Serine residue S362A which causes phosphorylation in endocytosis and helps determine the number of ROMK2, plays an important part in PDZ domain.

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Background: Bartter syndrome is renal tubular disorders that inhibit salt transport and increased renal salt wasting. Type II Bartter syndrome is caused by mutations in the KCNJ1 gene which encodes the inwardly rectifying ATP-sensitive potassium channel Kir1.1 (ROMK). They play a vital role in secretion of potassium into the tubule lumen. The effects of mutation at position 338 of ROMK2 (Kir1.1.b) was investigate. Materials and Methods: Site-directed mutagenesis was used to substitute of threonine for methionine at position 338 of ROMK2 (Kir1.1.b). M338T mutant ROMK2 expressed in oocytes of Xenopus laevis, and in a non-polarized mammalian cell line (MDCK). Two electrode voltage clamp and were used to measure oocyte ROMK-dependent currents. Confocal microscopy of EGFP-tagged ROMK2 determined express and distribution of these channels in MDCK cells. Results: The M338T mutant ROMK2 protein expressed in oocytes was functionally identical to wild type. Its cellular distribution was different in polarized and non-polarized MDCK cells. Conclusion: The M338T mutation is altered residue interactions within the carboxyl terminus of ROMK2 channels. Thus mistargeting of ROMK2 in vivo reduces the driving force for potassium secretion in the TAL and reduces salt reabsorption by this nephron segment.

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Background: Gentamicin is an aminoglycoside antibiotic that broadly is used to treat gram negative bacteria infections, although it has side effects such as nephrotoxicity. According to antioxidant, anti-inflammatory and vasodilatory properties of Zataria Multiflora, the effects of co-treatment with zataria Multiflora and hydroalcholic extract on gentamicin induced nephrotoxicitj were investigated.

Materials and Methods: In this study, male rats of Vistar race were divided into 4 groups: control group, co-treatment with gentamicin and vehicle group, co-treatment with gentamicin and zataria Multifiora extract group, and co-treatment with zataria Multiflora extract and normal saline solution group. Zataria Multiflora hydroalcoholic extract was added to drinking water as 800 PPm concentration. They, systolic blood pressure and renal blood flow (RBF) were measured. Also, the amounts of urea, creatinine, sodium, potassium and osmolarity were measured in plasma and urine samples

 Results: In co-treatment group with zataria Multiflora extract, the amounts of urea, creatinine, absolute sodium excretion and relative sodium and potassium excretion and malondialdehyde (MDA) that have been inceased in treatment with gentamicin, significantly were reduced. Creatinine clearance, urine osmolarity, RBF and FRAP that was decreased in gentamicin group in compare to control group, significantly increased.

Conclusion: Co-treatment prevents nephrotoxicity induced by gentamicin and attenuates oxidative-stress associated renal injury by reducing oxygen free radicals and lipid peroxidation, So it can be effective to cure rats receiving gentamicin.

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Background: Gentamicin (GM) is one the aminoglycoside antibiotics which isroutinelyused to treatinfections gram-negative, either alone or insynergistic withbeta-lactamantibioticsused. However, frequent useleads toserious side effectssuch asrenal toxicity, ototoxicity. Coenzyme Q₁₀ has antioxidant, anti-inflammatory and vasodilatory properties. According to these properties of Coenzyme Q₁₀ and tissue damage mechanism in GM induced-nephrotoxicity, in this study, the effects of these two substances for the co-treatment and post -treatment on renal injury induced by gentamicin were investigated.

Materials and Methods:  Experiments has been done on 77 male Wistar rats in weight range of 200 to 250 g. Animals were divided randomly into 5 groups of 7 numbers. Renal nephrotoxicity induced by i.p injection of gentamicin (100mg/kg) Therapeutic effect of coenzyme Q₁₀ (10mg/kg)in the two protocols co-treatment  and post-treatmentwas investigated.The animals after the last injectionon the ninth day of co-treatment andthe seventeenth day of post-treatmentwere placed into individual metabolic cages so as to collection urine and urine volume was measured gravimetrically. Afteranesthesia, systolic blood pressure and renal blood flow was measured. Then blood sampling was done. Amount of urea, creatinin, sodium, potassium and osmolarity was measured in plasma and urine samples. Left kidney, for doing histological experiments in 10% buffered formaldehyde and right kidney for biochemical experiments in fluid nitrogen was preserved.

Results: Co-treatment with Coenzyme Q10 significantly decreased fractional excretion of sodium (6.37±1.33 %; p<0.001) and decreased fractional excretion of potassium(219.14±83.8 %; p<0.001) MDA levels (2.13 ±0.24µmol/gkw; p<0.001), and significantly increased renal blood flow (6.38 ±0.1ml/min: p<0.01) and FRAP levels (24.44±0.42mmol/gkw; p<0.001). Post-treatment with coenzyme Q10 significantly decreased fractional excretion of sodium (3.58 ±0.57 %; p<0.001), potassium (111.77±29.4%; p<0.001) and MDA levels (3.08 ±0.12µmol/gkw; p<0.001) and significantly increased renal blood flow (6.74±0.15ml/min: p<0.001) and FRAP levels (24.34±0.75mmol/gkw; p<0.001) that is reduced by gentamicin.

Conclusion: According to the results, this study showed thatpost- treatment with coenzyme Q₁₀more protective effect on the kidney tissue andAnda greater increase inantioxidant defensecreated.

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