KCNJ11 Research Articles

DISTRIBUTION OF KCNJ11 RS5219 POLYMORPHISM IN VIETNAMESE POPULATION AND ITS ASSOCIATION WITH HYPERGLYCEMIA

Aims: KCNJ11 gene, encoding ATP-sensitive channel subunits, involves in insulin secretion. The study aimed at investigating the distribution of the KCNJ11 E23K (rs5219) polymorphism and its association with prediabetes and type 2 diabetes (T2D) in Vietnamese population. Methods: A cross-sectional study randomly recruited 2.676 participants aged 40-64 years from a general population in Ha Nam province, Vietnam. Glycemic status of the subjects was classified based on fasting plasma glucose and oral glucose tolerance test. PCR-restriction fragment length polymorphism method was applied to detect the KCNJ11-rs5219 polymorphism. Genotype frequencies were compared to find the distribution difference among normoglycemic, prediabetes, and T2D groups. Generalized linear models and multinomial logistic regression analysis were used to determine the associations of the KCNJ11-rs5219 polymorphism with prediabetes and T2D. Results: The frequencies of minor K allele in normoglycemic, prediabetes and T2D groups were 33.2, 32.6 and 35.4%, respectively. Genotype distribution of the E23K polymorphism was in Hardy-Weinberg equilibrium and not significantly different among the three glucose groups (p > 0.05). Fasting plasma glucose and 2-h glucose levels were not significantly different among three genotypes EE, EK, and KK (p > 0.05). After adjusted for social-economic status, lifestyle and clinical patterns, no significant association was found between the KCNJ11-rs5219 polymorphism and hyperglycemia with ORs from 0.80 to 1.29. Conclusions: The minor K allele was predominant and genotype frequency in the population was remained constant among generations. The study suggests no significant association between the KCNJ11-rs5219 polymorphism and hyperglycemia in the Vietnamese population.

GJB2 and GJB6 gene transcripts in the human cochlea: A study using RNAscope, confocal, and super-resolution structured illumination microscopy.

BackgroundGap junction (GJ) proteins, connexin26 and 30, are highly prevalent in the human cochlea (HC), where they are involved in transcellular signaling, metabolic supply, and fluid homeostasis. Their genes, GJB2 and GJB6, are both located at the DFNB1 locus on chromosome 13q12. Mutations in GJB2 may cause mild to profound non-syndromic deafness. Here, we analyzed for the first time the various expressions of GJB2 and GJB6 gene transcripts in the different cell networks in the HC using the RNAscope technique.Materials and methodsArchival paraformaldehyde-fixed sections of surgically obtained HC were used to label single mRNA oligonucleotides using the sensitive multiplex RNAscope® technique with fluorescent-tagged probes. Positive and negative controls also included the localization of ATP1A1, ATP1A2, and KCNJ10 gene transcripts in order to validate the specificity of labeling.ResultsConfocal and super-resolution structured illumination microscopy (SR-SIM) detected single gene transcripts as brightly stained puncta. The GJB2 and GJB6 gene transcripts were distributed in the epithelial and connective tissue systems in all three cochlear turns. The largest number of GJB2 and GJB6 gene transcripts was in the outer sulcus, spiral ligament, and stria vascularis (SV). Oligonucleotides were present in the supporting cells of the organ of Corti (OC), spiral limbus fibrocytes, and the floor of the scala vestibuli. Multiplex gene data suggest that cells in the cochlear lateral wall contain either GJB2 or GJB6 gene transcripts or both. The GJB6, but not GJB2, gene transcripts were found in the intermediate cells but none were found in the marginal cells. There were no GJB2 or GJB6 gene transcripts found in the hair cells and only a few in the spiral ganglion cells.ConclusionBoth GJB2 and GJB6 mRNA gene transcripts were localized in cells in the adult HC using RNAscope® in situ hybridization (ISH) and high resolution microscopy. Generally, GJB6 dominated over GJB2, except in the basal cells. Results suggest that cells may contain either GJB2 or GJB6 gene transcripts or both. This may be consistent with specialized GJ plaques having separate channel permeability and gating properties. A reduction in the number of GJB2 gene transcripts was found in the basal turn. Such information may be useful for future gene therapy.

Twelve exonic variants in the SLC12A1 and CLCNKB genes alter RNA splicing in a minigene assay.

Background: Bartter syndrome (BS) is a rare renal tubular disease caused by gene variants in SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND or MAGED2 genes. There is growing evidence that many exonic mutations can affect the pre-mRNA normal splicing and induce exon skipping by altering various splicing regulatory signals. Therefore, the aim of this study was to gain new insights into the consequences of exonic mutations associated with BS on pre-mRNA splicing. Methods: We analyzed all the missense, nonsense and synonymous variants described in six pathogenic genes by bioinformatics programs and identified candidate mutations that may promote exon skipping through a minigene system. Results: Results of the study showed that 12 of 14 candidate variants distributed in SLC12A1 (c.728G>A, C.735C>G, c.904C>T, c.905G>A, c.1304C>T, c.1493C>T, c.2221A>T) and CLCNKB (c.226C>T, c.228A>C, c.229G>A, c.229G>C, c.1979C>A) were identified to induce splicing alterations. These variants may not only disrupt exonic splicing enhancers (ESEs) but also generate new exonic splicing silencers (ESSs), or disturb the classic splicing sites. Conclusion: To our knowledge, this is a comprehensive study regarding alterations in pre-mRNA of exonic variants in BS pathogenic genes. Our results reinforce the necessity of assessing the consequences of exonic variants at the mRNA level.

Functional characterization of ion channels expressed in kidney organoids derived from human induced pluripotent stem cells.

Kidney organoids derived from human or rodent pluripotent stem cells have glomerular structures and differentiated/polarized nephron segments. Although there is an increasing understanding of the patterns of expression of transcripts and proteins within kidney organoids, there is a paucity of data regarding functional protein expression, in particular on transporters that mediate the vectorial transport of solutes. Using cells derived from kidney organoids, we examined the functional expression of key ion channels that are expressed in distal nephron segments: the large-conductance Ca2+-activated K+ (BKCa) channel, the renal outer medullary K+ (ROMK, Kir1.1) channel, and the epithelial Na+ channel (ENaC). RNA-sequencing analyses showed that genes encoding the pore-forming subunits of these transporters, and for BKCa channels, key accessory subunits, are expressed in kidney organoids. Expression and localization of selected ion channels was confirmed by immunofluorescence microscopy and immunoblot analysis. Electrophysiological analysis showed that BKCa and ROMK channels are expressed in different cell populations. These two cell populations also expressed other unidentified Ba2+-sensitive K+ channels. BKCa expression was confirmed at a single channel level, based on its high conductance and voltage dependence of activation. We also found a population of cells expressing amiloride-sensitive ENaC currents. In summary, our results show that human kidney organoids functionally produce key distal nephron K+ and Na+ channels.NEW & NOTEWORTHY Our results show that human kidney organoids express key K+ and Na+ channels that are expressed on the apical membranes of cells in the aldosterone-sensitive distal nephron, including the large-conductance Ca2+-activated K+ channel, renal outer medullary K+ channel, and epithelial Na+ channel.

Primary aldosteronism caused by a pI157S somatic KCNJ5 mutation in a black adolescent female with aldosterone-producing adenoma.

Aldosterone-producing adenoma is a rare cause of hypertension in children. Only a limited number of cases of aldosterone-producing adenomas with somatic KCNJ5 gene mutations have been described in children. Blacks are particularly more susceptible to developing long-standing cardiovascular effects of aldosterone-induced severe hypertension. Somatic CACNA1D gene mutations are particularly more prevalent in black males whereas KCNJ5 gene mutations are most frequently present in black females. We present here a novel somatic KCNJ5 p.I157S mutation in an aldosterone-producing adenoma from a 16-year-old black female whose severe drug-resistant hypertension significantly improved following unilateral adrenalectomy. Prompt diagnosis of aldosterone-producing adenoma and early identification of gene mutation would enable appropriate therapy and significantly reduce cardiovascular sequelae.

KCNJ11 and KCNQ1 Gene Polymorphisms and Placental Expression in Women with Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) represents carbohydrate intolerance in pregnant women. The pathogenesis of GDM is very complex, but abnormalities in insulin production and secretion underlie the disease. Potassium channels play an important role in insulin production and secretion. The family of potassium channels includes (among others) the potassium inwardly rectifying channel, subfamily J, member 11 (KCNJ11) and voltage-gated K+ channel (KCNQ1). The aim of the study was to examine the distribution of the KCNJ11 rs5219 and KCNQ1 rs151290 and rs2237892 gene polymorphisms in women with GDM and pregnant women with normal carbohydrate tolerance, to verify whether these polymorphisms are risk factors for GDM. This study included 204 Caucasian pregnant women with GDM and 207 pregnant women with normal glucose tolerance (NGT) from the West Pomeranian region of Poland. The diagnosis of GDM was based on a 75 g oral glucose tolerance test (OGTT) at 24–28 weeks gestation. There were no statistically significant differences in distribution of the KCNJ11 rs5219 and KCNQ1 rs151290 and rs2237892 gene polymorphisms between women with GDM and pregnant women with normal carbohydrate tolerance. Moreover, there were no statistically significant associations between the studied genotypes and the selected clinical parameters in women with GDM. The results of our study suggest that the KCNJ11 rs5219 and KCNQ1 rs2237892 and rs151290 gene polymorphisms are not significant risk factors associated with the development of GDM in our population. There were also no differences in the expression of KCNJ11 and KCNQ1 genes in the placenta of women with GDM and normal carbohydrate tolerance. However, an association between KCNJ11 gene expression in placenta and APGAR score in newborns was found.

Kir5.1 channels: potential role in epilepsy and seizure disorders.

Inwardly rectifying potassium (Kir) channels are broadly expressed in many mammalian organ systems, where they contribute to critical physiological functions. However, the importance and function of the Kir5.1 channel (encoded by the KCNJ16 gene) have not been fully recognized. This review focuses on the recent advances in understanding the expression patterns and functional roles of Kir5.1 channels in fundamental physiological systems vital to potassium homeostasis and neurological disorders. Recent studies have described the role of Kir5.1-forming Kir channels in mouse and rat lines with mutations in the Kcnj16 gene. The animal research reveals distinct renal and neurological phenotypes, including pH and electrolyte imbalances, blunted ventilatory responses to hypercapnia/hypoxia, and seizure disorders. Furthermore, it was confirmed that these phenotypes are reminiscent of those in patient cohorts in which mutations in the KCNJ16 gene have also been identified, further suggesting a critical role for Kir5.1 channels in homeostatic/neural systems health and disease. Future studies that focus on the many functional roles of these channels, expanded genetic screening in human patients, and the development of selective small-molecule inhibitors for Kir5.1 channels, will continue to increase our understanding of this unique Kir channel family member.

Update on the Genetics of Primary Aldosteronism and Aldosterone-Producing Adenomas.

Primary aldosteronism (PA) is the leading cause of secondary hypertension, accounting for over 10% of patients with high blood pressure. It is characterized by autonomous production of aldosterone from the adrenal glands leading to low-renin levels. The two most common forms arise from bilateral adrenocortical hyperplasia (BAH) and aldosterone-producing adenoma (APA). We discuss recent discoveries in the genetics of PA. Most APAs harbor variants in the KCNJ5, CACNA1D, ATP1A1, ATP2B3, and CTNNB1 genes. With the exception of β-catenin (CTNNB1), all other causative genes encode ion channels; pathogenic variants found in PA lead to altered ion transportation, cell membrane depolarization, and consequently aldosterone overproduction. Some of these genes are found mutated in the germline state (CYP11B2, CLCN2, KCNJ5, CACNA1H, and CACNA1D), leading then to familial hyperaldosteronism, and often BAH rather than single APAs. Several genetic defects in the germline or somatic state have been identified in PA. Understanding how these molecular abnormalities lead to excess aldosterone contributes significantly to the elucidation of the pathophysiology of low-renin hypertension. It may also lead to new and more effective therapies for this disease acting at the molecular level.

Using low-dose octreotide with diazoxide-resistant congenital hyperinsulinism resulting from compound heterozygous mutations in the ABCC8 gene.

Congenital hyperinsulinism (CHI) is an over-secretion of insulin by pancreatic ß-cells, causing hypoglycemia which can inhibit brain development in infants. CHI is primarily associated with mutations in the ABCC8 or KCNJ11 genes, which encode the SUR1 and KIR 6.2 subunits of the ATP-sensitive potassium (KATP) channel. Here, we report a case of hyperinsulinemic hypoglycemia with ABCC8 gene mutation in a full term, female, Korean infant who developed early onset hypoglycemia but was not subject to either genetic or metabolic workup. This infant was later admitted to the ER because of a hypoglycemic seizure, but her metabolic work up revealed that both her fasting insulin and C-peptide levels were within the normal range. Despite this glucagon stimulation produced positive results and the genetic workup revealed c.[298G>T(;)4252C>T] mutations in the ABCC8 gene. This indicated diazoxide treatment, but following an unsuccessful course of treatment we switched to octreotide which helped stabilize her glucose. Over 5 years of follow-up, the patient treated with a low dose of octreotide has had no hypoglycemic event, and her growth and psychomotor development remained within normal ranges. However, she is severely obese (BMI over 97 %) despite using octreotide. Thus, we report a mild case of CHI with octreotide treatment, wherein the patient has normal insulin and C-peptide levels and normal development, but is severe obese.

Neuromyotonia, myokymia and spinocerebellar ataxia in two Patterdale terrier littermates

AbstractTwo Patterdale terrier siblings presented with a progressive history of a spinocerebellar ataxia present from 15 weeks of age. Both dogs had daily episodes of myokymia affecting all four limbs. The female dog had a history of three neuromyotonic attacks and was euthanased due to extreme hyperthermia during one attack. Both were homozygous for the genetic mutation of the KCNJ10 gene, which was previously reported in the Jack Russell terrier. This case highlights the clinical reasoning of abnormal gaits and abnormal episodes. This is the first report of this mutation in the Patterdale terrier.

Pathogenesis of Primary Aldosteronism: Impact on Clinical Outcome.

Primary aldosteronism (PA) is the most common form of secondary arterial hypertension, with a prevalence of approximately 20% in patients with resistant hypertension. In the last decade, somatic pathogenic variants in KCNJ5, CACNA1D, ATP1A1 and ATP2B3 genes, which are involved in maintaining intracellular ionic homeostasis and cell membrane potential, were described in aldosterone-producing adenomas (aldosteronomas). All variants in these genes lead to the activation of calcium signaling, the major trigger for aldosterone production. Genetic causes of familial hyperaldosteronism have been expanded through the report of germline pathogenic variants in KCNJ5, CACNA1H and CLCN2 genes. Moreover, PDE2A and PDE3B variants were associated with bilateral PA and increased the spectrum of genetic etiologies of PA. Of great importance, the genetic investigation of adrenal lesions guided by the CYP11B2 staining strongly changed the landscape of somatic genetic findings of PA. Furthermore, CYP11B2 staining allowed the better characterization of the aldosterone-producing adrenal lesions in unilateral PA. Aldosterone production may occur from multiple sources, such as solitary aldosteronoma or aldosterone-producing nodule (classical histopathology) or clusters of autonomous aldosterone-producing cells without apparent neoplasia denominated aldosterone-producing micronodules (non-classical histopathology). Interestingly, KCNJ5 mutational status and classical histopathology of unilateral PA (aldosteronoma) have emerged as relevant predictors of clinical and biochemical outcome, respectively. In this review, we summarize the most recent advances in the pathogenesis of PA and discuss their impact on clinical outcome.

Kir7.1 disease mutant T153I within the inner pore affects K+ conduction.

Inward-rectifier potassium channel 7.1 (Kir7.1) is present in the polarized epithelium, including the retinal pigmented epithelium. A single amino acid change at position 153 in the KCNJ13 gene, a substitution of threonine to isoleucine in the Kir7.1 protein, causes blindness. We hypothesized that the disease caused by this single amino acid substitution within the transmembrane protein domain could alter the translation, localization, or ion transport properties. We assessed the effects of amino acid side-chain length, arrangement, and polarity on channel structure and function. We showed that the T153I mutation yielded a full-length protein localized to the cell membrane. Whole cell patch-clamp recordings and chord conductance analyses revealed that the T153I mutant channel had negligible K+ conductance and failed to hyperpolarize the membrane potential. However, the mutant channel exhibited enhanced inward current when rubidium was used as a charge carrier, suggesting that an inner pore had formed and the channel was dysfunctional. Substituting with a polar, nonpolar, or short side-chain amino acid did not affect the localization of the protein. Still, it had an altered channel function due to differences in pore radius. Polar side chains (cysteine and serine) with inner pore radii comparable to wildtype exhibited normal inward K+ conductance. Short side chains (glycine and alanine) produced a channel with wider than expected inner pore size and lacked the biophysical characteristics of the wild-type channel. Leucine substitution produced results similar to the T153I mutant channel. This study provides direct electrophysiological evidence for the structure and function of the Kir7.1 channel's narrow inner pore in regulating conductance.

Genetics for the pediatric endocrinologists – 1

The advancement in genetic laboratory technology has helped immensely in the diagnosis of many genetic disorders which could not hitherto be diagnosed. Monogenic diabetes among children and adolescents is not uncommon and needs a high index of clinical suspicion to diagnose. With the availability of genetic diagnostic laboratories with the latest technology, more and more patients should benefit from early diagnosis, specific targeted treatment, and better outcomes. The pediatricians and pediatric endocrinologists managing children with diabetes need to clinically suspect and advise appropriate genetic tests to confirm the diagnosis of monogenic diabetes. Neonatal diabetes mellitus is one of the most rewarding diagnoses, if we pick up a specific genetic abnormality that could respond to sulfonylurea. The child with KCNJ11 or ABCC8 gene mutation responding to sulfonylurea could escape from the life-long insulin injections and complications of diabetes. It is equally important to identify other forms of monogenic diabetes as the specific diagnosis can have implications in the treatment, genetic counseling, and identifying other family members harboring the same gene mutation.

Identification of Variants Responsible for Monogenic Forms of Diabetes in Brazil.

Monogenic forms of diabetes mellitus may affect a significant number of patients of this disease, and it is an important molecular cause to be investigated. However, studies of the genetic causes of monogenic diabetes, especially in populations with mixed ethnic backgrounds, such as the one in Brazil, are scarce. The aim of this study was to screen several genes associated with monogenic diabetes in fifty-seven Brazilian patients with recurrence of the disease in their families and thirty-four relatives. Inclusion criteria were: Age of onset ≤ 40 years old, BMI < 30 kg/m², at least two affected generations and negative anti-GAD and anti-IA2 antibodies. MODY genes HNF4A, GCK, HNF1A, HNF1B, NEUROD1, KLF11, PAX4, INS, KCNJ11, and MT-TL1 were sequenced by Sanger sequencing. We identified a total of 20 patients with variants, 13 GCK-MODY, four HNF1A-MODY, and one variant in each of the following genes, HNF4A, HNF1B and MT-TL1. Segregation analysis was performed in 13 families. Four variants were novel, two in GCK (p.(Met115Val) [c.343A>G] and p.(Asp365GlufsTer95) [c.1094_1095insGCGA]) and two in HNF1A (p.(Tyr163Ter) [c.489C>G] and p.(Val380CysfsTer39) [c.1136_1137insC]). Here we highlight the importance of screening for monogenic diabetes in admixed populations.

Phosphorylation‐Dependent Endocytosis of ROMK Limits Urinary Potassium Loss

ROMK channels in the aldosterone‐sensitive distal nephron are tightly regulated to maintain potassium balance. In response to potassium restriction, ROMK channels are internalized from the apical membrane, safeguarding against extensive potassium loss. Previously, we found the clathrin adaptor protein, ARH, directly binds to ROMK to stimulate endocytosis (Fang et al, JCI ‘09). WNK signaling pathway has been implicated in activating ROMK endocytosis, but the mechanism is not clear, and physiological significance of ARH‐mediated ROMK regulation remains uncertain. To tackle these questions, we explored how WNK kinases activate ARH and compared the homeostatic response to dietary potassium restriction in wild type and ARH KO mice.In vitro phosphorylation studies revealed ARH is phosphorylated by ERK, which in turn is activated by L‐WNK1 or WNK4. Examination of the phosphorylation cascade revealed that dietary potassium restriction increases phosphorylation of ERK and ARH‐S14 in the kidney, coincident with phospho‐activation of the WNKs, and reduction of ROMK apical surface expression. We found phosphorylation of ARH at S14 prevents its degradation, stimulates its membrane association, and this enhances ROMK endocytosis. ARH KO mice exhibit an increase in glycosylated ROMK in the renal cortex without changes in the abundance of the core‐glycosylated or unglycosylated forms, consistent with inhibition of post‐endocytic degradation. In dietary potassium deficiency, ARH KO mice are unable to reduce ROMK protein to the same extent as WT mice, and thus exhibit excessive urinary potassium loss compared to WT mice.These studies identify ARH as a downstream effector of the WNK/ERK pathway that stimulates ROMK endocytosis in potassium deficiency.

AP-517-03 WHOLE EXOME SEQUENCING IDENTIFIES TWO NOVEL EXTREMELY RARE CANDIDATE VARIANTS ASSOCIATED WITH THE SHORT QT SYNDROME IN TWO LARGE PEDIGREES

Short QT syndrome (SQTS) is caused by pathogenic variants predominantly in the KCNQ1, KCNH2, KCNJ2 genes and genes encoding for different subunits of the L-type calcium channel. Recently, a variant in the SLC4A3 gene has been associated with the SQTS as well.

Effects of Potassium Supplementation and Kir7.1 Knockout on Renal Function During the Progression of Salt‐Sensitive Hypertension

The inward‐rectifying potassium channel (Kir) family has been shown to play an important role in the kidney’s ability to maintain electrolyte homeostasis as well as blood pressure. Kir7.1 (encoded by the Kcnj13 gene) is expressed in the kidney, though its role in renal function is rudimentary. To examine the role of Kir7.1 in renal electrolyte handling and the development of salt‐induced hypertension, we generated a knockout of Kcnj13 on the Dahl salt‐sensitive (SS) rat background. Rats with the knockout of Kcnj13 were embryonically lethal; therefore, to study the Kir7.1 channel, we used heterozygous (SSKcnj13+/‐) rats. Immunohistochemistry staining for Kir7.1 revealed basolateral staining in the cortical sections of the kidneys in wild‐type littermates (WT), while SSKcnj13+/‐ rats showed little to no staining. In addition, immunofluorescence analysis confirmed that Kir7.1 was localized on the basolateral side in tubules and revealed that Kir7.1 co‐localized with aquaporin 2 and sodium‐chloride cotransporter (NCC). This data indicates that Kir7.1 is expressed in the cortical collecting duct (CCD) and the distal convoluted tubule (DCT). SSKcnj13+/‐ rats were further used to measure blood pressure and electrolyte handling changes during the development of salt‐induced hypertension. DSI telemeters were implanted in animals at 8 weeks of age; after a week to recover from surgery, animals were fed a high salt 4% NaCl diet (HS) for three weeks. Both WT and SSKcnj13+/‐rats become hypertensive after three weeks of HS; no difference between mean arterial pressures (153.1 ± 5.7 vs. 159.2 ± 6.9 mmHg for WT and SSKcnj13+/‐rats, respectively) was identified. Serum electrolytes were measured and also showed no difference in the amount of potassium (3.4 ± 0.1 vs. 3.5 ± 0.1 mmol/L) or sodium (142 ± 0.8 vs. 142 ± 0.4 mmol/L). Furthermore, urinary electrolyte excretion was measured and revealed no differences between WT and SSKcnj13+/‐ animals for potassium (14.0 ± 0.6 vs. 15.6 ± 1.9 K+/Cre) or sodium excretion (91 ± 11 vs. 117 ± 16 Na+/Cre). Potassium supplementation has previously been shown to augment renal Kir7.1 expression. Thus, we repeated the previous protocol but instead using a high salt and high potassium 4% NaCl and 2% KCl (HSHK) diet. After three weeks on this diet, animals exhibited no difference in blood pressure (154.2 ± 9.1 vs 145.6 ± 4.1 mmHg) and no differences in plasma potassium (4.0 ± 0.1 vs 4.0 ± 0.1 mmol/L) or sodium (139 ± 1 vs 139 ± 1 mmol/L). Urinary electrolyte excretion showed no differences in potassium excretion (60.9 ± 3.3 vs 54.8 ± 1.1 K+/Cre), but SSKcnj13+/‐ animals did have a significant decrease in sodium excretion (130 ± 8 vs 103 ± 3 Na+/Cre). Altogether, we have confirmed the presence of Kir7.1 in the CCD and DCT, found that heterozygous knockout of Kir7.1 does not affect the development of blood pressure on a HS or HSHK diet. However, we do see a reduction in sodium excretion in SSKcnj13+/‐rats on the HSHK diet, suggesting that Kir7.1’s role may be more pronounced under conditions of higher potassium.

The E23K Polymorphism of KCNJ11 and Diabetic Retinopathy in Northern Iran

Background: Diabetic Retinopathy (DR) is one of the most severe micro-vascular complications of diabetes mellitus (DM), involving interactions between environmental and genetic risk factors. KCNJ11 gene has a key role in insulin secretion and is of substantial interest in various populations. Methods: A population-based association of 524 T2DM patients was performed to delineate the genetic influence of KCNJ11 polymorphisms (rs5219, c.67A>G or E23K) on the risk of DR in an Iranian population. Genotyping was performed using TaqMan assay. Univariate and MLR analysis controlling for confounders was conducted to evaluate the association between rs5219 and DR. Results: No significant difference was observed in either genotypes distribution (p = 0.83) or allele frequency (p = 0.66) between T2DM individuals with and without DR in any models of inheritance. Genotype-phenotype association showed that DR group carrying GA genotypes, a significantly higher mean age was observed compared with two other genotypes (p = 0.04). MLR analysis indicated that HbAlc with adjusted OR of 1.84 (95% CI, 1.46–2.33, p = 0.00) and first-degree relatives of family history with adjusted OR of 2.85 (95% CI, 1.45–5.58, p = 0.002) were significantly associated with DR, but the c.67A>G genotype is not an independent predictor of retinopathy. Conclusion: Collectively, rs5219 was not associated with DR among Iranians with T2DM.

Pattern Recognition versus Pathogenesis: Electrolytes in a Patient with Adrenal Insufficiency.

Pattern Recognition versus Pathogenesis: Electrolytes in a Patient with Adrenal Insufficiency.

Effect of inward rectifier potassium 2.1 channel on the osteogenic differentiation of human dental follicle cells and its mechanism.

This study aims to explore the effect of inward rectifier potassium (Kir) 2.1 channel on the osteogenic differentiation of human dental follicle cells (hDFCs) and its mechanism. hDFCs were isolated and cultured, and their source was verified by flow cytometry. Osteogenic differentiation ability of hDFCs was evaluated by osteogenic induction. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect the gene expression of Kir2.1 gene (KCNJ2) in hDFCs. Real-time quantitative PCR (RT-qPCR) was performed to detect the expression of the Kir2.1 gene (KCNJ2) in hDFCs before and after osteogenic induction. Patch clamp technique was conducted to record the membrane potential changes of hDFCs before and after osteogenic induction. Moreover, the effect on the osteogenic differentiation of hDFCs was confirmed by increasing the concentration of extracellular potassium ions (50 mmol·L-1). Kir2.1 channel blockers cesium chloride (CsCl) and C19H20CINO (ML133) were applied to determine the effect of the Kir2.1 potassium channel on the osteogenic differentiation of hDFCs. At the same time, RT-qPCR was used to observe the expression changes of osteogenic differentiation related genes Runx related transcription factor 2 (Runx2) and osteocalcin (OCN) before and after the two intervention measures. Calcium imaging was performed to observe the effect of membrane potential hyperpolarization caused by decreased extracellular potassium level (2 mmol·L-1) on intracellular calcium concentration. RT-PCR results showed that hDFCs expressed the Kir2.1 channel gene (KCNJ2). The RT-qPCR results showed that the KCNJ2 gene expression in hDFCs was upregulated 7 days after osteogenic induction. The patch clamp results showed that the membrane potential of hDFCs hyperpolarized to (-47±5.2) mV from (-12±3.2) mV. Alizarin red and alkaline phosphatase staining results showed that increasing the concentration of the extracellular potassium or blocking the function of the Kir2.1 channel significantly inhibited the osteogenic mineralization ability of hDFCs. The membrane potential hyperpolarization increased the intracellular calcium concentration in hDFCs. Membrane potential hyperpolarization mediated by the Kir2.1 channel plays an important role in the osteogenic differentiation of hDFCs.