Clinical and genetic analysis of a case of Gitelman syndrome accompanied with Graves disease and adrenocortical adenoma: A case report.

RATIONALE: Gitelman syndrome (GS), also known as familial hypokalemia and hypomagnesemia, is a rare autosomal recessive inherited disease caused by primary renal desalinization caused by impaired reabsorption of sodium and chloride ions in the distal renal tubules. We report a case of clinical and genetic characteristics of GS accompanied with Graves disease and adrenocorticotrophic hormone (ACTH)-independent adrenocortical adenoma. PATIENT CONCERNS: The patient is a 45 year old female, was admitted to our hospital, due to a left adrenal gland occupying lesion as the chief complaint. DIAGNOSIS: The patient was finally diagnosed as GS with Graves disease and adrenocortical adenoma. INTERVENTIONS: Potassium magnesium aspartate (1788 mg/d, taken orally 3 times a day (supplement a few times a day, intake method, treatment duration). Contains 217.2 mg of potassium and 70.8 mg of magnesium, and potassium chloride (4.5 g/d, taken orally 3 times a day (supplement a few times a day, intake method, and treatment duration); Potassium 2356 mg), spironolactone (20 mg/d, taken orally once a day (supplement a few times a day, intake method, treatment duration). After 3 months of treatment, the patient's blood potassium fluctuated between 3.3-3.6 mmol/L, and blood magnesium fluctuated between 0.5-0.7 mmol/L, indicating a relief of fatigue symptoms. OUTCOMES: On the day 6 of hospitalization, the symptoms of dizziness, limb fatigue, fatigue and pain were completely relieved on patient. In the follow-up of the following year, no recurrence of the condition was found. LESSONS: The novel c.1444-10(IVS11)G > A variation may be a splicing mutation. The compound heterozygous mutations of the SLC12A3 gene may be the pathogenic cause of this GS pedigree.

Potassium-Switch Signaling Pathway Dictates Acute Blood Pressure Response to Dietary Potassium.

BACKGROUND: Potassium (K+)-deficient diets, typical of modern processed foods, increase blood pressure (BP) and NaCl sensitivity. A K+-dependent signaling pathway in the kidney distal convoluted tubule, coined the K+ switch, that couples extracellular K+ sensing to activation of the thiazide-sensitive NaCl cotransporter (NCC) and NaCl retention has been implicated, but causality has not been established. METHODS: To test the hypothesis that small, physiological changes in plasma K+ (PK+) are translated to BP through the switch pathway, a genetic approach was used to activate the downstream switch kinase, SPAK (SPS1-related proline/alanine-rich kinase), within the distal convoluted tubule. The CA-SPAK (constitutively active SPS1-related proline/alanine-rich kinase mice) were compared with control mice over a 4-day PK+ titration (3.8-5.1 mmol) induced by changes in dietary K+. Arterial BP was monitored using radiotelemetry, and renal function measurements, NCC abundance, phosphorylation, and activity were made. RESULTS: As PK+ decreased in control mice, BP progressively increased and became sensitive to dietary NaCl and hydrochlorothiazide, coincident with increased NCC phosphorylation and urinary sodium retention. By contrast, BP in CA-SPAK mice was elevated, resistant to the PK+ titration, and sensitive to hydrochlorothiazide and salt at all PK+ levels, concomitant with sustained and elevated urinary sodium retention and NCC phosphorylation and activity. Thus, genetically locking the switch on drives NaCl sensitivity and prevents the response of BP to potassium. CONCLUSIONS: Low K+, common in modern ultraprocessed diets, presses the K+-switch pathway to turn on NCC activity, increasing sodium retention, BP, and salt sensitivity.

[Genetic analysis of two patients with Gitelman syndrome].

OBJECTIVE: To explore the genetic etiology of two patients with Gitelman syndrome (GS). METHODS: Two patients who had presented at the Linyi People's Hospital in January and June 2022 respectively were selected as the study subjects. Peripheral blood samples of them were collected and subjected to whole exome sequencing (WES). Electrolyte levels in their serum and urine were detected. Candidate variants were verified by Sanger sequencing. PyMOL software was used to predict the impact of the variants on the protein structure. RESULTS: Patient 1 was a 27-year-old female with decreased serum levels of sodium, potassium, chloride and magnesium, along with decreased urine chloride and calcium. WES revealed that she has harbored compound heterozygous variants of the SLC12A3 gene, namely c.1456G>A (p.D486N) and c.179C>T (p.T60M). The former was inherited from her mother and known to be pathogenic. Patient 2 was a 4-year-old male with lower serum sodium, chloride and magnesium levels, and his serum potassium level was found to be critically low. He was found to harbor compound heterozygous variants of c.602-16G>A and c.805_806insTTGGCGTGGTCTCGGTCA (p.V268_T269insIGVVSV) of the SLC12A3 gene, which were inherited from his mother and father, respectively. Based on the guidelines from the American College of Medical Genetics and Genomics, both variants were predicted to be pathogenic (PVS1+PM2_Supporting+PP3; PVS1+PM2_Supporting+PM4). CONCLUSION: The above heterozygous variants of the SLC12A3 gene probably underlay the GS in these patients.

Several first-line anti-hypertensives act on fibrosarcoma progression and PD1ab blockade therapy.

PURPOSE: Patients are typically diagnosed with both hypertension and fibrosarcoma. Medical oncologists must prescribe suitable anti-hypertensive medications while considering anti-tumor drugs. Recently, immunotherapy has become prominent in cancer treatment. Nonetheless, it is unknown what role anti-hypertensive medications will play in immunotherapy. METHODS: We examined the effects of six first-line anti-hypertensive medications on programmed cell death protein 1 antibody (PD1ab) in tumor treatment using a mouse model of subcutaneous fibrosarcoma. The drugs examined were verapamil, losartan, furosemide, spironolactone, captopril, and hydrochlorothiazide (HCTZ). The infiltration of CD8+ T cells was examined by immunohistochemistry. Additionally, several in vitro and in vivo assays were used to study the effects of HCTZ on human fibrosarcoma cancer cells to explore its mechanism. RESULTS: Verapamil suppressed tumor growth and showed an improved effect on the tumor inhibition of PD1ab. Captopril did not affect tumor growth but brought an unexpected benefit to PD1ab treatment. In contrast, spironolactone and furosemide showed no effect on tumor growth but had an offset effect on the PD1ab therapy. Consequently, the survival time of mice was also significantly reduced. Notably, losartan and HCTZ, especially HCTZ, promoted tumor growth and weakened the effect of PD1ab treatment. Consistent results were observed in vivo and in vitro using the human fibrosarcoma cell line HT1080. We determined that the Solute Carrier Family 12 Member 3 (SLC12A3), a known target of HCTZ, may be the principal factor underlying its effect-enhancing properties through mechanism studies employing The Cancer Genome Atlas (TCGA) data and in vivo and in vitro assays. CONCLUSION: Verapamil and captopril potentiated the anti-tumor effect of PD1ab, whereas spironolactone and furosemide weakened the effect of PD1ab on tumor inhibition. Alarmingly, losartan and HCTZ promoted tumor growth and impaired the effect of PD1ab. Furthermore, we preliminarily found that HCTZ may promote tumor progression through SLC12A3. Based on this study, futher mechanism researches and clinical trials should be conducted in the future.

Kinase Scaffold Cab39 Is Necessary for Phospho-Activation of the Thiazide-Sensitive NCC.

BACKGROUND: Potassium regulates the WNK (with no lysine kinase)-SPAK (STE20/SPS1-related proline/alanine-rich kinase) signaling axis, which in turn controls the phosphorylation and activation of the distal convoluted tubule thiazide-sensitive NCC (sodium-chloride cotransporter) for sodium-potassium balance. Although their roles in the kidney have not been investigated, it has been postulated that Cab39 (calcium-binding protein 39) or Cab39l (Cab39-like) is required for SPAK/OSR1 (oxidative stress response 1) activation. This study demonstrates how they control the WNK-SPAK/OSR1-NCC pathway. METHODS: We created a global knockout of Cab39l and a tamoxifen-inducible, NCC-driven, Cab39 knockout. The 2 lines were crossed to generate Cab39-DKO (Cab39 double knockout) animals. Mice were studied under control and low-potassium diet, which activates WNK-SPAK/OSR1-NCC phosphorylation. Western blots were used to assess the expression and phosphorylation of proteins. Blood and urine electrolytes were measured to test for compromised NCC function. Immunofluorescence studies were conducted to localize SPAK and OSR1. RESULTS: Both Cab39l and Cab39 are expressed in distal convoluted tubule, and only the elimination of both leads to a striking absence of NCC phosphorylation. Cab39-DKO mice exhibited a loss-of-NCC function, like in Gitelman syndrome. In contrast to the apical membrane colocalization of SPAK with NCC in wild-type mice, SPAK and OSR1 become confined to intracellular puncta in the Cab39-DKO mice. CONCLUSIONS: In the absence of Cab39 proteins, NCC cannot be phosphorylated, resulting in a Gitelman-like phenotype. Cab39 proteins function to localize SPAK at the apical membrane with NCC, reminiscent of the Cab39 yeast homolog function, translocating kinases during cytokinesis.

Interpreting epigenetic causes of recurrent hypokalemia and seizures: Gitelman syndrome co-exist with pseudohypoparathyroidism type 1B.

We describe a unique case of 27-year-old male with Gitelman syndrome (GS) co-exist with pseudohypoparathyroidism type 1B (PHP1B). The patient presented with a 5-year history of seizures, tetany, and numbness of the extremities. Further examinations showed recurrent hypokalemia, inappropriate kaliuresis, hypocalcemia, hyperphosphatemia, and elevated PTH levels. A novel variant of autosomal recessive GS (p.Val287Met SLC12A3) and a novel 492.3Kb deletion containing the whole of STX16, were discovered by a whole-exome sequencing. Following the diagnosis, calcitriol, calcium, and potassium supplements were started. Hematuria calcium and phosphorus levels, as well as blood potassium levels, have recovered and remained within normal ranges after 3 years of follow-up. Our findings have important consequences for supporting the idea that heterozygosity for variants have effects on the patients' clinical performance with autosomal recessive inheritance disorders. Further study is need for the putative effects of the variant. Likewise, further investigation with regards to the gene-gene interaction relations between GS and other electrolyte imbalance disorders is warranted.

Dietary sodium alters aldosterone's effect on renal sodium transporter expression and distal convoluted tubule remodelling.

Aldosterone is responsible for maintaining volume and potassium homeostasis. Although high salt consumption should suppress aldosterone production, individuals with hyperaldosteronism lose this regulation, leading to a state of high aldosterone despite dietary sodium consumption. The present study examines the effects of elevated aldosterone, with or without high salt consumption, on the expression of key Na+ transporters and remodelling in the distal nephron. Epithelial sodium channel (ENaC) α-subunit expression was increased with aldosterone regardless of Na+ intake. However, ENaC ß- and γ-subunits unexpectedly increased at both a transcript and protein level with aldosterone when high salt was present. Expression of total and phosphorylated Na+ Cl- cotransporter (NCC) significantly increased with aldosterone, in association with decreased blood [K+ ], but the addition of high salt markedly attenuated the aldosterone-dependent NCC increase, despite equally severe hypokalaemia. We hypothesized this was a result of differences in distal convoluted tubule length when salt was given with aldosterone. Imaging and measurement of the entire pNCC-positive tubule revealed that aldosterone alone caused a shortening of this segment, although the tubule had a larger cross-sectional diameter. This was not true when salt was given with aldosterone because the combination was associated with a lengthening of the tubule in addition to increased diameter, suggesting that differences in the pNCC-positive area are not responsible for differences in NCC expression. Together, our results suggest the actions of aldosterone, and the subsequent changes related to hypokalaemia, are altered in the presence of high dietary Na+ . KEY POINTS: Aldosterone regulates volume and potassium homeostasis through effects on transporters in the kidney; its production can be dysregulated, preventing its suppression by high dietary sodium intake. Here, we examined how chronic high sodium consumption affects aldosterone's regulation of sodium transporters in the distal nephron. Our results suggest that high sodium consumption with aldosterone is associated with increased expression of all three epithelial sodium channel subunits, rather than just the alpha subunit. Aldosterone and its associated decrease in blood [K+ ] lead to an increased expression of Na-Cl cotransporter (NCC); the addition of high sodium consumption with aldosterone partially attenuates this NCC expression, despite similarly low blood [K+ ]. Upstream kinase regulators and tubule remodelling do not explain these results.

Dysregulation of the WNK4-SPAK/OSR1 pathway has a minor effect on baseline NKCC2 phosphorylation.

The with-no-lysine kinase 4 (WNK4)-sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway mediates activating phosphorylation of the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and the thiazide-sensitive NaCl cotransporter (NCC). The commonly used pT96/pT101-pNKCC2 antibody cross-reacts with pT53-NCC in mice on the C57BL/6 background due to a five amino acid deletion. We generated a new C57BL/6-specific pNKCC2 antibody (anti-pT96-NKCC2) and tested the hypothesis that the WNK4-SPAK/OSR1 pathway strongly regulates the phosphorylation of NCC but not NKCC2. In C57BL/6 mice, anti-pT96-NKCC2 detected pNKCC2 and did not cross-react with NCC. Abundances of pT96-NKCC2 and pT53-NCC were evaluated in Wnk4-/-, Osr1-/-, Spak-/-, and Osr1-/-/Spak-/- mice and in several models of the disease familial hyperkalemic hypertension (FHHt) in which the CUL3-KLHL3 ubiquitin ligase complex that promotes WNK4 degradation is dysregulated (Cul3+/-/Δ9, Klhl3-/-, and Klhl3R528H/R528H). All mice were on the C57BL/6 background. In Wnk4-/- mice, pT53-NCC was almost absent but pT96-NKCC2 was only slightly lower. pT53-NCC was almost absent in Spak-/- and Osr1-/-/Spak-/- mice, but pT96-NKCC2 abundance did not differ from controls. pT96-NKCC2/total NKCC2 was slightly lower in Osr1-/- and Osr1-/-/Spak-/- mice. WNK4 expression colocalized not only with NCC but also with NKCC2 in Klhl3-/- mice, but pT96-NKCC2 abundance was unchanged. Consistent with this, furosemide-induced urinary Na+ excretion following thiazide treatment was similar between Klhl3-/- and controls. pT96-NKCC2 abundance was also unchanged in the other FHHt mouse models. Our data show that disruption of the WNK4-SPAK/OSR1 pathway only mildly affects NKCC2 phosphorylation, suggesting a role for other kinases in NKCC2 activation. In FHHt models NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide sensitivity of FHHt.NEW & NOTEWORTHY The renal cation cotransporters NCC and NKCC2 are activated following phosphorylation mediated by the WNK4-SPAK/OSR1 pathway. While disruption of this pathway strongly affects NCC activity, effects on NKCC2 activity are unclear since the commonly used phospho-NKCC2 antibody was recently reported to cross-react with phospho-NCC in mice on the C57BL/6 background. Using a new phospho-NKCC2 antibody specific for C57BL/6, we show that inhibition or activation of the WNK4-SPAK/OSR1 pathway in mice only mildly affects NKCC2 phosphorylation.

From Fish Physiology to Human Disease: The Discovery of the NCC, NKCC2, and the Cation-Coupled Chloride Cotransporters.

The renal Na-K-2Cl and Na-Cl cotransporters are the major salt reabsorption pathways in the thick ascending limb of Henle loop and the distal convoluted tubule, respectively. These transporters are the target of the loop and thiazide type diuretics extensively used in the world for the treatment of edematous states and arterial hypertension. The diuretics appeared in the market many years before the salt transport systems were discovered. The evolving of the knowledge and the cloning of the genes encoding the Na-K-2Cl and Na-Cl cotransporters were possible thanks to the study of marine species. This work presents the history of how we came to know the mechanisms for the loop and thiazide type diuretics actions, the use of marine species in the cloning process of these cotransporters and therefore in the whole solute carrier cotransproters 12 (SLC12) family of electroneutral cation chloride cotransporters, and the disease associated with each member of the family.

Potential low toxic alternative for Na-Cl cotransporter inhibition: A diuretic effect and mechanism study of Pyrrosia petiolosa.

BACKGROUND: Hydrochlorothiazide, a diuretic commonly used for the treatment of hypertension, is often associated with serious metabolic side effects. Pyrrosia petiolosa (Christ) Ching is a traditional Chinese medicine that possesses diuretic properties, without any obvious side effects. AIM: To evaluate the diuretic effect of P. petiolosa (Christ) Ching and to elucidate its underlying mechanism of action. METHODS: Extracts obtained from different polar components of P. petiolosa (Christ) Ching were analyzed for toxicity in a Kunming mouse model. The diuretic effects of the extracts were compared to that of hydrochlorothiazide in rats. In addition, compound isolation procedures, cell assays of Na-Cl cotransporter inhibition and rat diuretic test of monomeric compounds were conducted to identify the active ingredients in the extract. Subsequently, homology modeling and molecular docking were performed to explain the reason behind the diuretic activity observed. Finally, LC-MS analysis was used to elucidate the underlying mechanism of action of P. petiolosa (Christ) Ching. RESULTS: No toxicity was observed in mice administered P. petiolosa (Christ) Ching extracts. The ethyl acetate fraction showed the most significant diuretic effect. Similar results were obtained during the analysis for Na+ content in rat urine. Further separation of P. petiolosa (Christ) Ching components led to the isolation of methyl chlorogenate, 2',3'-dihydroxy propyl pentadecanoate, and ß-carotene. Results from cell assays showed that the Na-Cl cotransporter inhibitory activity of methyl chlorogenate was greater than that of hydrochlorothiazide. This result was again confirmed by the diuresis tests of monomeric compounds in rats. The molecular simulations explain the stronger interactions between the methyl chlorogenate and Na-Cl cotransporter. Of the compounds determined using LC-MS analysis, 185 were identified to be mostly organic acids. CONCLUSIONS: P. petiolosa possesses significant diuretic activities without any obvious toxicity, with least two possible mechanisms of action. Further study on this herb is warranted.

Pharmacology of Compounds Targeting Cation-Chloride Cotransporter Physiology.

Transporters of the solute carrier family 12 (SLC12) carry inorganic cations such as Na+ and/or K+ alongside Cl across the plasma membrane of cells. These tightly coupled, electroneutral, transporters are expressed in almost all tissues/organs in the body where they fulfil many critical functions. The family includes two key transporters participating in salt reabsorption in the kidney: the Na-K-2Cl cotransporter-2 (NKCC2), expressed in the loop of Henle, and the Na-Cl cotransporter (NCC), expressed in the distal convoluted tubule. NCC and NKCC2 are the targets of thiazides and "loop" diuretics, respectively, drugs that are widely used in clinical medicine to treat hypertension and edema. Bumetanide, in addition to its effect as a loop diuretic, has recently received increasing attention as a possible therapeutic agent for neurodevelopmental disorders. This chapter also describes how over the past two decades, the pharmacology of Na+ independent transporters has expanded significantly to provide novel tools for research. This work has indeed led to the identification of compounds that are 100-fold to 1000-fold more potent than furosemide, the first described inhibitor of K-Cl cotransport, and identified compounds that possibly directly stimulate the function of the K-Cl cotransporter. Finally, the recent cryo-electron microscopy revolution has begun providing answers as to where and how pharmacological agents bind to and affect the function of the transporters.

Arginine vasopressin regulates the renal Na+-Cl- and Na+-K+-Cl- cotransporters through with-no-lysine kinase 4 and inhibitor 1 phosphorylation.

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

Long-Term Indomethacin Treatment in a Chinese Child with Gitelman Syndrome: Case Report and Literature Review on its Efficacy and Tolerance.

BACKGROUND Gitelman syndrome (GS) is a rare inherited autosomal recessive salt-losing renal tubulopathy. Early-onset GS is difficult to differentiate from Bartter syndrome (BS). It has been reported in some cases that cyclooxygenase (COX) inhibitors, which pharmacologically reduce prostaglandin E2(PGE2) synthesis, are helpful for GS patients, especially in children, but the long-term therapeutic effect has not yet been revealed. CASE REPORT A 4-year-old boy was first brought to our hospital for the chief concern of short stature and growth retardation. Biochemical tests demonstrated severe hypokalemia, hyponatremia, and hypochloremic metabolic alkalosis. The patient's serum magnesium was normal. He was diagnosed with BS and treated with potassium supplementation and indomethacin and achieved stable serum potassium levels and slow catch-up growth. At 11.8 years of age, the patient showed hypomagnesemia and a genetic test confirmed that he had GS with compound heterozygous mutations in the SLC12A3 gene. At the age of 14.8 years, when indomethacin had been taken for nearly 10 years, the boy reported having chronic stomachache, while his renal function remained normal. After proton pump inhibitor and acid inhibitor therapy, the patient's symptoms were ameliorated, and he continued to take a low dose of indomethacin (37.5 mg/d divided tid) with good tolerance. CONCLUSIONS Early-onset GS in childhood can be initially misdiagnosed as BS, and gene detection can confirm the final diagnosis. COX inhibitors, such as indomethacin, might be tolerated by pediatric patients, and long-term therapy can improve the hypokalemia and growth retardation without significant adverse effects.

Gitelman syndrome combined with diabetes mellitus: A case report and literature review.

RATIONALE: Gitelman syndrome (GS) is an uncommon autosomal recessive tubulopathy resulting from a functional deletion mutation in the SLC12A3 gene. Its onset is typically insidious and challenging to discern, and it is characterized by hypokalemia, metabolic alkalosis, and reduced urinary calcium excretion. There is limited literature on the diagnosis and management of GS in individuals with concomitant diabetes. PATIENT CONCERNS: A 36-year-old male patient with a longstanding history of diabetes exhibited suboptimal glycemic control. Additionally, he presented with concurrent findings of hypokalemia, hypomagnesemia, hypocalciuria, and metabolic alkalosis. DIAGNOSIS: Building upon the patient's clinical manifestations and extensive laboratory evaluations, we conducted thorough genetic testing, leading to the identification of a compound heterozygous mutation within the SLC12A3 gene. This definitive finding confirmed the diagnosis of GS. INTERVENTIONS: We have formulated a detailed medication regimen for patients, encompassing personalized selection of hypoglycemic medications and targeted electrolyte supplementation. OUTCOMES: Following 1 week of comprehensive therapeutic intervention, the patient's serum potassium level effectively normalized to 3.79 mmol/L, blood glucose parameters stabilized, and there was significant alleviation of clinical symptoms. LESSONS: GS has a hidden onset and requires early diagnosis and intervention based on patient related symptoms and laboratory indicators in clinical practice, and personalized medication plans need to be provided according to the specific situation of the patient.

Identification of key genes and validation of key gene aquaporin 1 on Wilms' tumor metastasis.

Background: Wilms' tumor (WT) is one of the most common solid tumors in children with unsatisfactory prognosis, but few molecular prognostic markers have been discovered for it. Many genes are associated with the occurrence and prognosis of WT. This study aimed to explore the key genes and potential molecular mechanisms through bioinformatics and to verify the effects of aquaporin 1 (AQP1) on WT metastasis. Methods: Differentially expressed genes (DEGs) were generated from WT gene expression data sets from the Gene Expression Omnibus (GEO) database. Gene functional enrichment analysis was carried out with the Database for Annotation, Visualization and Integrated Discovery (DAVID). A protein-protein interaction network (PPI) was constructed and visualized by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database and Cytoscape software. Minimal Common Oncology Data Elements (MCODE) was used to detect the important modules in the PPI network, and the important nodes (genes) in the PPI module were sorted by CytoHubba. RT-qPCR was performed to validate the expression of the key genes in WT. Wound healing and Transwell assays were used to detect the cell migration and invasion abilities of AQP1-overexpressing cells. Phalloidin-iFlour 488 was used to stain the cytoskeleton to observe how AQP1 overexpression affects cytoskeletal microfilament structure. Results: A total of 73 co-expressed DEGs were chosen for further investigation. The importance of homeostasis and transmembrane transport of ions and water were highlighted by functional analysis. Gene regulatory network and PPI network were predicted. MCODE plug identified two important modules. Finally, top five key genes were identified using CytoHubba, including Renin (REN), nephrosis 2 (NPHS2), Solute Carrier Family 12 Member 3 (SLC12A3), Solute Carrier Family 12 Member 1 (SLC12A1) and AQP1. The five key genes were mainly enriched in cell volume and ion homeostasis. RT-qPCR confirmed the expression of the five key genes in WT. AQP1 was validated to be expressed at significantly lower levels in WT than in normal tissue. AQP1 overexpression significantly reduced the migratory and invasive capacity of Wit-49 cells, as evidenced by reducing the scratch healing rate and the number of perforated control cells by Wit-49 cells. AQP1 overexpression also reduced the expression of biomarkers of epithelial-mesenchymal transformation, decreased levels of vimentin and N-cadherin and increased expression of E-cadherin, resulting in decreased formation of conspicuous lamellipodial protrusions, characteristic of diminished WT cell invasion and migration. Conclusion: Our study reveals the key genes of WT. These key genes may provide novel insight for the mechanism and diagnosis of WT. AQP1 overexpression inhibited invasion, migration, EMT, and cytoskeletal rearrangement of WT cells, indicating that AQP1 plays a role in the pathogenesis of WT.

[Clinical and genetic analysis of a case of Gitelman syndrome with comorbid Graves disease and adrenocortical adenoma].

OBJECTIVE: To report the clinical and genetic characteristics of a rare case of Gitelman syndrome with comorbid Graves disease and ACTH-independent adrenocortical adenoma. METHODS: A patient who had presented at the Nanchong Central Hospital on December 21, 2020 was selected as the study subject. Clinical data of the patient was collected. Whole-exome sequencing was carried out on DNA extracted from peripheral venous blood samples from the patient and her family members. RESULTS: The patient, a 45-year-old woman, was found to have Graves disease, ACTH-independent Cushing syndrome, hypokalemia and hypomagnesemia following the discovery of an adrenal incidentaloma. MRI scan had revealed a 3.8 cm × 3.2 cm mass in the left adrenal gland. The mass was removed by surgery and confirmed as adrenocortical adenoma. DNA sequencing revealed that the patient and her sister have both harbored compound heterozygous variants of the SLC12A3 gene, namely c.1444-10(IVS11)G>A and c.179(exon1)C>T (p.T60M), which were respectively inherited from their father and mother. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.1444-10(IVS11)G>A and c.179(exon1)C>T (p.T60M) were respectively classified as a variant of uncertain significance (PM2_Supporting+PP3) and a likely pathogenic variant (PM3_Strong+PM1+PP3). CONCLUSION: The conjunction of Gitelman syndrome with Graves disease and adrenal cortex adenoma is rather rare. The newly discovered c.1444-10(IVS11)G>A variant of the SLC12A3 gene, together with the heterozygous variant of c.179(exon1)C>T (p.T60M), probably underlay the pathogenesis in this patient.

Characteristics of sodium and water retention in rats with nephrotic syndrome induced by puromycin aminonucleoside.

INTRODUCTION: Nephrotic syndrome (NS) is characterized by renal sodium and water retention. The mechanisms are not fully elucidated. METHODS: The NS rat model was established by single intraperitoneal injection of 100 mg/kg puromycin aminonucleoside (PAN). The plasma electrolyte level and urinary sodium excretion were monitored dynamically. The changes of some sodium transporters, including epithelial Na+ channel (ENaC), Na+/H+ exchanger 3 (NHE3), Na+-K+-2Cl- cotransporter 2 (NKCC2) and Na+-Cl- cotransporter (NCC) in renal cortex at different time points and the level of peripheral circulation factors were detected. RESULTS: The urinary sodium excretion of the model group increased significantly on the first day, then decreased compared with the control group, and there was no significant difference between the model group and the control group on the 12th day. The changes of peripheral circulation factors were not obvious. Some sodium transporters in renal cortex increased in varying degrees, while NKCC2 decreased significantly compared with the control group. CONCLUSIONS: The occurrence of NS edema may not be related to the angiotensin system. The decrease of urinary sodium excretion is independent of the development of albuminuria. During the 18 days of observation, it can be divided into three stages: sodium retention, sodium compensation, and simple water retention. The mechanism is related to the increased expression of α-ENaC, γ-ENaC, NHE3 and NCC in a certain period of time, the compensatory decrease of NKCC2 expression and the continuous increase of aquaporin 2 (AQP2) expression.

Novel heterozygous mutations of SLC12A3 gene in a Chinese pedigree with Gitelman syndrome: A care-compliant case report.

RATIONALE: The diagnosis of Gentleman syndrome (GS) is usually delayed because the clinical symptoms are easily mistaken. PATIENT CONCERNS: A 19-year-old male patient was referred to endocrinology due to intermittent twitch of extremities for approximately 7 years. DIAGNOSES: The diagnosis of GS was made based on the laboratory and gene detection results. We identified 2 new variants in the SLC12A3 gene [c.857 A > C (exon7) and c.2089_2095del (exon17)] in his Asian family. INTERVENTIONS: The patient received the treatment of potassium chloride sustained release tablets, potassium magnesium aspartate and spironolactone. After given potassium supplement through enema, his serum potassium level was corrected to normal. OUTCOMES: The electrolyte imbalance including hypokalemia and hypomagnesemia were improved with a remission of the clinical manifestations. But the patient's condition still could not remain stable for his irregular oral potassium supplementation during the follow-up of nearly 3 months. LESSONS: Our finding broadens the variant spectrum of SLC12A3 and contributes to a more quickly genetic counseling. As a result, when a patient presents with persistent, unspecified, and inadequately treated hypokalemia, tests for GS should indeed be considered. For suspected cases of GS, genetic testing should always be considered in the diagnosis.

Identification of potential biomarkers and candidate therapeutic drugs for clear cell renal cell carcinoma by bioinformatic analysis and reverse network pharmacology.

This study aims to analyze the potential biomarkers using bioinformatics technology, explore the pathogenesis, and investigate potential Chinese herbal ingredients for the Clear cell renal cell carcinoma (ccRCC), which could provide theoretical basis for early diagnosis and effective treatment of ccRCC. The gene expression datasets GSE6344 and GSE53757 were obtained from the Gene Expression Omnibus database to screen differentially expressed genes (DEGs) involved in ccRCC carcinogenesis and disease progression. Enrichment analyses, protein-protein interaction networks construction, survival analysis and herbal medicines screening were performed with related software and online analysis platforms. Moreover, network pharmacology analysis has also been performed to screen potential target drugs of ccRCC and molecular docking analysis has been used to validate their effects. Total 274 common DEGs were extracted through above process, including 194 up-regulated genes and 80 down-regulated genes. The enrichment analysis revealed that DEGs were significantly focused on multiple amino acid metabolism and HIF signaling pathway. Ten hub genes, including FLT1, BDNF, LCP2, AGXT2, PLG, SLC13A3, SLC47A2, SLC22A8, SLC22A7, and SLC13A3, were screened. Survival analysis showed that FLT1, BDNF, AGXT2, PLG, SLC47A2, SLC22A8, and SLC12A3 were closely correlated with the overall survival of ccRCC, and AGXT2, SLC47A2, SLC22A8, and SLC22A7 were closely associated with DFS. The potential therapeutic herbs that have been screened were Danshen, Baiguo, Yinxing, Huangqin and Chuanshanlong. The active compounds which may be effective in ccRCC treatment were kaempferol, Scillaren A and (-)-epigallocatechin-3-gallate.

Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure.

Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine kinase/STE20/SPS1-related proline-alanine-rich protein kinase (WNK/SPAK) pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high-potassium "DASH-like" diets (dietary approaches to stop hypertension) inactivate the cotransporter and whether this decreases BP. A transcriptomics screen identified Ppp1Ca, encoding PP1A, as a potassium-upregulated gene, and its negative regulator Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK/SPAK kinase cascade, we confirmed that PP1A dephosphorylated NCC directly in a potassium-regulated manner. Prior adaptation to a high-potassium diet was required to maximally dephosphorylate NCC and lower BP in constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a and dephosphorylation of its cognate protein, inhibitory subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drove NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK/SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.