Genetic and Physiological Effects of Insulin-Like Growth Factor-1 (IGF-1) on Human Urate Homeostasis.

SIGNIFICANCE STATEMENT: Hyperinsulinemia induces hyperuricemia by activating net renal urate reabsorption in the renal proximal tubule. The basolateral reabsorptive urate transporter GLUT9a appears to be the dominant target for insulin. By contrast, IGF-1 infusion reduces serum urate (SU), through mechanisms unknown. Genetic variants of IGF1R associated with reduced SU have increased IGF-1R expression and interact with genes encoding the GLUT9 and ABCG2 urate transporters, in a sex-specific fashion, which controls the SU level. Activation of IGF-1/IGF-1R signaling in Xenopus oocytes modestly activates GLUT9a and inhibits insulin's stimulatory effect on the transporter, which also activates multiple secretory urate transporters-ABCG2, ABCC4, OAT1, and OAT3. The results collectively suggest that IGF-1 reduces SU by activating secretory urate transporters and inhibiting insulin's action on GLUT9a. BACKGROUND: Metabolic syndrome and hyperinsulinemia are associated with hyperuricemia. Insulin infusion in healthy volunteers elevates serum urate (SU) by activating net urate reabsorption in the renal proximal tubule, whereas IGF-1 infusion reduces SU by mechanisms unknown. Variation within the IGF1R gene also affects SU levels. METHODS: Colocalization analyses of a SU genome-wide association studies signal at IGF1R and expression quantitative trait loci signals in cis using COLOC2, RT-PCR, Western blotting, and urate transport assays in transfected HEK 293T cells and in Xenopus laevis oocytes. RESULTS: Genetic association at IGF1R with SU is stronger in women and is mediated by control of IGF1R expression. Inheritance of the urate-lowering homozygous genotype at the SLC2A9 locus is associated with a differential effect of IGF1R genotype between men and women. IGF-1, through IGF-1R, stimulated urate uptake in human renal proximal tubule epithelial cells and transfected HEK 293T cells, through activation of IRS1, PI3/Akt, MEK/ERK, and p38 MAPK; urate uptake was inhibited in the presence of uricosuric drugs, specific inhibitors of protein tyrosine kinase, PI3 kinase (PI3K), ERK, and p38 MAPK. In X. laevis oocytes expressing ten individual urate transporters, IGF-1 through endogenous IGF-1R stimulated urate transport mediated by GLUT9, OAT1, OAT3, ABCG2, and ABCC4 and inhibited insulin's stimulatory action on GLUT9a and OAT3. IGF-1 significantly activated Akt and ERK. Specific inhibitors of PI3K, ERK, and PKC significantly affected IGF-1 stimulation of urate transport in oocytes. CONCLUSIONS: The combined results of infusion, genetics, and transport experiments suggest that IGF-1 reduces SU by activating urate secretory transporters and inhibiting insulin's action.

Zoledronic Acid-Associated Fanconi Syndrome in Patients With Cancer.

Zoledronic acid (ZA) is an antiresorptive agent typically used for fracture prevention in postmenopausal osteoporosis, malignancy-associated metastatic bone lesions, and as a treatment for hypercalcemia. ZA is excreted almost entirely by the kidney; as a result, a reduction in renal clearance can lead to its accumulation and potential renal toxicity. Although uncommon, acute kidney injury (AKI) from intravenous bisphosphonates has been described, with different patterns including tubulointerstitial nephritis, acute tubular necrosis, as well as focal segmental glomerulosclerosis. Here we present 4 patients with an underlying malignancy who each developed evidence of generalized proximal tubular dysfunction, also known as Fanconi syndrome, approximately 1 week after receiving treatment with ZA. On presentation, all patients had AKI, low serum bicarbonate levels, abnormal urinary acidification, hypophosphatemia, hypokalemia, and increased urine amino acid excretion or renal glycosuria. Based on the temporal association between ZA infusion and the development of these electrolyte abnormalities, each case is highly suggestive of ZA-associated Fanconi syndrome. Due to the severity of presentation, all required discontinuation of ZA and ongoing electrolyte repletion. Nephrologists and oncologists should be aware of this complication and consider ZA as a possible trigger of new-onset Fanconi syndrome.

Association of acidic urine pH with impaired renal function in primary gout patients: a Chinese population-based cross-sectional study.

BACKGROUND: Patients with gout frequently have low urinary pH, which is associated with the nephrolithiasis. However, the specific distribution of urinary pH and potential relationship of acidic urine pH to broader manifestations of kidney disease in gout are still poorly understood. METHODS: A 2016-2020 population-based cross-sectional study was conducted among 3565 gout patients in the dedicated gout clinic of the Affiliated Hospital of Qingdao University to investigate the association between low urinary pH and kidney disease. We studied patients that we defined to have "primary gout", based on the absence of > stage 2 CKD. All subjects underwent 14 days of medication washout and 3-day standardized metabolic diet. We obtained general medical information, blood and urine biochemistries, and renal ultrasound examination on the day of the visit. The primary readouts were urine pH, eGFR, nephrolithiasis, renal cysts, microhematuria, and proteinuria. Patients were assigned into 5 subgroups (urine pH ≤5.0, 5.0 6.9), aligning with the clinical significance of urine pH. RESULTS: Overall, the median urine pH and eGFR of all patients was 5.63 (IQR 5.37~6.09), and 98.32 (IQR 86.03~110.6), with acidic urine in 46.5% of patients. The prevalence of nephrolithiasis, microhematuria, and proteinuria were 16.9%, 49.5%, and 6.9%, respectively. By univariate analysis, eGFR was significantly associated with age, sex, duration of gout, tophus, body mass index, systolic blood pressure, diastolic blood pressure, fasting blood glucose, total cholesterol, serum utare, hypertension, diabetes, and urine pH. On multivariable analysis, eGFR was associated with age, sex, diastolic blood pressure, serum uric acid, hypertension, diabetes, and urine pH. Acidic urine pH, especially urine pH < 5.0, was significantly associated with the prevalence of kidney disease, including > stage 1 CKD, nephrolithiasis, kidney cyst, and microhematuria. Patients with 6.2 ≤ urine pH ≤ 6.9 and SU ≤ 480 µmol/L had the highest eGFR with the lowest prevalence of nephrolithiasis, microhematuria, and proteinuria. CONCLUSIONS: Approximately half of gout subjects had acidic urine pH. Urine pH < 5.0 was associated with significantly increased nephrolithiasis, renal cyst, microhematuria, and proteinuria. The results support prospective clinical investigation of urinary alkalinization in selected gout patients with acidic urine pH.

Incidence of Hyponatremia in Patients With Indwelling Peritoneal Catheters for Drainage of Malignant Ascites.

Importance: Indwelling peritoneal catheters (IPCs) are frequently used to drain tense, symptomatic, malignant ascites. Large-volume drainage may lead to hyponatremia owing to massive salt depletion. To date, no studies have examined the epidemiology of hyponatremia after placement of an IPC. Objective: To evaluate the incidence of hyponatremia after IPC placement, the risk factors associated with its development, and how it is managed. Design, Setting, and Participants: This cohort study retrospectively reviewed the medical records of 461 patients who had IPCs placed during the period between 2006 and 2016 at a tertiary care hospital in Boston, Massachusetts, of whom 309 patients met the inclusion criteria. Data analysis was performed from June to November 2019. Main Outcomes and Measures: Main outcomes were the incidence of hyponatremia (with a serum sodium level <135 mEq/L) after IPC placement, the risk factors for its development, and how it was managed. We also examined the clinical course of a subset of 21 patients with hypovolemic hyponatremia. Results: Of the 309 eligible patients with laboratory results both before IPC placement and 2 days or more after IPC placement, 189 (72.1%) were female, and the mean (SD) age was 59 (12) years. The overall incidence of hyponatremia after IPC placement was 84.8% (n = 262), of whom 21 patients (8.0%) had severe hyponatremia. The mean (SD) decrease in serum sodium level before vs after IPC placement was 5 (5.1) mEq/L and decreased by 10 mEq/L or more among 52 patients (16.8%). Patients with hyponatremia prior to IPC placement had an 8-fold higher adjusted odds of having persistent hyponatremia after IPC placement (odds ratio, 7.9; 95% CI, 2.9-21.7). Patients with hepatopancreatobiliary malignant neoplasms were more likely to develop hyponatremia (78 of 262 patients with hyponatremia [29.8%] vs 7 of 47 patients without hyponatremia [14.9%]). Hyponatremia was either unrecognized or untreated in 189 patients (72.1%). Conclusions and Relevance: Although the placement of an IPC is often a palliative measure, hyponatremia is common and is often untreated or unrecognized. Patients at highest risk, such as those with hyponatremia at baseline and those with hepatopancreatobiliary malignant neoplams, should be evaluated carefully prior to IPC placement and may warrant closer monitoring after placement. In all cases, hyponatremia should be evaluated and managed within the context of a patient's overall goals of care.

Molecular Pathophysiology of Uric Acid Homeostasis.

Uric acid, the end product of purine metabolism, plays a key role in the pathogenesis of gout and other disease processes. The circulating serum uric acid concentration is governed by the relative balance of hepatic production, intestinal secretion, and renal tubular reabsorption and secretion. An elegant synergy between genome-wide association studies and transport physiology has led to the identification and characterization of the major transporters involved with urate reabsorption and secretion, in both kidney and intestine. This development, combined with continued analysis of population-level genetic data, has yielded an increasingly refined mechanistic understanding of uric acid homeostasis as well as greater understanding of the genetic and acquired influences on serum uric acid concentration. The continued delineation of novel and established regulatory pathways that regulate uric acid homeostasis promises to lead to a more complete understanding of uric acid-associated diseases and to identify new targets for treatment.

The Management of Gout in Renal Disease.

Gout, a debilitating inflammatory arthritis, currently affects more than 9 million Americans. Hyperuricemia, the laboratory abnormality associated with the development of gout, also occurs in a significant number of patients with chronic kidney disease (CKD), a condition that affects approximately 14% of the US population. Several recent studies have attempted to provide a definitive link between the presence of hyperuricemia and progression of CKD; however, the treatment of asymptomatic hyperuricemia in CKD is not supported by recent randomized controlled trials. The pharmacology of acute gout flares and urate lowering is complicated in patients who also have evidence of CKD, primarily because of an increased risk of medication toxicity. Recipients of kidney transplants are particularly at risk of debilitating gout and medication toxicity. We review the available data linking CKD, gout, and hyperuricemia, providing practice guidelines on managing gout in CKD patients and kidney transplant recipients. We advocate for much greater involvement of nephrologists in the management of gout in renal patients.

"Pleurex Desalination" in Malignancy-related Ascites: A Novel Mechanism of Hyponatremia.

OBJECTIVE: Pleurex catheters are a form of palliative therapy for patients, offering relief from symptomatic ascites while also affording greater independence and flexibility; however, aggressive drainage can lead to significant total body sodium losses. We describe the course of patients with "Pleurex desalination," an under-appreciated cause of hypovolemic hyponatremia, highlighting its unique pathophysiology and providing recommendations on how to manage these complex patients. PATIENTS AND METHODS: We included representative patients with "Pleurex desalination" who were evaluated and treated by the renal consult service at Brigham and Women's between 2017 and 2019. RESULTS: We identified 3 patients who were hospitalized with "Pleurex desalination" and had complete data on serum and urine studies, as well as treatment course. We demonstrate that patients with "Pleurex desalination" were removing up to 1 to 2 L of ascitic fluid a day and were admitted with signs and symptoms of profound hypovolemia and hyponatremia. Patients worsened with administration of diuretics and salt restriction and improved with aggressive fluid resuscitation in the form of hypertonic saline, normal saline, and/or intravenous albumin. CONCLUSION: "Pleurex desalination" is an under-recognized cause of hyponatremia; at-risk patients require close observation and periodic resuscitation with intravenous, volume-expanding fluids.

Hyperuricemia, Acute and Chronic Kidney Disease, Hypertension, and Cardiovascular Disease: Report of a Scientific Workshop Organized by the National Kidney Foundation.

Urate is a cause of gout, kidney stones, and acute kidney injury from tumor lysis syndrome, but its relationship to kidney disease, cardiovascular disease, and diabetes remains controversial. A scientific workshop organized by the National Kidney Foundation was held in September 2016 to review current evidence. Cell culture studies and animal models suggest that elevated serum urate concentrations can contribute to kidney disease, hypertension, and metabolic syndrome. Epidemiologic evidence also supports elevated serum urate concentrations as a risk factor for the development of kidney disease, hypertension, and diabetes, but differences in methodologies and inpacts on serum urate concentrations by even subtle changes in kidney function render conclusions uncertain. Mendelian randomization studies generally do not support a causal role of serum urate in kidney disease, hypertension, or diabetes, although interpretation is complicated by nonhomogeneous populations, a failure to consider environmental interactions, and a lack of understanding of how the genetic polymorphisms affect biological mechanisms related to urate. Although several small clinical trials suggest benefits of urate-lowering therapies on kidney function, blood pressure, and insulin resistance, others have been negative, with many trials having design limitations and insufficient power. Thus, whether uric acid has a causal role in kidney and cardiovascular diseases requires further study.

Insulin and SGK1 reduce the function of Na+/monocarboxylate transporter 1 (SMCT1/SLC5A8).

SMCTs move several important fuel molecules that are involved in lipid, carbohydrate, and amino acid metabolism, but their regulation has been poorly studied. Insulin controls the translocation of several solutes that are involved in energetic cellular metabolism, including glucose. We studied the effect of insulin on the function of human SMCT1 expressed in Xenopus oocytes. The addition of insulin reduced α-keto-isocaproate (KIC)-dependent 22Na+ uptake by 29%. Consistent with this result, the coinjection of SMCT1 with SGK1 cRNA decreased the KIC-dependent 22Na+ uptake by 34%. The reduction of SMCT1 activity by SGK1 depends on its kinase activity, and it was observed that the coinjection of SMCT1 with S442D-SGK1 (a constitutively active mutant) decreased the KIC-dependent 22Na+ uptake by 50%. In contrast, an SMCT1 coinjection with K127M-SGK1 (an inactive mutant) had no effect on the KIC-dependent Na+ uptake. The decreasing SMCT1 function by insulin or SGK1 was corroborated by measuring [1-14C]acetate uptake and the electric currents of SMCT1-injected oocytes. Previously, we found that SMCT2/Slc5a12-mRNA, but not SMCT1/Slc5a8-mRNA, is present in zebrafish pancreas (by in situ hybridization); however, SLC5a8 gene silencing was associated with the development of human pancreatic cancer. We confirmed that the mRNA and protein of both transporters were present in rat pancreas using RT-PCR with specific primers, Western blot analysis, and immunohistochemistry. Additionally, significant propionate-dependent 22Na+ uptake occurred in pancreatic islets and was reduced by insulin treatment. Our data indicate that human SMCT1 is regulated by insulin and SGK1 and that both SMCTs are present in the mammalian pancreas.

The molecular physiology of uric acid homeostasis.

Uric acid, generated from the metabolism of purines, has proven and emerging roles in human disease. Serum uric acid is determined by production and the net balance of reabsorption or secretion by the kidney and intestine. A detailed understanding of epithelial absorption and secretion of uric acid has recently emerged, aided in particular by the results of genome-wide association studies of hyperuricemia. Novel genetic and regulatory networks with effects on uric acid homeostasis have also emerged. These developments promise to lead to a new understanding of the various diseases associated with hyperuricemia and to novel, targeted therapies for hyperuricemia.

Conformational change in transfer RNA is an early indicator of acute cellular damage.

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.

Glycosylation regulates the function and membrane localization of KCC4.

Glycosylation plays a role in regulating many biological activities, including protein folding and cell surface expression of biomolecules. However, the importance of glycosylation for KCC4 function has not previously been demonstrated. Site-directed mutagenesis was performed on the four putative extracellular N-linked glycosylation sites of KCC4 to determine the role of these sites in KCC4 half-life, cell surface expression, and transporter activity, as well as in KCC4-dependent tumor formation. We showed that triple (N312/331/344/Q) and quadruple (N312/331/344/360/Q) mutations of N-linked glycosylation sites disrupt the N-linked glycosylation of KCC4, resulting in the accumulation of KCC4, predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Further investigation indicated that mutations of the central two (N331/344/Q) N-linked glycosylation sites inhibit the membrane trafficking of KCC4. Our data suggest that the glycan moieties at the N331 and N344 sites were Endo H-resistant, complex-form structures, and that the N312 and N360 sites were Endo H-sensitive, high mannose-containing structures. Under hypotonic stress conditions, the ability to adapt to changes in intracellular chloride ion concentrations and RVD (regulatory volume decrease) activities were less efficient in cells containing the deglycosylated form of KCC4 that were not expressed at the cell surface. Deglycosylated forms of KCC4 also demonstrated decreased tumor formation and lung colonization in mouse xenografts. The difference in glycan complexity may account for the differential impact of each branch on the biological effects of KCC4. We propose that glycosylation is essential for the surface expression, stabilization, and bioactivity of KCC4.

The genetics of hyperuricaemia and gout.

Gout is a common and very painful inflammatory arthritis caused by hyperuricaemia. This review provides an update on the genetics of hyperuricaemia and gout, including findings from genome-wide association studies. Most of the genes that associated with serum uric acid levels or gout are involved in the renal urate-transport system. For example, the urate transporter genes SLC2A9, ABCG2 and SLC22A12 modulate serum uric acid levels and gout risk. The net balance between renal urate absorption and secretion is a major determinant of serum uric acid concentration and loss-of-function mutations in SLC2A9 and SLC22A12 cause hereditary hypouricaemia due to reduced urate absorption and unopposed urate secretion. However, the variance in serum uric acid explained by genetic variants is small and their clinical utility for gout risk prediction seems limited because serum uric acid levels effectively predict gout risk. Urate-associated genes and genetically determined serum uric acid levels were largely unassociated with cardiovascular-metabolic outcomes, challenging the hypothesis of a causal role of serum uric acid in the development of cardiovascular disease. Strong pharmacogenetic associations between HLA-B*5801 alleles and severe allopurinol-hypersensitivity reactions were shown in Asian and European populations. Genetic testing for HLA-B*5801 alleles could be used to predict these potentially fatal adverse effects.

K-Cl cotransporter gene expression during human and murine erythroid differentiation.

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.

Differences in the large extracellular loop between the K(+)-Cl(-) cotransporters KCC2 and KCC4.

K(+)Cl(-) cotransporters (KCCs) play fundamental physiological roles in processes such as inhibitory neurotransmission and cell volume regulation. Mammalian genomes encode four distinct KCC paralogs, which share basic transport characteristics but differ significantly in ion affinity, pharmacology, and relative sensitivity to cell volume. Studies to identify divergence in functional characteristics have thus far focused on the cytoplasmic termini. Here, we investigated sequence requirements of the large extracellular loop (LEL) for function in KCC2 and KCC4. Mutation of all four evolutionarily conserved cysteines abolished KCC2 transport activity. This behavior differs from that of its closest relative, KCC4, which is insensitive to this mutation. Chimeras supported the differences in the LEL of the two cotransporters, because swapping wild-type LEL resulted in functional KCC2 but rendered KCC4 inactive. Insertion of the quadruple cysteine substitution mutant of the KCC4 loop, which was functional in the parental isoform, abolished transport activity in KCC2. Dose-response curves of wild-type and chimeric KCCs revealed that the LEL contributes to the different sensitivity to loop diuretics; a KCC2 chimera containing the KCC4 LEL displayed an IC(50) of 396.5 mum for furosemide, which was closer to KCC4 (548.8 mum) than to KCC2 (184.4 mum). Cell surface labeling and immunocytochemistry indicated that mutations do not affect trafficking to the plasma membrane. Taken together, our results show a dramatic and unexpected difference in the sequence requirements of the LEL between the closely related KCC2 and KCC4. Furthermore, they demonstrate that evolutionarily highly conserved amino acids can have different functions within KCC members.

Genetics of gout.

PURPOSE OF REVIEW: This review provides an update on recent findings with regards to the genetics of hyperuricemia and gout, including recent data from genome-wide association studies (GWAS). RECENT FINDINGS: Five GWAS around the same time reported that genetic variants of SLC2A9/GLUT9 were associated with lower serum uric acid (SUA) levels and the effects were stronger among women (e.g. SUA level difference per copy of a minor allele, -0.46 mg/dl in women vs. -0.22 mg/dl in men). One study involving four cohorts and one meta-analysis of 14 genome-wide scans found that genetic variants of ABCG2 were associated with higher SUA concentrations and these effects were stronger among men (e.g. uric acid level difference per copy of the minor allele, 0.32 mg/dl in men vs. 0.18 mg/dl in women). Limited data indicate that these associations likely translate into those with the risk of gout. Functional determination that GLUT9 and ABCG2 can transport urate at the apical border of proximal tubules implicates them as substantial players in the renal excretion of urate. Furthermore, five novel genetic loci have been reported in the meta-analysis of 14 genome-wide scans. SUMMARY: Combined with their activities as urate transporters and their strong associations with serum uric acid concentrations, GLUT9 and ABCG2 appeared to be important modulators of uric acid levels and likely of the risk of gout. Together with a growing list of environmental risk factors, these genetic data add considerably to our understanding of the pathogenesis of hyperuricemia and gout.

Motor protein-dependent membrane trafficking of KCl cotransporter-4 is important for cancer cell invasion.

The KCl cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. This study stresses the important role of KCC4 in tumor malignant behavior. Real-time reverse transcription-PCR on samples collected by laser microdissection and immunofluorescent stainings with different KCC isoform antibodies indicate that KCC4 is abundant in metastatic cervical and ovarian cancer tissues. Insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulate KCC4 recruitment from a presumably inactive cytoplasmic pool of endoplasmic reticulum and Golgi to plasma membrane along actin cytoskeleton that is significantly inhibited by LY294002 and wortmannin. Throughout the trafficking process, KCC4 is incorporated into lipid rafts that function as a platform for the association between KCC4 and myosin Va, an actin-dependent motor protein. KCC4 and ezrin, a membrane cytoskeleton linker, colocalize at lamellipodia of migratory cancer cells. Interference with KCC activity by either an inhibitor or a dominant-negative loss-of-function mutant profoundly suppressed the IGF-I-induced membrane trafficking of KCC4 and the structural interaction between KCC4 and ezrin near the cell surface. Endogenous cancer cell invasiveness was significantly attenuated by small interfering RNA targeting KCC4, and the residual invasiveness was much less sensitive to IGF-I or EGF stimulation. In the metastatic cancer tissues, KCC4 colocalizes with IGF-I or EGF, indicating a likely in vivo stimulation of KCC4 function by growth factors. Thus, blockade of KCC4 trafficking and surface expression may provide a potential target for the prevention of IGF-I- or EGF-dependent cancer spread.

Mortality after hospitalization with mild, moderate, and severe hyponatremia.

BACKGROUND: Hyponatremia is the most common electrolyte abnormality in hospitalized individuals. METHODS: To investigate the association between serum sodium concentration and mortality, we conducted a prospective cohort study of 98,411 adults hospitalized between 2000 and 2003 at 2 teaching hospitals in Boston, Massachusetts. The main outcome measures were in-hospital, 1-year, and 5-year mortality. Multivariable logistic regression and Cox proportional hazards models were used to compare outcomes in patients with varying degrees of hyponatremia against those with normal serum sodium concentration. RESULTS: Hyponatremia (serum sodium concentration <135 mEq/L) was observed in 14.5% of patients on initial measurement. Compared with patients with normonatremia (135-144 mEq/L), those with hyponatremia were older (67.0 vs 63.1 years, P <.001) and had more comorbid conditions (mean Deyo-Charlson Index 1.9 vs 1.4, P <.001). In multivariable-adjusted models, patients with hyponatremia had an increased risk of death in hospital (odds ratio 1.47, 95% confidence interval [CI], 1.33-1.62), at 1 year (hazard ratio 1.38, 95% CI, 1.32-1.46), and at 5 years (hazard ratio 1.25, 95% CI, 1.21-1.30). The increased risk of death was evident even in those with mild hyponatremia (130-134 mEq/L; odds ratio 1.37, 95% CI, 1.23-1.52). The relationship between hyponatremia and mortality was pronounced in patients admitted with cardiovascular disease, metastatic cancer, and those admitted for procedures related to the musculoskeletal system. Resolution of hyponatremia during hospitalization attenuated the increased mortality risk conferred by hyponatremia. CONCLUSION: Hyponatremia, even when mild, is associated with increased mortality.

Slc26a9 is inhibited by the R-region of the cystic fibrosis transmembrane conductance regulator via the STAS domain.

SLC26 proteins function as anion exchangers, channels, and sensors. Previous cellular studies have shown that Slc26a3 and Slc26a6 interact with the R-region of the cystic fibrosis transmembrane conductance regulator (CFTR), (R)CFTR, via the Slc26-STAS (sulfate transporter anti-sigma) domain, resulting in mutual transport activation. We recently showed that Slc26a9 has both nCl(-)-HCO(3)(-) exchanger and Cl(-) channel function. In this study, we show that the purified STAS domain of Slc26a9 (a9STAS) binds purified (R)CFTR. When Slc26a9 and (R)CFTR fragments are co-expressed in Xenopus oocytes, both Slc26a9-mediated nCl(-)-HCO(3)(-) exchange and Cl(-) currents are almost fully inhibited. Deletion of the Slc26a9 STAS domain (a9-DeltaSTAS) virtually eliminated the Cl(-) currents with only a modest affect on nCl(-)-HCO(3)(-) exchange activity. Co-expression of a9-DeltaSTAS and the (R)CFTR fragment did not alter the residual a9-DeltaSTAS function. Replacing the Slc26a9 STAS domain with the Slc26a6 STAS domain (a6-a9-a6) does not change Slc26a9 function and is no longer inhibited by (R)CFTR. These data indicate that the Slc26a9-STAS domain, like other Slc26-STAS domains, binds CFTR in the R-region. However, unlike previously reported data, this binding interaction inhibits Slc26a9 ion transport activity. These results imply that Slc26-STAS domains may all interact with (R)CFTR but that the physiological outcome is specific to differing Slc26 proteins, allowing for dynamic and acute fine tuning of ion transport for various epithelia.

Short-term regulation of the Cl-/HCO3(-) exchanger in immortalized SHR proximal tubular epithelial cells.

The present study evaluated the activity of Cl(-)/HCO(3)(-) exchanger and the abundance of Slc26a6 in immortalized renal proximal tubular epithelial (PTE) cells from the Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) and identified the signaling pathways that regulate the activity of the transporter. The affinity for HCO(3)(-) was identical in WKY and SHR PTE cells, but V(max) values (in pH units/min) in SHR PTE cells (0.4016) were significantly higher than in WKY PTE cells (0.2304). The expression of Slc26a6 in SHR PTE cells was sevenfold that in WKY PTE cells. Dibutyryl-cAMP (db-cAMP) or forskolin, which increased endogenous cAMP, phorbol-12,13-dibutyrate (PDBu) and anisomycin, significantly (P<0.05) increased the Cl(-)/HCO(3)(-) exchanger activity in WKY and SHR PTE cells to a similar extent. The stimulatory effects of db-cAMP and forskolin were prevented by the PKA inhibitor H89, but not by chelerythrine. The stimulatory effects of PDBu were prevented by both chelerythrine and SB 203580, but not by H89 or the MEK inhibitor PD 98059. The stimulatory effect of anisomycin was prevented by SB 203580, but not by chelerythrine. Increases in phospho-p38 MAPK by anisomycin were identical in WKY and SHR PTE cells, this being sensitive to SB 203580 but not to chelerythrine. It is concluded that SHR PTE cells, which overexpress the Slc26a6 protein, are endowed with an enhanced activity of the Cl(-)/HCO(3)(-) exchanger. The Cl(-)/HCO(3)(-) exchanger is an effector protein for PKA, PKC and p38 MAPK in both WKY and SHR PTE cells.