Case report: disease mechanisms and medical management of calcium nephrolithiasis in rheumatologic diseases.

BACKGROUND: Nephrolithiasis as a feature of rheumatologic diseases is under recognized. Understanding presenting features, diagnostic testing is crucial to proper management. CASE PRESENTATION: A 32 year old woman with a history of recurrent complicated nephrolithiasis presented to a rheumatologist for a several month history of fatigue, dry eyes, dry mouth, arthralgias. She had a positive double-stranded DNA, positive SSA and SSB antibodies. She was diagnosed with Systemic Lupus erythematosus (SLE) and Sjogren's syndrome and was started on mycophenalate mofetil. Of relevance was a visit to her local emergency room 4 years earlier with profound weakness with unexplained marked hypokalemia and a non-anion gap metabolic acidosis. Approximately one year after that episode she developed flank pain and nephrocalcinosis. She had multiple issues over the ensuing years with stones and infections on both sides. Interventions included extracorporeal shockwave lithotripsy as well as open lithotomy and eventual auto-transplantation of left kidney for recurrent ureteric stenosis. 24 h stone profile revealed marked hypocitraturia, normal urine calcium, normal urine oxalate and uric acid. She was treated with potassium citrate. Mycophenolate was eventually stopped due to recurrent urinary tract infections and she was started on Belimumab. Because of recurrent SLE flares, treatment was changed to Rituximab (every 6 months) with clinical and serologic improvement. Her kidney stone frequency gradually improved and no further interventions needed although she continued to require citrate repletion for hypocitraturia. CONCLUSIONS: Nephrolithiasis can be a prominent and even presenting feature in Sjogrens syndrome as well as other rheumatologic diseases. Prompt recognition and understanding disease mechanisms is important for best therapeutic interventions for kidney stone prevention as well as treatment of underlying bone mineral disease.

Loss of the Na+/H+ Exchange Regulatory Factor 1 Increases Susceptibility to Cisplatin-Induced Acute Kidney Injury.

Na+/H+ exchange regulatory cofactor (NHERF)-1, a scaffolding protein, anchors multiple membrane proteins in renal proximal tubules. Cultured proximal tubule cells deficient in Nherf1 and proximal tubules from Nherf1-deficient mice exhibit aberrant trafficking. Nherf1-deficient cells also exhibit an altered transcription pattern and worse survival. These observations suggest that NHERF1 loss increases susceptibility to acute kidney injury (AKI). Male and female wild-type C57BL/6J and Nherf1 knockout mice were treated with saline or cisplatin (20 mg/kg dose i.p.) to induce AKI and were euthanized after 72 hours. Blood and urine were collected for assessments of blood urea nitrogen and neutrophil gelatinase-associated lipocalin, respectively. Kidneys were harvested for histology (hematoxylin and eosin, periodic acid-Schiff) and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, Kim1 mRNA assessment, and Western blot analysis for cleaved caspase 3. Cisplatin treatment was associated with significantly greater severity of AKI in knockout compared with wild-type mice, as demonstrated by semiquantitative injury score (2.8 versus 1.89, P < 0.001), blood urea nitrogen (151.8 ± 17.2 mg/dL versus 97.8 ± 10.1 mg/dL, P < 0.05), and neutrophil gelatinase-associated lipocalin urine protein (55.6 ± 21.3 µg/mL versus 2.7 ± 0.53 µg/mL, P < 0.05). Apoptosis markers were significantly increased in cisplatin-treated Nherf1 knockout and wild-type mice compared to respective controls. These data suggest that NHERF1 loss increases susceptibility to AKI.

A Novel Pediatric Multisystem Inflammatory Syndrome During the COVID-19 Pandemic.

In late March and early April, New York City was an epicenter of the COVID-19 pandemic. Citizens were ordered to stay at home to flatten the curve. The adult population was affected with a severe respiratory illness as well as acute kidney injury, cardiomyopathy, arrhythmia, and thromboembolism. Although children were not affected in the same manner, weeks after the peak, reports from other countries emerged about cases of pediatric patients presenting with a novel inflammatory syndrome. We present 4 patients along with their emergency department course, so providers will have a better understanding of the identification and workup of these patients. Currently, it is unclear when this inflammatory syndrome develops in respect to a COVID-19 infection. The clinical features of this syndrome seem to overlap between Kawasaki disease, toxic shock syndrome, and myocarditis. All patients presenting to our emergency department had fever, variable rash, abdominal pain, vomiting, and/or diarrhea. Patients remained persistently tachycardic and febrile despite being given proper doses of antipyretics. Severity of presentations varied among the 4 cases. All 4 patients were found to have antibodies to COVID-19. All patients required admission, but 2 required the pediatric intensive care unit for cardiac and/or respiratory support or closer monitoring. Upon follow-up on our patients, it seems that most patients are recovering with treatment, and overall, there is a low reported mortality rate.

Current knowledge on the acute regulation of steroidogenesis.

How rapid induction of steroid hormone biosynthesis occurs in response to trophic hormone stimulation of steroidogenic cells has been a subject of intensive investigation for approximately six decades. A key observation made very early was that acute regulation of steroid biosynthesis required swift and timely synthesis of a new protein whose role appeared to be involved in the delivery of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane where the process of steroidogenesis begins. It was quickly learned that this transfer of cholesterol to the inner mitochondrial membrane was the regulated and rate-limiting step in steroidogenesis. Following this observation, the quest for this putative regulator protein(s) began in earnest in the late 1950s. This review provides a history of this quest, the candidate proteins that arose over the years and facts surrounding their rise or decline. Only two have persisted-translocator protein (TSPO) and the steroidogenic acute regulatory protein (StAR). We present a detailed summary of the work that has been published for each of these two proteins, the specific data that has appeared in support of their role in cholesterol transport and steroidogenesis, and the ensuing observations that have arisen in recent years that have refuted the role of TSPO in this process. We believe that the only viable candidate that has been shown to be indispensable is the StAR protein. Lastly, we provide our view on what may be the most important questions concerning the acute regulation of steroidogenesis that need to be asked in future.

Nuclear receptors and nonalcoholic fatty liver disease.

Nuclear receptors are transcription factors which sense changing environmental or hormonal signals and effect transcriptional changes to regulate core life functions including growth, development, and reproduction. To support this function, following ligand-activation by xenobiotics, members of subfamily 1 nuclear receptors (NR1s) may heterodimerize with the retinoid X receptor (RXR) to regulate transcription of genes involved in energy and xenobiotic metabolism and inflammation. Several of these receptors including the peroxisome proliferator-activated receptors (PPARs), the pregnane and xenobiotic receptor (PXR), the constitutive androstane receptor (CAR), the liver X receptor (LXR) and the farnesoid X receptor (FXR) are key regulators of the gut:liver:adipose axis and serve to coordinate metabolic responses across organ systems between the fed and fasting states. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and may progress to cirrhosis and even hepatocellular carcinoma. NAFLD is associated with inappropriate nuclear receptor function and perturbations along the gut:liver:adipose axis including obesity, increased intestinal permeability with systemic inflammation, abnormal hepatic lipid metabolism, and insulin resistance. Environmental chemicals may compound the problem by directly interacting with nuclear receptors leading to metabolic confusion and the inability to differentiate fed from fasting conditions. This review focuses on the impact of nuclear receptors in the pathogenesis and treatment of NAFLD. Clinical trials including PIVENS and FLINT demonstrate that nuclear receptor targeted therapies may lead to the paradoxical dissociation of steatosis, inflammation, fibrosis, insulin resistance, dyslipidemia and obesity. Novel strategies currently under development (including tissue-specific ligands and dual receptor agonists) may be required to separate the beneficial effects of nuclear receptor activation from unwanted metabolic side effects. The impact of nuclear receptor crosstalk in NAFLD is likely to be profound, but requires further elucidation. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

Polychlorinated biphenyls disrupt hepatic epidermal growth factor receptor signaling.

1. Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that disrupt hepatic xenobiotic and intermediary metabolism, leading to metabolic syndrome and nonalcoholic steatohepatitis (NASH). 2. Since phenobarbital indirectly activates Constitutive Androstane Receptor (CAR) by antagonizing growth factor binding to the epidermal growth factor receptor (EGFR), we hypothesized that PCBs may also diminish EGFR signaling. 3. The effects of the PCB mixture Aroclor 1260 on the protein phosphorylation cascade triggered by EGFR activation were determined in murine (in vitro and in vivo) and human models (in vitro). EGFR tyrosine residue phosphorylation was decreased by PCBs in all models tested. 4. The IC50 values for Aroclor 1260 concentrations that decreased Y1173 phosphorylation of EGFR were similar in murine AML-12 and human HepG2 cells (∼2-4 µg/mL). Both dioxin and non-dioxin-like PCB congeners decreased EGFR phosphorylation in cell culture. 5. PCB treatment reduced phosphorylation of downstream EGFR effectors including Akt and mTOR, as well as other phosphoprotein targets including STAT3 and c-RAF in vivo. 6. PCBs diminish EGFR signaling in human and murine hepatocyte models and may dysregulate critical phosphoprotein regulators of energy metabolism and nutrition, providing a new mechanism of action in environmental diseases.

Selective VIP Receptor Agonists Facilitate Immune Transformation for Dopaminergic Neuroprotection in MPTP-Intoxicated Mice.

Vasoactive intestinal peptide (VIP) mediates a broad range of biological responses by activating two related receptors, VIP receptor 1 and 2 (VIPR1 and VIPR2). Although the use of native VIP facilitates neuroprotection, clinical application of the hormone is limited due to VIP's rapid metabolism and inability to distinguish between VIPR1 and VIPR2 receptors. In addition, activation of both receptors by therapeutics may increase adverse secondary toxicities. Therefore, we developed metabolically stable and receptor-selective agonists for VIPR1 and VIPR2 to improve pharmacokinetic and pharmacodynamic therapeutic end points. Selective agonists were investigated for their abilities to protect mice against MPTP-induced neurodegeneration used to model Parkinson's disease (PD). Survival of tyrosine hydroxylase neurons in the substantia nigra was determined by stereological tests after MPTP intoxication in mice pretreated with either VIPR1 or VIPR2 agonist or after adoptive transfer of splenic cell populations from agonist-treated mice administered to MPTP-intoxicated animals. Treatment with VIPR2 agonist or splenocytes from agonist-treated mice resulted in increased neuronal sparing. Immunohistochemical tests showed that agonist-treated mice displayed reductions in microglial responses, with the most pronounced effects in VIPR2 agonist-treated, MPTP-intoxicated mice. In parallel studies, we observed reductions in proinflammatory cytokine release that included IL-17A, IL-6, and IFN-γ and increases in GM-CSF transcripts in CD4(+) T cells recovered from VIPR2 agonist-treated animals. Moreover, a phenotypic shift of effector to regulatory T cells was observed. These results support the use of VIPR2-selective agonists as neuroprotective agents for PD treatment. SIGNIFICANCE STATEMENT: Vasoactive intestinal peptide receptor 2 can elicit immune transformation in a model of Parkinson's disease (PD). Such immunomodulatory capabilities can lead to neuroprotection by attenuating microglial activation and by slowing degradation of neuronal cell bodies and termini in MPTP-intoxicated mice. The protective mechanism arises from altering a Th1/Th2 immune cytokine response into an anti-inflammatory and neuronal sparing profile. These results are directly applicable for the development of novel PD therapies.

Identification of an RNA-binding protein that is phosphorylated by PTH and potentially mediates PTH-induced destabilization of Npt2a mRNA.

Parathyroid hormone (PTH) is a key regulator of the expression and function of the type IIa sodium-phosphate cotransporter (Npt2a), the protein responsible for regulated renal phosphate reabsorption. We previously showed that PTH induces rapid decay of Npt2a mRNA through posttranscriptional mechanisms. We hypothesized that PTH-induced changes in RNA-binding protein (RBP) activity mediate the degradation of Npt2a mRNA. To address this aim, we treated opossum kidney (OK) cells, a PTH-sensitive proximal tubule cell culture model, with 100 nM PTH for 30 min and 2 h, followed by mass spectrometry characterization of the PTH-stimulated phosphoproteome. We identified 1,182 proteins differentially phosphorylated in response to PTH, including 68 RBPs. Preliminary analysis identified a phospho-RBP, hnRNPK-homology-type-splicing regulatory protein (KSRP), with predicted binding sites for the 3'-untranslated region (UTR) of Npt2a mRNA. Western blot analysis confirmed expression of KSRP in OK cells and showed PTH-dependent translocation to the nucleus. Immunoprecipitation of KSRP from control and PTH-treated cells followed by RNA isolation and RT-quantitative PCR analysis identified Npt2a mRNA from both control and PTH-treated KSRP pulldowns. Knockdown of KSRP followed by PTH treatment showed that KSRP is required for mediating PTH-stimulated reduction in sodium/hydrogen exchanger 3 mRNA, but not Npt2a mRNA. We conclude that 1) PTH is a major regulator of both transcription and translation, and 2) KSRP binds Npt2a mRNA but its role in PTH regulation of Npt2a mRNA is not clear.

Dehydroepiandrosterone Activation of G-protein-coupled Estrogen Receptor Rapidly Stimulates MicroRNA-21 Transcription in Human Hepatocellular Carcinoma Cells.

Little is known about the regulation of the oncomiR miR-21 in liver. Dehydroepiandrosterone (DHEA) regulates gene expression as a ligand for a G-protein-coupled receptor and as a precursor for steroids that activate nuclear receptor signaling. We report that 10 nm DHEA increases primary miR-21 (pri-miR-21) transcription and mature miR-21 expression in HepG2 cells in a biphasic manner with an initial peak at 1 h followed by a second, sustained response from 3-12 h. DHEA also increased miR-21 in primary human hepatocytes and Hep3B cells. siRNA, antibody, and inhibitor studies suggest that the rapid DHEA-mediated increase in miR-21 involves a G-protein-coupled estrogen receptor (GPER/GPR30), estrogen receptor α-36 (ERα36), epidermal growth factor receptor-dependent, pertussis toxin-sensitive pathway requiring activation of c-Src, ERK1/2, and PI3K. GPER antagonist G-15 attenuated DHEA- and BSA-conjugated DHEA-stimulated pri-miR-21 transcription. Like DHEA, GPER agonists G-1 and fulvestrant increased pri-miR-21 in a GPER- and ERα36-dependent manner. DHEA, like G-1, increased GPER and ERα36 mRNA and protein levels. DHEA increased ERK1/2 and c-Src phosphorylation in a GPER-responsive manner. DHEA increased c-Jun, but not c-Fos, protein expression after 2 h. DHEA increased androgen receptor, c-Fos, and c-Jun recruitment to the miR-21 promoter. These results suggest that physiological concentrations of DHEA activate a GPER intracellular signaling cascade that increases pri-miR-21 transcription mediated at least in part by AP-1 and androgen receptor miR-21 promoter interaction.

Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1.

BACKGROUND/AIMS: Phosphate homeostasis is controlled by the renal reabsorption of Pi by the type IIa sodium phosphate cotransporter, Npt2a, which is localized in the proximal tubule brush border membrane. Regulation of Npt2a expression is a key control point to maintain phosphate homeostasis with most studies focused on regulating protein levels in the brush border membrane. Molecular mechanisms that control Npt2a mRNA, however, remain to be defined. We have reported that Npt2a mRNA and protein levels correlate directly with the expression of the Na+/H+ exchanger regulatory factor 1 (NHERF-1) using opossum kidney (OK) cells and the NHERF-1-deficient OK-H cells. The goal of this study was to determine whether NHERF-1 contributes to transcriptional and/or post-transcriptional mechanisms controlling Npt2a mRNA levels. METHODS: Npt2a mRNA half-life was compared between OK and NHERF-1 deficient OK-H cell lines. oNpt2a promoter-reporter gene assays and electrophoretic mobility shift assays (EMSA) were used identify a NHERF-1 responsive region within the oNpt2a proximal promoter. RESULTS: Npt2a mRNA half-life is the same in OK and OK-H cells. The NHERF-1 responsive region lies within the proximal promoter in a region that contains a highly conserved CAATT box and G-rich element. Specific protein-DNA complex formation with the CAATT element is altered by the absence of NHERF-1 (OK v OK-H EMSA) although NHERF-1 does not directly contribute to complex formation. CONCLUSION: NHERF-1 helps maintain steady-state Npt2a mRNA levels in OK cells through indirect mechanisms that help promote protein-DNA interactions at the Npt2a proximal promoter.

Effect of intravenous iron on outcomes of acute kidney injury.

BACKGROUND: Little is known about benefit versus risk in treating iron deficiency anemia with intravenous (IV) iron in patients with acute kidney injury (AKI). Concerns about adverse outcomes may dissuade use and could contribute to greater use of red blood cell (RBC) transfusion. STUDY DESIGN AND METHODS: We performed a retrospective case-control study of patients with AKI who received IV iron (cases) compared to those with AKI without IV iron (controls). RESULTS: We identified 67 cases and 67 controls matched for age, stage of chronic kidney disease, and severity of anemia (hemoglobin [Hb], 7.7 ± 0.1 mg/dL vs. 7.5 ± 0.1 mg/dL; p = 0.47). Cases tended to be sicker with longer length of stay (27 + 4 days vs. 15 + 1.3 days; p = 0.003) and more intensive care unit days (13 + 2 days vs. 5 + 1 days; p = 0.003), more often with diagnosis of sepsis and greater number of antibiotics used (2.7 ± 0.3 vs. 1.8 ± 0.2; p = 0.02). Sepsis and AKI preceded use of IV iron. Despite greater illness severity, there was no difference in dialysis (38.8% vs. 34.3%; p = 0.59), mortality (24% vs. 21%; p = 0.679), or severity and/or recovery of AKI. Discharge Hb was similar (9.0 ± 0.1 mg/dL vs. 9.1 ± 0.1 mg/dL; p = 0.47). IV iron was used later in the stay and hence the cases also had more RBC transfusions. CONCLUSIONS: We were unable to find any adverse consequences of use of IV iron when used to treat anemia in patients with AKI in regard to recovery of AKI or mortality even in patients with a diagnosis of sepsis. Consideration of preemptive use of IV iron in AKI with severe anemia is warranted to determine if this would reduce RBC transfusion.

Structural determinants for the ouabain-stimulated increase in Na-K ATPase activity.

Recent studies suggest that at low concentrations, ouabain increases Na-K ATPase and NHE1 activity and activates the Src signaling cascade in proximal tubule cells. Our laboratory demonstrated that low concentrations of ouabain increase blood pressure in rats. We hypothesize that ouabain-induced increase in blood pressure and Na-K ATPase activity requires NHE1 activity and association. To test this hypothesis we treated rats with ouabain (1µgkg body wt(-1)day(-1)) for 9days in the presence or absence of the NHE1 inhibitor, zoniporide. Ouabain stimulated a significant increase in blood pressure which was prevented by zoniporide. Using NHE1-expressing Human Kidney cells 2 (HK2), 8 (HK8) and 11 (HK11) and Mouse Kidney cells from Wild type (WT) and NHE1 knock-out mice (SWE) cell lines, we show that ouabain stimulated Na-K ATPase activity and surface expression in a Src-dependent manner in NHE1-expressing cells but not in NHE1-deplete cells. Zoniporide prevented ouabain-induced stimulation of (86)Rb uptake in the NHE1-expressing cells. FRET and TIRF microscopy showed that ouabain increased association between GFP-NHE1 and mCherry-Na-K ATPase transfected into NHE1-deficient SWE cells. Mutational analysis demonstrated that the caveolin binding motif (CBM) of Na-K ATPase α1 is required for translocation of both Na-K ATPase α1 and NHE1 to the basolateral membrane. Mutations in activity or scaffold domains of NHE1 resulted in loss of ouabain-mediated regulation of Na-K ATPase. These results support that NHE1 is required for the ouabain-induced increase in blood pressure, and that the caveolin binding motif of Na-K ATPase α1 as well as the activity and scaffolding domains of NHE1 are required for their functional association.

Role of Na+/H+ exchanger regulatory factor 1 in forward trafficking of the type IIa Na+-Pi cotransporter.

Na+/H+ exchanger regulatory factor (NHERF1) plays a critical role in the renal transport of phosphate by binding to Na+-Pi cotransporter (NpT2a) in the proximal tubule. While the association between NpT2a and NHERF1 in the apical membrane is known, the role of NHERF1 to regulate the trafficking of NpT2a has not been studied. To address this question, we performed cell fractionation by sucrose gradient centrifugation in opossum kidney (OK) cells placed in low-Pi medium to stimulate forward trafficking of NpT2a. Immunoblot analysis demonstrated expression of NpT2a and NHERF1 in the endoplasmic reticulum (ER)/Golgi. Coimmunoprecipitation demonstrated a NpT2a-NHERF1 interaction in the ER/Golgi. Low-Pi medium for 4 and 8 h triggered a decrease in NHERF1 in the plasma membrane with a corresponding increase in the ER/Golgi. Time-lapse total internal reflection fluorescence imaging of OK cells placed in low-Pi medium, paired with particle tracking and mean square displacement analysis, indicated active directed movement of NHERF1 at early and late time points, whereas NpT2a showed active movement only at later times. Silence of NHERF1 in OK cells expressing green fluorescent protein (GFP)-NpT2a resulted in an intracellular accumulation of GFP-NpT2a. Transfection with GFP-labeled COOH-terminal (TRL) PDZ-binding motif deleted or wild-type NpT2a in OK cells followed by cell fractionation and immunoprecipitation confirmed that the interaction between NpT2a and NHERF1 was dependent on the TRL motif of NpT2a. We conclude that appropriate trafficking of NpT2a to the plasma membrane is dependent on the initial association between NpT2a and NHERF1 through the COOH-terminal TRL motif of NpT2a in the ER/Golgi and requires redistribution of NHERF1 to the ER/Golgi.

Vacuolar ATPase driven potassium transport in highly metastatic breast cancer cells.

Breast cancer is the second leading cause of death in women and thus has received a great deal of attention by researchers. Recent studies suggested decreased occurrence of cancer in patients treated with cardiac glycosides (CGs) for heart conditions. Because CGs induce their cellular effects via the Na(+), K(+) ATPase (Na-K), we treated four breast cancer cell lines (MCF-7, T47D, MDA-MB453, and MDA-MB231) and a non-cancerous breast ductal epithelial cell line (MCF-10A) with ouabain, a well-characterized CG, and measured cell proliferation by measuring bromodeoxyuridine incorporation. Ouabain (1µM) decreased cell proliferation in all cell lines studied except MDA-MB453 cells. Western blot of Na-K α and ß subunits showed α1, α3, and ß1 expression in all cell lines except MDA-MB453 cells where Na-K protein and mRNA were absent. Potassium uptake, measured as rubidium ((86)Rb) flux, and intracellular potassium were both significantly higher in MDA-MB453 cells compared to MCF-10A cells. RT-qPCR suggested a 7 fold increase in voltage-gated potassium channel (KCNQ2) expression in MDA-MB453 cells compared to MCF-10A cells. Inhibition of KCNQ2 prevented cell growth and (86)Rb uptake in MDA-MB453 cells but not in MCF-10A cells. All cancer cells had significantly higher vacuolar H-ATPase (V-ATPase) activity than MCF-10A cells. Inhibition of V-ATPase decreased (86)Rb uptake and intracellular potassium in MDA-MB453 cells but not in MCF-10A cells. The findings point to the absence of Na-K, high hERG and KCNQ2 expression, elevated V-ATPase activity and sensitivity to V-ATPase inhibitors in MDA-MB453. We conclude that cancer cells exhibit fundamentally different metabolic pathways for maintenance of intracellular ion homeostasis.

Aldosterone regulates Na(+), K(+) ATPase activity in human renal proximal tubule cells through mineralocorticoid receptor.

The mechanisms by which aldosterone increases Na(+), K(+) ATPase and sodium channel activity in cortical collecting duct and distal nephron have been extensively studied. Recent investigations demonstrate that aldosterone increases Na-H exchanger-3 (NHE-3) activity, bicarbonate transport, and H(+) ATPase in proximal tubules. However, the role of aldosterone in regulation of Na(+), K(+) ATPase in proximal tubules is unknown. We hypothesize that aldosterone increases Na(+), K(+) ATPase activity in proximal tubules through activation of the mineralocorticoid receptor (MR). Immunohistochemistry of kidney sections from human, rat, and mouse kidneys revealed that the MR is expressed in the cytosol of tubules staining positively for Lotus tetragonolobus agglutinin and type IIa sodium-phosphate cotransporter (NpT2a), confirming proximal tubule localization. Adrenalectomy in Sprague-Dawley rats decreased expression of MR, ENaC α, Na(+), K(+) ATPase α1, and NHE-1 in all tubules, while supplementation with aldosterone restored expression of above proteins. In human kidney proximal tubule (HKC11) cells, treatment with aldosterone resulted in translocation of MR to the nucleus and phosphorylation of SGK-1. Treatment with aldosterone also increased Na(+), K(+) ATPase-mediated (86)Rb uptake and expression of Na(+), K(+) ATPase α1 subunits in HKC11 cells. The effects of aldosterone on Na(+), K(+) ATPase-mediated (86)Rb uptake were prevented by spironolactone, a competitive inhibitor of aldosterone for the MR, and partially by Mifepristone, a glucocorticoid receptor (GR) inhibitor. These results suggest that aldosterone regulates Na(+), K(+) ATPase in renal proximal tubule cells through an MR-dependent mechanism.

Parathyroid hormone (PTH) decreases sodium-phosphate cotransporter type IIa (NpT2a) mRNA stability.

The acute inhibitory effects of parathyroid hormone (PTH) on proximal tubule Na(+)-K(+)-ATPase (Na-K) and sodium-dependent phosphate (NaPi) transport have been extensively studied, while little is known about the chronic effects of PTH. Patients with primary hyperparathyroidism, a condition characterized by chronic elevations in PTH, exhibit persistent hypophosphatemia but not significant evidence of salt wasting. We postulate that chronic PTH stimulation results in differential desensitization of PTH responses. To address this hypothesis, we compared the effects of chronic PTH stimulation on Na-P(i) cotransporter (Npt2a) expression and Na-K activity and expression in Sprague Dawley rats, transgenic mice featuring parathyroid-specific cyclin D1 overexpression (PTH-D1), and proximal tubule cell culture models. We demonstrated a progressive decrease in brush-border membrane (BBM) expression of Npt2a from rats treated with PTH for 6 h or 4 days, while Na-K expression and activity in the basolateral membranes (BLM) exhibited an initial decrease followed by recovery to control levels by 4 days. Npt2a protein expression in PTH-D1 mice was decreased relative to control animals, whereas levels of Na-K, NHERF-1, and PTH receptor remained unchanged. In PTH-D1 mice, NpT2a mRNA expression was reduced by 50% relative to control mice. In opossum kidney proximal tubule cells, PTH decreased Npt2a mRNA levels. Both actinomycin D and cycloheximide treatment prevented the PTH-mediated decrease in Npt2a mRNA, suggesting that the PTH response requires transcription and translation. These findings suggest that responses to chronic PTH exposure are selectively regulated at a posttranscriptional level. The persistence of the phosphaturic response to PTH occurs through posttranscriptional mechanisms.

Structure-function of DHEA binding proteins.

Dehydroepiandrosterone (3ß-hydroxy-5-androsten-17-one, DHEA) and its sulfated metabolite DHEA-S are the most abundant circulating steroids and are precursors for active sex steroid hormones, estradiol and testosterone. DHEA has a broad range of reported effects in the central nervous system (CNS), cardiovascular system, adipose tissue, kidney, liver, and in the reproductive system. The mechanisms by which DHEA and DHEA-S initiate their biological effects are diverse. DHEA and DHEA-S may directly bind to plasma membrane (PM) receptors, including a DHEA-specific, G-protein coupled receptor (GPCR) in endothelial cells; various neuroreceptors, e.g., aminobutyric-acid-type A (GABA(A)), N-methyl-d-aspartate (NMDA) and sigma-1 (S1R) receptors (NMDAR and SIG-1R). DHEA and DHEA-S directly bind the nuclear androgen and estrogen receptors (AR, ERα, or ERß) although with significantly lower binding affinities compared to the steroid hormones, e.g., testosterone, dihydrotestosterone, and estradiol, which are the cognate ligands for AR and ERs. Thus, extra-gonadal metabolism of DHEA to the sex hormones must be considered for many of the biological benefits of DHEA. DHEA also actives GPER1 (G protein coupled estrogen receptor 1). DHEA activates constitutive androstane receptor CAR (CAR) and proliferator activated receptor (PPARα) by indirect dephosphorylation. DHEA affects voltage-gated sodium and calcium ion channels and DHEA-2 activates TRPM3 (Transient Receptor Potential Cation Channel Subfamily M Member 3). This chapter updates our previous 2018 review pertaining to the physiological, biochemical, and molecular mechanisms of DHEA and DHEA-S activity.

Dopamine regulation of Na+-K+-ATPase requires the PDZ-2 domain of sodium hydrogen regulatory factor-1 (NHERF-1) in opossum kidney cells.

Na(+)-K(+)-ATPase activity in renal proximal tubule is regulated by several hormones including parathyroid hormone (PTH) and dopamine. The current experiments explore the role of Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) in dopamine-mediated regulation of Na(+)-K(+)-ATPase. We measured dopamine regulation of ouabain-sensitive (86)Rb uptake and Na(+)-K(+)-ATPase α1 subunit phosphorylation in wild-type opossum kidney (OK) (OK-WT) cells, OKH cells (NHERF-1-deficient), and OKH cells stably transfected with full-length human NHERF-1 (NF) or NHERF-1 constructs with mutated PDZ-1 (Z1) or PDZ-2 (Z2) domains. Treatment with 1 µM dopamine decreased ouabain-sensitive (86)Rb uptake, increased phosphorylation of Na(+)-K(+)-ATPase α1-subunit, and enhanced association of NHERF-1 with D1 receptor in OK-WT cells but not in OKH cells. Transfection with wild-type, full-length, or PDZ-1 domain-mutated NHERF-1 into OKH cells restored dopamine-mediated regulation of Na(+)-K(+)-ATPase and D1-like receptor association with NHERF-1. Dopamine did not regulate Na(+)-K(+)-ATPase or increase D1-like receptor association with NHERF-1 in OKH cells transfected with mutated PDZ-2 domain. Dopamine stimulated association of PKC-ζ with NHERF-1 in OK-WT and OKH cells transfected with full-length or PDZ-1 domain-mutated NHERF-1 but not in PDZ-2 domain-mutated NHERF-1-transfected OKH cells. These results suggest that NHERF-1 mediates Na(+)-K(+)-ATPase regulation by dopamine through its PDZ-2 domain.

Topiramate-induced severe electrolyte abnormalities and hypernatremia leading to central pontine myelinolysis.

Central pontine myelinolysis (CPM) develops due to acute hypernatremia from a normal baseline serum sodium in the setting of electrolyte abnormalities induced by topiramate use. Topiramate is a commonly used medication with several indications including migraines, myoclonic jerks and seizures. It has been reported to cause renal tubular acidosis and severe electrolyte abnormalities, which in turn predispose patients to neuropathology via renal concentration defects and osmotic shifts. Our patient is a 55-year-old woman with a history of multiple sclerosis and myoclonus on topiramate for several years who presented with weakness and was found to be profoundly hypokalemic. She went on to develop changes in mental status, motor deficits and evidence of CPM on MRI during her hospitalisation. Surprisingly, the patient never had hyponatremia; however, she had an acute rise in serum sodium from a normal baseline after fluid resuscitation with normal saline for hypotension during her admission.

Inhibition of endogenous ouabain by atrial natriuretic peptide is a guanylyl cyclase independent effect.

BACKGROUND: Endogenous ouabain (EO) and atrial natriuretic peptide (ANP) are important in regulation of sodium and fluid balance. There is indirect evidence that ANP may be involved in the regulation of endogenous cardenolides. METHODS: H295R are human adrenocortical cells known to release EO. Cells were treated with ANP at physiologic concentrations or vehicle (0.1% DMSO), with or without guanylyl cyclase inhibitor 1,2,4 oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Cyclic guanosine monophosphate (cGMP), the intracellular second messenger of ANP, was measured by a chemiluminescent immunoassay and EO was measured by radioimmunoassay of C18 extracted samples. RESULTS: EO secretion is inhibited by ANP treatment, with the most prolonged inhibition (90 min vs ≤ 60 min) occurring at physiologic ANP concentrations (50 pg/mL). Inhibition of guanylyl cyclase with ODQ, also reduces EO secretion. The inhibitory effects on EO release in response to cotreatment with ANP and ODQ appeared to be additive. CONCLUSIONS: ANP inhibits basal EO secretion, and it is unlikely that this is mediated through ANP-A or ANP-B receptors (the most common natriuretic peptide receptors) or their cGMP second messenger; the underlying mechanisms involved are not revealed in the current studies. The role of ANP in the control of EO synthesis and secretion in vivo requires further investigation.