Multicopy Crystallographic & Biophysical Analyses of the N-terminal Domain of NBCe1-A: Illumination of the Human R298S Mutation

Background: NBCe1-A membrane-embedded macromolecules cotransport sodium and bicarbonate ions across the bilayers that serve to maintain acid-base homeostasis throughout the body. Defects are linked to a number of disorders, including proximal renal tubule acidosis, mental retardation, dental defects, and cataracts. Previously, we demonstrated the N-terminal domain of NBCe1-A (Nt) is in pH-sensitive monomer-dimer equilibrium. At neutral pH, bicarbonate ions bind the Nt, stabilizing dimerization and intermolecular self-associations of dimers. Methods: We determine and analyze the X-ray crystal structure of the Nt as a dimer at 2.4-A resolution using molecular-replacement methods, and a multicopy crystallographic structure of the monomer using 5 atomic models and strict 4-fold NCS constraints in refinement procedures. We measure the pH-sensitivity of a truncated Nt mutant by light-scattering techniques, and bicarbonate, bisulfite, mutant self-association bindings by surface plasmon resonance techniques. Results: The structures reveal that R298 implicated in the disorders is part of a putative conduit that transverses the Nt. The conduit opens to the transmembrane domain (TMD) on one end and an apparent foyer entrance on the opposite end. The naturally occurring mutation R298S disrupts an electrostatic pocket within the conduit that disables substrate binding. We also report similar conducts in family member AE1 (Band 3) when exploring its crystal structure. Further, we identify by biophysical analyses on a truncated Nt that the autoregulatory domain (ARD) at the N-terminus of the Nt is responsible for self-associations. Conclusions: The Nt responds to changes in pH or bicarbonate fluctuations. In proximal tubule cells, we propose a model where the ARD is a gate for the foyer. When self-associated, the foyer entrance is accessible, allowing substrate entry into the conduit. During acid loads, the gates close entry into the foyer, preventing bicarbonate from leaving the cell. The R298S defect similarly prevents bicarbonate ions from being transported to blood, giving rise to metabolic acidosis that results from the renal tubule acidosis

Trimethylamine N-Oxide: The Good, the Bad and the Unknown

Trimethylamine N-oxide (TMAO) is a small colorless amine oxide generated from choline, betaine, and carnitine by gut microbial metabolism. It accumulates in the tissue of marine animals in high concentrations and protects against the protein-destabilizing effects of urea. Plasma level of TMAO is determined by a number of factors including diet, gut microbial flora and liver flavin monooxygenase activity. In humans, a positive correlation between elevated plasma levels of TMAO and an increased risk for major adverse cardiovascular events and death is reported. The atherogenic effect of TMAO is attributed to alterations in cholesterol and bile acid metabolism, activation of inflammatory pathways and promotion foam cell formation. TMAO levels increase with decreasing levels of kidney function and is associated with mortality in patients with chronic kidney disease. A number of therapeutic strategies are being explored to reduce TMAO levels, including use of oral broad spectrum antibiotics, promoting the growth of bacteria that utilize TMAO as substrate and the development of target-specific molecules with varying level of success. Despite the accumulating evidence, it is questioned whether TMAO is the mediator of a bystander in the disease process. Thus, it is important to undertake studies examining the cellular signaling in physiology and pathological states in order to establish the role of TMAO in health and disease in humans

Gut Microbiota and Cardiovascular Uremic Toxicities

Cardiovascular disease (CVD) remains a major cause of high morbidity and mortality in patients with chronic kidney disease (CKD). Numerous CVD risk factors in CKD patients have been described, but these do not fully explain the high pervasiveness of CVD or increased mortality rates in CKD patients. In CKD the loss of urinary excretory function results in the retention of various substances referred to as “uremic retention solutes”. Many of these molecules have been found to exert toxicity on virtually all organ systems of the human body, leading to the clinical syndrome of uremia. In recent years, an increasing body of evidence has been accumulated that suggests that uremic toxins may contribute to an increased cardiovascular disease (CVD) burden associated with CKD. This review examined the evidence from several clinical and experimental studies showing an association between uremic toxins and CVD. Special emphasis is addressed on emerging data linking gut microbiota with the production of uremic toxins and the development of CKD and CVD. The biological toxicity of some uremic toxins on the myocardium and the vasculature and their possible contribution to cardiovascular injury in uremia are also discussed. Finally, various therapeutic interventions that have been applied to effectively reduce uremic toxins in patients with CKD, including dietary modifications, use of prebiotics and/or probiotics, an oral intestinal sorbent that adsorbs uremic toxins and precursors, and innovative dialysis therapies targeting the protein-bound uremic toxins are also highlighted. Future studies are needed to determine whether these novel therapies to reduce or remove uremic toxins will reduce CVD and related cardiovascular events in the long-term in patients with chronic renal failure

Hypertonicity: Pathophysiologic Concept and Experimental Studies

Disturbances in tonicity (effective osmolarity) are the major clinical disorders affecting cell volume. Cell shrinking secondary to hypertonicity causes severe clinical manifestations and even death. Quantitative management of hypertonic disorders is based on formulas computing the volume of hypotonic fluids required to correct a given level of hypertonicity. These formulas have limitations. The major limitation of the predictive formulas is that they represent closed system calculations and have been tested in anuric animals. Consequently, the formulas do not account for ongoing fluid losses during development or treatment of the hypertonic disorders. In addition, early comparisons of serum osmolality changes predicted by these formulas and observed in animals infused with hypertonic solutions clearly demonstrated that hypertonicity creates new intracellular solutes causing rises in serum osmolality higher than those predicted by the formulas. The mechanisms and types of intracellular solutes generated by hypertonicity and the effects of the solutes have been studied extensively in recent times. The solutes accumulated intracellularly in hypertonic states have potentially major adverse effects on the outcomes of treatment of these states. When hypertonicity was produced by the infusion of hypertonic sodium chloride solutions, the predicted and observed changes in serum sodium concentration were equal. This finding justifies the use of the predictive formulas in the management of hypernatremic states

Management of severe hyponatremia: Infusion of hypertonic saline and desmopressin or infusion of vasopressin inhibitors?

Rapid correction of severe hyponatremia carries the risk of osmotic demyelination. Two recently introduced methods of correction of hyponatremia have diametrically opposite effects on aquaresis. Inhibitors of vasopressin V2 receptor (vaptans) lead to the production of dilute urine, whereas infusion of desmopressin causes urinary concentration. Identification of the category of hyponatremia that will benefit from one or the other treatment is critical. In general, vaptans are effective in hyponatremias presenting with concentrated urine and, with the exception of hypovolemic hyponatremia, can be used as their primary treatment. Desmopressin is effective in hyponatremias presenting with dilute urine or developing urinary dilution after saline infusion. In this setting, desmopressin infusion helps prevent overcorrection of the hyponatremia. Monitoring of the changes in serum sodium concentration as a guide to treatment changes is imperative regardless of the initial treatment of severe hyponatremia

Principles of Management of Severe Hyponatremia

Hyponatremia represents a serious health hazard.1 Hospitalized patients,2 nursing home residents,3 women,4,5 and children6 exhibit high frequency and/or severity of hyponatremia. Hyponatremia developing during the course of other morbid conditions increases their severity.7–10 Estimates of direct costs for treating hyponatremia in the United States ranged between $1.61 and$3.6 billion.11 Clinical manifestations of hyponatremia are universal12,13 and range from subtle (disturbances of balance, problems in cognition detected only during specific testing) to life-threatening manifestations of increased intracranial pressure with life-threatening hypoxia14–16 and noncardiac pulmonary edema.17 Although the treating physicians must make an accurate diagnosis based on well-established and described clinical criteria,1 treatment is also guided by the severity of these manifestations. The magnitude and rate of increase in serum sodium concentration ([Na]) during treatment are critical. Overcorrection of chronic hyponatremia may lead to osmotic myelinolysis,18–21 whereas undercorrection may fail to prevent life-threatening manifestations.1,2

Higher plasma CXCL12 levels predict incident myocardial infarction and death in chronic kidney disease: findings from the Chronic Renal Insufficiency Cohort study

Aims Genome-wide association studies revealed an association between a locus at 10q11, downstream from CXCL12, and myocardial infarction (MI). However, the relationship among plasma CXCL12, cardiovascular disease (CVD) risk factors, incident MI, and death is unknown. Methods and Results We analysed study-entry plasma CXCL12 levels in 3687 participants of the Chronic Renal Insufficiency Cohort (CRIC) Study, a prospective study of cardiovascular and kidney outcomes in chronic kidney disease (CKD) patients. Mean follow-up was 6 years for incident MI or death. Plasma CXCL12 levels were positively associated with several cardiovascular risk factors (age, hypertension, diabetes, hypercholesterolaemia), lower estimated glomerular filtration rate (eGFR), and higher inflammatory cytokine levels (P \u3c 0.05). In fully adjusted models, higher study-entry CXCL12 was associated with increased odds of prevalent CVD (OR 1.23; 95% confidence interval 1.14, 1.33, P \u3c 0.001) for one standard deviation (SD) increase in CXCL12. Similarly, one SD higher CXCL12 increased the hazard of incident MI (1.26; 1.09,1.45, P \u3c 0.001), death (1.20; 1.09,1.33, P \u3c 0.001), and combined MI/death (1.23; 1.13–1.34, P \u3c 0.001) adjusting for demographic factors, known CVD risk factors, and inflammatory markers and remained significant for MI (1.19; 1.03,1.39, P = 0.01) and the combined MI/death (1.13; 1.03,1.24, P = 0.01) after further controlling for eGFR and urinary albumin:creatinine ratio. Conclusions In CKD, higher plasma CXCL12 was associated with CVD risk factors and prevalent CVD as well as the hazard of incident MI and death. Further studies are required to establish if plasma CXCL12 reflect causal actions at the vessel wall and is a tool for genomic and therapeutic trials

Alkali Therapy in Lactic Acidosis

This report attempts to frame the debate about clinical administration of sodium bicarbonate in the setting of lactic acidosis in terms of simple questions. Specifically, we address why we develop lactic acidosis in some circumstances, how acute lactic acidosis impairs cardiovascular function and why sodium bicarbonate may have deleterious effects which limit its utility. We also attempt to explore treatment alternatives to sodium bicarbonate

Interleukin-6 Is a Risk Factor for Atrial Fibrillation in Chronic Kidney Disease: Findings from the CRIC Study.

Atrial fibrillation (AF) is the most common sustained arrhythmia in patients with chronic kidney disease (CKD). In this study, we examined the association between inflammation and AF in 3,762 adults with CKD, enrolled in the Chronic Renal Insufficiency Cohort (CRIC) study. AF was determined at baseline by self-report and electrocardiogram (ECG). Plasma concentrations of interleukin(IL)-1, IL-1 Receptor antagonist, IL-6, tumor necrosis factor (TNF)-α, transforming growth factor-β, high sensitivity C-Reactive protein, and fibrinogen, measured at baseline. At baseline, 642 subjects had history of AF, but only 44 had AF in ECG recording. During a mean follow-up of 3.7 years, 108 subjects developed new-onset AF. There was no significant association between inflammatory biomarkers and past history of AF. After adjustment for demographic characteristics, comorbid conditions, laboratory values, echocardiographic variables, and medication use, plasma IL-6 level was significantly associated with presence of AF at baseline (Odds ratio [OR], 1.61; 95% confidence interval [CI], 1.21 to 2.14; P = 0.001) and new-onset AF (OR, 1.25; 95% CI, 1.02 to 1.53; P = 0.03). To summarize, plasma IL-6 level is an independent and consistent predictor of AF in patients with CKD

Hypoxia Inducible Factor 1: A Urinary Biomarker of Kidney Disease.

Identifying noninvasive biomarkers of kidney disease is valuable for diagnostic and therapeutic purposes. Hypoxia inducible factor 1 (HIF-1) expression is known to be elevated in the kidneys in several renal disease pathologies. We hypothesized that the urinary HIF-1a mRNA level may be a suitable biomarker for expression of this protein in chronic kidney disease (CKD). We compared HIF-1a mRNA levels from urine pellets of CKD and healthy subjects. To ensure that urinary HIF-1a mRNA is of kidney origin, we examined colocalization of HIF-1a mRNA with two kidney specific markers in urine cells. We found that HIF-1a mRNA is readily quantifiable in urine pellets and its expression was significantly higher in CKD patients compared with healthy adults. We also showed that the urinary HIF-1a mRNA comes primarily from cells of renal origin. Our data suggest that urinary HIF-1a mRNA is a potential biomarker in CKD and can be noninvasively assessed in patients