Molecular dynamics simulations of lipid-protein interactions in SLC4 proteins.

The SLC4 family of secondary bicarbonate transporters is responsible for the transport of HCO3-, CO32-, Cl-, Na+, K+, NH3, and H+, which are necessary for regulation of pH and ion homeostasis. They are widely expressed in numerous tissues throughout the body and function in different cell types with different membrane properties. Potential lipid roles in SLC4 function have been reported in experimental studies, focusing mostly on two members of the family: AE1 (Cl-/HCO3- exchanger) and NBCe1 (Na+-CO32-cotransporter). Previous computational studies of the outward-facing state of AE1 with model lipid membranes revealed enhanced protein-lipid interactions between cholesterol (CHOL) and phosphatidylinositol bisphosphate (PIP2). However, the protein-lipid interactions in other members of the family and other conformation states are still poorly understood and this precludes the detailed studies of a potential regulatory role for lipids in the SLC4 family. In this work, we performed coarse-grained and atomistic molecular dynamics simulations on three members of the SLC4 family with different transport modes: AE1, NBCe1, and NDCBE (an Na+-CO32-/Cl- exchanger), in model HEK293 membranes consisting of CHOL, PIP2, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. The recently resolved inward-facing state of AE1 was also included in the simulations. Lipid-protein contact analysis of the simulated trajectories was performed with the ProLint server, which provides a multitude of visualization tools for illustration of areas of enhanced lipid-protein contact and identification of putative lipid binding sites within the protein matrix. We observed enrichment of CHOL and PIP2 around all proteins with subtle differences in their distribution depending on the protein type and conformation state. Putative binding sites were identified for CHOL, PIP2, phosphatidylcholine, and sphingomyelin in the three studied proteins, and their potential roles in the SLC4 transport function, conformational transition, and protein dimerization are discussed.

Base (HCO3-/CO32-) Transport Properties of SLC4 Proteins: New Insights in Acid-Base Kidney Physiology.

H+ or base transporters and channels in the mammalian genome play important roles in the maintenance of numerous cellular biochemical and physiologic processes throughout the body. Among the known base transporters, those within the SLC4 and SLC26 gene families are involved in cell, transepithelial, and whole organ function. Whether the functional properties of these transporters involve HCO3-, CO32-, or HCO3-/CO32- stimulated H+ (or OH-) transport has not received widespread attention in the literature. Accordingly, "bicarbonate" is the term typically used in most textbooks without greater specificity. Moreover, clinicians and physiologists have historically focused on the blood HCO3- concentration as the base term in the Henderson-Hasselbalch equation in the analysis of clinical acid-base abnormalities, thus, bicarbonate has been assumed to be the species reabsorbed along the nephron as required to maintain the blood [HCO3-] at approximately 25 mM. However, accumulating data in the literature suggest that carbonate, rather than bicarbonate, is the species absorbed across the proximal tubule basolateral membrane, whereas in the collecting duct, bicarbonate is indeed transported. Various experimental approaches leading to this new concept are herein reviewed.

Peroxidasin-mediated bromine enrichment of basement membranes.

Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine (79Br, 81Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by ∼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.

Identification of multiple substrate binding sites in SLC4 transporters in the outward-facing conformation: Insights into the transport mechanism.

Solute carrier family 4 (SLC4) transporters mediate the transmembrane transport of HCO3-, CO32-, and Cl- necessary for pH regulation, transepithelial H+/base transport, and ion homeostasis. Substrate transport with varying stoichiometry and specificity is achieved through an exchange mechanism and/or through coupling of the uptake of anionic substrates to typically co-transported Na+. Recently solved outward-facing structures of two SLC4 members (human anion exchanger 1 [hAE1] and human electrogenic sodium bicarbonate cotransporter 1 [hNBCe1]) with different transport modes (Cl-/HCO3- exchange versus Na+-CO32- symport) revealed highly conserved three-dimensional organization of their transmembrane domains. However, the exact location of the ion binding sites and their protein-ion coordination motifs are still unclear. In the present work, we combined site identification by ligand competitive saturation mapping and extensive molecular dynamics sampling with functional mutagenesis studies which led to the identification of two substrate binding sites (entry and central) in the outward-facing states of hAE1 and hNBCe1. Mutation of residues in the identified binding sites led to impaired transport in both proteins. We also showed that R730 in hAE1 is crucial for anion binding in both entry and central sites, whereas in hNBCe1, a Na+ acts as an anchor for CO32- binding to the central site. Additionally, protonation of the central acidic residues (E681 in hAE1 and D754 in hNBCe1) alters the ion dynamics in the permeation cavity and may contribute to the transport mode differences in SLC4 proteins. These results provide a basis for understanding the functional differences between hAE1 and hNBCe1 and may facilitate potential drug development for diseases such as proximal and distal renal tubular acidosis.

Intravitreal vascular endothelial growth factors hypertension, proteinuria, and renal injury: a concise review.

PURPOSE OF REVIEW: Nearly 20 years ago, vascular endothelial growth factor (VEGF)inhibitors (VEGFi) were adapted from systemic use from antiangiogenesis roles to intravitreal uses. Initially bevacizumab a murine immunoglobulin was injected 'off label' as a treatment for diabetic macular edema and age-related macular degeneration. Throughout the following decade aflibercept and finally ranibizumab were adapted and obtained Food and Drug Administration approval for intravitreal use. Initially systemic absorption was thought to be quite low after intravitreal injections and was quoted as being 200-fold lower than levels postulated to induce significant VEGF inhibition. Pharmacodynamic studies obtained in 2014 and again in 2017 revealed significant systemic absorption and detectable VEGF inhibition, this has since been confirmed in multiple subsequent studies. RECENT FINDINGS: A few case reports of renal dysfunction and glomerular disease related to VEGFi were initially identified. Mixed findings on effects on blood pressure were noted in studies. More recently, 32 cases of de-novo glomerular disease and/or proteinuria exacerbation were identified. New studies have corroborated increased blood pressure, proteinuria exacerbation in patients with pre-existing nephrotic syndrome, and systemic VEGF depletion. Further, the most common lesion of systemic VEGFi nephrotoxicity, thrombotic microangiopathy, has recently been reported by our group. SUMMARY: We will review the pharmacokinetic, translational, and epidemiological data that year upon year establish the finite-yet real risk of intravitreal VEGFi.

SLC4A11 function: evidence for H+(OH-) and NH3-H+ transport.

Whether SLC4A11 transports ammonia and its potential mode of ammonia transport (NH4+, NH3, or NH3-2H+ transport have been proposed) are controversial. In the absence of ammonia, whether SLC4A11 mediates significant conductive H+(OH-) transport is also controversial. The present study was performed to determine the mechanism of human SLC4A11 ammonia transport and whether the transporter mediates conductive H+(OH-) transport in the absence of ammonia. We quantitated H+ flux by monitoring changes in intracellular pH (pHi) and measured whole cell currents in patch-clamp studies of HEK293 cells expressing the transporter in the absence and presence of NH4Cl. Our results demonstrate that SLC4A11 mediated conductive H+(OH-) transport that was stimulated by raising the extracellular pH (pHe). Ammonia-induced HEK293 whole cell currents were also stimulated by an increase in pHe. In studies using increasing NH4Cl concentrations with equal NH4+ extracellular and intracellular concentrations, the shift in the reversal potential (Erev) due to the addition of ammonia was compatible with NH3-H+ transport competing with H+(OH-) rather than NH3-nH+ (n ≥ 2) transport. The increase in equivalent H+(OH-) flux observed in the presence of a transcellular H+ gradient was also compatible with SLC4A11-mediated NH3-H+ flux. The NH3 versus Erev data fit a theoretical model suggesting that NH3-H+ and H+(OH-) competitively interact with the transporter. Studies of mutant SLC4A11 constructs in the putative SLC4A11 ion coordination site showed that both H+(OH-) transport and ammonia-induced whole cell currents were blocked suggesting that the H+(OH-) and NH3-H+ transport processes share common features involving the SLC4A11 transport mechanism.

Host Genetic Background and Gut Microbiota Contribute to Differential Metabolic Responses to Fructose Consumption in Mice.

BACKGROUND: It is unclear how high fructose consumption induces disparate metabolic responses in genetically diverse mouse strains. OBJECTIVE: We aimed to investigate whether the gut microbiota contributes to differential metabolic responses to fructose. METHODS: Eight-week-old male C57BL/6J (B6), DBA/2J (DBA), and FVB/NJ (FVB) mice were given 8% fructose solution or regular water (control) for 12 wk. The gut microbiota composition in cecum and feces was analyzed using 16S ribosomal DNA sequencing, and permutational multivariate ANOVA (PERMANOVA) was used to compare community across mouse strains, treatments, and time points. Microbiota abundance was correlated with metabolic phenotypes and host gene expression in hypothalamus, liver, and adipose tissues using Biweight midcorrelation. To test the causal role of the gut microbiota in determining fructose response, we conducted fecal transplants from B6 to DBA mice and vice versa for 4 wk, as well as gavaged antibiotic-treated DBA mice with Akkermansia for 9 wk, accompanied with or without fructose treatment. RESULTS: Compared with B6 and FVB, DBA mice had significantly higher Firmicutes to Bacteroidetes ratio and lower baseline abundance of Akkermansia and S24-7 (P < 0.05), accompanied by metabolic dysregulation after fructose consumption. Fructose altered specific microbial taxa in individual mouse strains, such as a 7.27-fold increase in Akkermansia in B6 and 0.374-fold change in Rikenellaceae in DBA (false discovery rate <5%), which demonstrated strain-specific correlations with host metabolic and transcriptomic phenotypes. Fecal transplant experiments indicated that B6 microbes conferred resistance to fructose-induced weight gain in DBA mice (F = 43.1, P < 0.001), and Akkermansia colonization abrogated the fructose-induced weight gain (F = 17.8, P < 0.001) and glycemic dysfunctions (F = 11.8, P = 0.004) in DBA mice. CONCLUSIONS: Our findings support that differential microbiota composition between mouse strains is partially responsible for host metabolic sensitivity to fructose, and that Akkermansia is a key bacterium that confers resistance to fructose-induced metabolic dysregulation.

The apical Na+ -HCO3 - cotransporter Slc4a7 (NBCn1) does not contribute to bicarbonate transport by mouse salivary gland ducts.

The HCO3 - secretion mechanism in salivary glands is unclear but is thought to rely on the co-ordinated activity of multiple ion transport proteins including members of the Slc4 family of bicarbonate transporters. Slc4a7 was immunolocalized to the apical membrane of mouse submandibular duct cells. In contrast, Slc4a7 was not detected in acinar cells, and correspondingly, Slc4a7 disruption did not affect fluid secretion in response to cholinergic or ß-adrenergic stimulation in the submandibular gland (SMG). Much of the Na + -dependent intracellular pH (pH i ) regulation in SMG duct cells was insensitive to 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, S0859, and to the removal of extracellular HCO 3 - . Consistent with these latter observations, the Slc4a7 null mutation had no impact on HCO 3 - secretion nor on pH i regulation in duct cells. Taken together, our results revealed that Slc4a7 targets to the apical membrane of mouse SMG duct cells where it contributes little if any to pH i regulation or stimulated HCO 3 - secretion.

Nephrotoxicity induced by intravitreal vascular endothelial growth factor inhibitors: emerging evidence.

Vascular endothelial growth factor (VEGF) inhibitors have emerged as powerful tools to treat malignant neoplasms and ocular diseases by virtue of their ability to inhibit angiogenesis. Recent data indicate that intravitreal injections of VEGF inhibitors can lead to significant systemic absorption as well as a measurable reduction of plasma VEGF activity. There is increasing evidence showing that vitreal absorption of these drugs is associated with cases of accelerated hypertension, worsening proteinuria, glomerular disease, thrombotic microangiopathy, and possible chronic renal function decline. In this review, the 3 most commonly used anti-VEGF agents-bevacizumab, ranibizumab, and aflibercept-are discussed, highlighting their intravitreal absorption and associated effects on the kidney as a target organ system. We provide clinical suggestions for clinicians to both better manage patients receiving anti-VEGF agents intravitreally and detect any putative systemic renal effects of these agents. While acknowledging the risks of aberrant retinal angiogenesis, it is important for clinicians to be aware of the potential for adverse renal risks with use of these agents.

Atypical hemolytic uremic syndrome and complement blockade: established and emerging uses of complement inhibition.

PURPOSE OF REVIEW: Atypical hemolytic uremic syndrome (aHUS) is a diagnosis that has captured the interest of specialists across multiple fields. The hallmark features of aHUS are microangiopathic hemolysis and thrombocytopenia, which creates a diagnostic dilemma because of the occurrence of these findings in a wide variety of clinical disorders. RECENT FINDINGS: In most of the instances, aHUS is a diagnosis of exclusion after ruling out causes such as Shigella toxin, acquired or genetic a disintegrin and metalloproteinase thrombospondin motif 13 deficiency (thrombotic thrombocytopenic purpura), and vitamin B12 deficiency. In the purest sense, aHUS is a genetic condition that is activated (or unmasked) by an environmental exposure. However, it is now evident that complement activation is a feature of many diseases. Variants in complement regulatory genes predispose to microangiopathic hemolysis in many rheumatologic, oncologic, and drug-induced vascular, obstetric, peritransplant, and infectious syndromes. SUMMARY: Many 'hemolysis syndromes' overlap clinically with aHUS, and we review the literature on the treatment of these conditions with complement inhibition. New reports on the treatment of C3 glomerulopathy, Shiga toxin-related classic hemolytic uremic syndrome, and medication-related thrombotic microangiopathy will be reviewed as well.

Successful use of eculizumab to treat atypical hemolytic uremic syndrome in patients with inflammatory bowel disease.

BACKGROUND: Atypical hemolytic uremic syndrome is a rare group of disorders that have in common underlying complement amplifying conditions. These conditions can accelerate complement activation that results in a positive feedback cycle. The known triggers for complement activation can be diverse and include, infection, autoimmune disease, and malignancy. Recent reports suggest that certain autoimmune and rheumatological triggers of complement activation may result in atypical hemolytic uremic syndrome that does not resolve despite treating the underlying disorder. Specifically, patients with systemic lupus erythematosus and microangiopathic hemolysis may not respond to treatment of their underlying rheumatological trigger but responded to complement blockade. CASE PRESENTATIONS: We report two patients with inflammatory bowel disease complicated by development of atypical hemolytic uremic syndrome. In both cases, patients were on treatment for inflammatory bowel disease, that was not well controlled/flaring at the time. The first patient is a male who developed Crohn's disease and microangiopathic hemolysis at age 5 and was treated with eculizumab successfully. Discontinuation of the medication led to multiple relapses, and the patient currently is being treated with eculizumab and has normal hematological and stable renal parameters. The second patient is a 49-year-old female with Ulcerative Colitis treated with 6-Mercaptopurine. She developed acute kidney injury and microangiopathic hemolysis. Prompt diagnosis and treatment with eculizumab resulted in the recovery of kidney injury along with a complete hematological response. CONCLUSIONS: These two cases are the fifth and sixth patients to be published in the literature with atypical hemolytic uremic syndrome and inflammatory bowel disease treated with complement blockade. This confirms that C5 complement blockade is effective in treating complement mediated thrombotic microangiopathy/atypical hemolytic uremic syndrome when it is triggered in patients with inflammatory bowel disease.

Intracellular Cl- as a signaling ion that potently regulates Na+/HCO3- transporters.

Cl(-) is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Surprisingly, a role of intracellular Cl(-) (Cl(-) in) as a signaling ion has not been previously evaluated. Here we report that Cl(-) in functions as a regulator of cellular Na(+) and HCO3 (-) concentrations and transepithelial transport through modulating the activity of several electrogenic Na(+)-HCO3 (-) transporters. We describe the molecular mechanism(s) of this regulation by physiological Cl(-) in concentrations highlighting the role of GXXXP motifs in Cl(-) sensing. Regulation of the ubiquitous Na(+)-HCO3(-) co-transport (NBC)e1-B is mediated by two GXXXP-containing sites; regulation of NBCe2-C is dependent on a single GXXXP motif; and regulation of NBCe1-A depends on a cryptic GXXXP motif. In the basal state NBCe1-B is inhibited by high Cl(-) in interacting at a low affinity GXXXP-containing site. IP3 receptor binding protein released with IP3 (IRBIT) activation of NBCe1-B unmasks a second high affinity Cl(-) in interacting GXXXP-dependent site. By contrast, NBCe2-C, which does not interact with IRBIT, has a single high affinity N-terminal GXXP-containing Cl(-) in interacting site. NBCe1-A is unaffected by Cl(-) in between 5 and 140 mM. However, deletion of NBCe1-A residues 29-41 unmasks a cryptic GXXXP-containing site homologous with the NBCe1-B low affinity site that is involved in inhibition of NBCe1-A by Cl(-) in. These findings reveal a cellular Cl(-) in sensing mechanism that plays an important role in the regulation of Na(+) and HCO3 (-) transport, with critical implications for the role of Cl(-) in cellular ion homeostasis and epithelial fluid and electrolyte secretion.

Multifunctional ion transport properties of human SLC4A11: comparison of the SLC4A11-B and SLC4A11-C variants.

Congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (CHED with progressive sensorineural deafness), and potentially a subset of individuals with late-onset Fuchs' endothelial corneal dystrophy are caused by mutations in the SLC4A11 gene that results in corneal endothelial cell abnormalities. Originally classified as a borate transporter, the function of SLC4A11 as a transport protein remains poorly understood. Elucidating the transport function(s) of SLC4A11 is needed to better understand how its loss results in the aforementioned posterior corneal dystrophic disease processes. Quantitative PCR experiments demonstrated that, of the three known human NH2-terminal variants, SLC4A11-C is the major transcript expressed in human corneal endothelium. We studied the expression pattern of the three variants in mammalian HEK-293 cells and demonstrated that the SLC4A11-B and SLC4A11-C variants are plasma membrane proteins, whereas SLC4A11-A is localized intracellularly. SLC4A11-B and SLC4A11-C were shown to be multifunctional ion transporters capable of transporting H+ equivalents in both a Na+-independent and Na+-coupled mode. In both transport modes, SLC4A11-C H+ flux was significantly greater than SLC4A11-B. In the presence of ammonia, SLC4A11-B and SLC4A11-C generated inward currents that were comparable in magnitude. Chimera SLC4A11-C-NH2-terminus-SLC4A11-B experiments demonstrated that the SLC4A11-C NH2-terminus functions as an autoactivating domain, enhancing Na+-independent and Na+-coupled H+ flux without significantly affecting the electrogenic NH3-H(n)+ cotransport mode. All three modes of transport were significantly impaired in the presence of the CHED causing p.R109H (SLC4A11-C numbering) mutation. These complex ion transport properties need to be addressed in the context of corneal endothelial disease processes caused by mutations in SLC4A11.

Interplay between disulfide bonding and N-glycosylation defines SLC4 Na+-coupled transporter extracellular topography.

The extracellular loop 3 (EL-3) of SLC4 Na(+)-coupled transporters contains 4 highly conserved cysteines and multiple N-glycosylation consensus sites. In the electrogenic Na(+)-HCO3(-) cotransporter NBCe1-A, EL-3 is the largest extracellular loop and is predicted to consist of 82 amino acids. To determine the structural-functional importance of the conserved cysteines and the N-glycosylation sites in NBCe1-A EL-3, we analyzed the potential interplay between EL-3 disulfide bonding and N-glycosylation and their roles in EL-3 topological folding. Our results demonstrate that the 4 highly conserved cysteines form two intramolecular disulfide bonds, Cys(583)-Cys(585) and Cys(617)-Cys(642), respectively, that constrain EL-3 in a folded conformation. The formation of the second disulfide bond is spontaneous and unaffected by the N-glycosylation state of EL-3 or the first disulfide bond, whereas formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected by N-glycosylation. Importantly, EL-3 from each monomer is adjacently located at the NBCe1-A dimeric interface. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. This unique adjacent parallel location of two symmetrically folded EL-3 loops from each monomer resembles a domain-like structure that is potentially important for NBCe1-A function in vivo. Moreover, the formation of this unique structure is critically dependent on the finely tuned interplay between disulfide bonding and N-glycosylation in the membrane processed NBCe1-A dimer.

Human SLC4A11-C functions as a DIDS-stimulatable H⁺(OH⁻) permeation pathway: partial correction of R109H mutant transport.

The SLC4A11 gene mutations cause a variety of genetic corneal diseases, including congenital hereditary endothelial dystrophy 2 (CHED2), Harboyan syndrome, some cases of Fuchs' endothelial dystrophy (FECD), and possibly familial keratoconus. Three NH2-terminal variants of the human SLC4A11 gene, named SLC4A11-A, -B, and -C are known. The SLC4A11-B variant has been the focus of previous studies. Both the expression of the SLC4A11-C variant in the cornea and its functional properties have not been characterized, and therefore its potential pathophysiological role in corneal diseases remains to be explored. In the present study, we demonstrate that SLC4A11-C is the predominant SLC4A11 variant expressed in human corneal endothelial mRNA and that the transporter functions as an electrogenic H(+)(OH(-)) permeation pathway. Disulfonic stilbenes, including 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS), and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonate (SITS), which are known to bind covalently, increased SLC4A11-C-mediated H(+)(OH(-)) flux by 150-200% without having a significant effect in mock-transfected cells. Noncovalently interacting 4,4'-diaminostilbene-2,2'-disulfonate (DADS) was without effect. We tested the efficacy of DIDS on the functionally impaired R109H mutant (SLC4A11-C numbering) that causes CHED2. DIDS (1 mM) increased H(+)(OH(-)) flux through the mutant transporter by ∼40-90%. These studies provide a basis for future testing of more specific chemically modified dilsulfonic stilbenes as potential therapeutic agents to improve the functional impairment of specific SLC4A11 mutant transporters.

Topology of NBCe1 protein transmembrane segment 1 and structural effect of proximal renal tubular acidosis (pRTA) S427L mutation.

In the kidney proximal tubule, NBCe1-A plays a critical role in absorbing HCO3(-) from cell to blood. NBCe1-A transmembrane segment 1 (TM1) is involved in forming part of the ion permeation pathway, and a missense mutation S427L in TM1 impairs ion transport, causing proximal renal tubular acidosis. In the present study, we examined the topology of NBCe1-A-TM1 in detail and its structural perturbation induced by S427L. We analyzed the N-terminal cytoplasmic region (Cys-389-Gln-424) of NBCe1-A-TM1 using the substituted cysteine scanning accessibility method combined with extensive chemical stripping, in situ chemical probing, and functional transport assays. NBCe1-A-TM1 was previously modeled on the anion exchanger 1 TM1 (AE1-TM1); however, our data demonstrated that the topology of AE1-TM1 differs significantly from NBCe1-A-TM1. Our findings revealed that NBCe1-A-TM1 is unusually long, consisting of 31 membrane-embedded amino acids (Phe-412 to Thr-442). The linker region (Arg-394-Pro-411) between the N terminus of TM1 and the cytoplasmic domain is minimally exposed to aqueous and is potentially folded in a helical structure that intimately interacts with the NBCe1-A cytoplasmic domain. In contrast, AE1-TM1 contains 25 amino acids connected to an aqueous-exposed cytoplasmic region. Based on our new NBCe1-A-TM1 model, Ser-427 resides in the middle of TM1. Leucine substitution at Ser-427 blocks the normal aqueous access to Thr-442, Ala-435, and Lys-404, implying a significant alteration of NBCe1-TM1 orientation. Our study provides novel structural insights into the pathogenic mechanism of S427L in mediating proximal renal tubular acidosis.

NBCe1 as a model carrier for understanding the structure-function properties of Na⁺ -coupled SLC4 transporters in health and disease.

SLC4 transporters are membrane proteins that in general mediate the coupled transport of bicarbonate (carbonate) and share amino acid sequence homology. These proteins differ as to whether they also transport Na(+) and/or Cl(-), in addition to their charge transport stoichiometry, membrane targeting, substrate affinities, developmental expression, regulatory motifs, and protein-protein interactions. These differences account in part for the fact that functionally, SLC4 transporters have various physiological roles in mammals including transepithelial bicarbonate transport, intracellular pH regulation, transport of Na(+) and/or Cl(-), and possibly water. Bicarbonate transport is not unique to the SLC4 family since the structurally unrelated SLC26 family has at least three proteins that mediate anion exchange. The present review focuses on the first of the sodium-dependent SLC4 transporters that was identified whose structure has been most extensively studied: the electrogenic Na(+)-base cotransporter NBCe1. Mutations in NBCe1 cause proximal renal tubular acidosis (pRTA) with neurologic and ophthalmologic extrarenal manifestations. Recent studies have characterized the important structure-function properties of the transporter and how they are perturbed as a result of mutations that cause pRTA. It has become increasingly apparent that the structure of NBCe1 differs in several key features from the SLC4 Cl(-)-HCO3 (-) exchanger AE1 whose structural properties have been well-studied. In this review, the structure-function properties and regulation of NBCe1 will be highlighted, and its role in health and disease will be reviewed in detail.

Defining the role of albumin infusion in cirrhosis-associated hyponatremia.

The presence of negatively charged, impermeant proteins in the plasma space alters the distribution of diffusible ions in the plasma and interstitial fluid (ISF) compartments to preserve electroneutrality and is known as Gibbs-Donnan equilibrium. In patients with hypoalbuminemia due to underlying cirrhosis, the decrease in the plasma water albumin concentration ([Alb-]pw) would be expected to result in a decrease in the plasma water sodium concentration ([Na+]pw) due to an alteration in the distribution of Na+ between the plasma and ISF. In addition, cirrhosis-associated hyponatremia may be due to the renal diluting defect resulting from the intravascular volume depletion due to gastrointestinal losses and overdiuresis and/or decreased effective circulatory volume secondary to splanchnic vasodilatation. Therefore, albumin infusion may result in correction of the hyponatremia in cirrhotic patients either by modulating the Gibbs-Donnan effect due to hypoalbuminemia or by restoring intravascular volume in patients with intravascular volume depletion due to gastrointestinal losses and overdiuresis. However, the differential role of albumin infusion in modulating the [Na+]pw in these patients has not previously been analyzed quantitatively. In the present study, we developed an in vitro assay system to examine for the first time the quantitative effect of changes in albumin concentration on the distribution of Na+ between two compartments separated by a membrane that allows the free diffusion of Na+. Our findings demonstrated that changes in [Alb-]pw are linearly related to changes in [Na+]pw as predicted by Gibbs-Donnan equilibrium. However, based on our findings, we predict that the improvement in cirrhosis-associated hyponatremia due to intravascular volume depletion results predominantly from the restoration of intravascular volume rather than alterations in Gibbs-Donnan equilibrium.

Data-driven prediction of continuous renal replacement therapy survival.

Continuous renal replacement therapy (CRRT) is a form of dialysis prescribed to severely ill patients who cannot tolerate regular hemodialysis. However, as the patients are typically very ill to begin with, there is always uncertainty whether they will survive during or after CRRT treatment. Because of outcome uncertainty, a large percentage of patients treated with CRRT do not survive, utilizing scarce resources and raising false hope in patients and their families. To address these issues, we present a machine learning-based algorithm to predict short-term survival in patients being initiated on CRRT. We use information extracted from electronic health records from patients who were placed on CRRT at multiple institutions to train a model that predicts CRRT survival outcome; on a held-out test set, the model achieves an area under the receiver operating curve of 0.848 (CI = 0.822-0.870). Feature importance, error, and subgroup analyses provide insight into bias and relevant features for model prediction. Overall, we demonstrate the potential for predictive machine learning models to assist clinicians in alleviating the uncertainty of CRRT patient survival outcomes, with opportunities for future improvement through further data collection and advanced modeling.

Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis.

Mutations in SLC4A4, the gene encoding the electrogenic Na(+)-HCO3(-) cotransporter NBCe1, cause severe proximal renal tubular acidosis (pRTA), growth retardation, decreased IQ, and eye and teeth abnormalities. Among the known NBCe1 mutations, the disease-causing mechanism of the T485S (NBCe1-A numbering) mutation is intriguing because the substituted amino acid, serine, is structurally and chemically similar to threonine. In this study, we performed intracellular pH and whole cell patch-clamp measurements to investigate the base transport and electrogenic properties of NBCe1-A-T485S in mammalian HEK 293 cells. Our results demonstrated that Ser substitution of Thr485 decreased base transport by ~50%, and importantly, converted NBCe1-A from an electrogenic to an electroneutral transporter. Aqueous accessibility analysis using sulfhydryl reactive reagents indicated that Thr485 likely resides in an NBCe1-A ion interaction site. This critical location is also supported by the finding that G486R (a pRTA causing mutation) alters the position of Thr485 in NBCe1-A thereby impairing its transport function. By using NO3(-) as a surrogate ion for CO3(2-), our result indicated that NBCe1-A mediates electrogenic Na(+)-CO3(2-) cotransport when functioning with a 1:2 charge transport stoichiometry. In contrast, electroneutral NBCe1-T485S is unable to transport NO3(-), compatible with the hypothesis that it mediates Na(+)-HCO3(-) cotransport. In patients, NBCe1-A-T485S is predicted to transport Na(+)-HCO3(-) in the reverse direction from blood into proximal tubule cells thereby impairing transepithelial HCO3(-) absorption, possibly representing a new pathogenic mechanism for generating human pRTA.