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.

Review of intravitreal VEGF inhibitor toxicity and report of collapsing FSGS with TMA in a patient with age-related macular degeneration.

Intravitreal vascular endothelial growth factor (VEGF) receptor blockade is used for a variety of retinal pathologies. These include age-related macular degeneration (AMD), diabetic macular edema (DME) and central retinal vein obstruction. Reports of absorption of intravitreal agents into systemic circulation have increased in number and confirmation of depletion of VEGF has been confirmed. Increasingly there are studies and case reports showing worsening hypertension, proteinuria, renal dysfunction and glomerular disease. The pathognomonic findings of systemic VEGF blockade, thrombotic microangiopathies (TMAs), are also being increasingly reported. One lesion that occurs in conjunction with TMAs that has been described is collapsing focal segmental glomerulosclerosis (cFSGS). cFSGS has been postulated to occur due to TMA-induced chronic glomerular hypoxia. In this updated review we discuss the mechanistic, pharmacological, epidemiological and clinical evidence of intravitreal VEGF toxicity. We review cases of biopsy-proven toxicity presented by our group and other investigators. We also present the third reported case of cFSGS in the setting of intravitreal VEGF blockade with a chronic TMA component that was crucially found on biopsy. This patient is a 74-year-old nondiabetic male receiving aflibercept for AMD. Of the two prior cases of cFSGS in the setting of VEGF blockade, one had AMD and the other had DME. This case solidifies the finding of cFSGS and its association with chronic TMA as a lesion that may be frequently encountered in patients receiving intravitreal VEGF inhibitors.

Refractory scleroderma renal crisis precipitated after high-dose oral corticosteroids and concurrent intravitreal injection of bevacizumab.

Scleroderma renal crisis is a serious complication that can develop in certain patients with systemic sclerosis. Some risks have been identified as potential triggers of scleroderma renal crisis, including the high-dose oral corticosteroids. Here, we present a patient who developed clinically severe systemic sclerosis and scleroderma renal crisis after exposure to oral corticosteroids and intravitreal vascular endothelial growth factor blockade with bevacizumab for cotton wool spots. The patient's scleroderma renal crisis was severe, progressive, and refractory to the standard of care therapy: oral captopril. Biopsy showed a diffuse thrombotic microangiopathy and findings consistent with scleroderma renal crisis. We hypothesize that depletion of systemic vascular endothelial growth factor with intravitreal anti-vascular endothelial growth factor injections likely contributed to the particularly severe presentation seen in this case. Though the finding of a monoclonal gammopathy of undetermined significance is another complicating factor, this case suggests that vascular endothelial growth factor inhibition may be a newly recognized trigger of scleroderma renal crisis.

Energy Shortage in Human and Mouse Models of SLC4A11-Associated Corneal Endothelial Dystrophies.

Purpose: To elucidate the molecular events in solute carrier family 4 member 11 (SLC4A11)-deficient corneal endothelium that lead to the endothelial dysfunction that characterizes the dystrophies associated with SLC4A11 mutations, congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy 4. Methods: Comparative transcriptomic analysis (CTA) was performed in primary human corneal endothelial cells (pHCEnC) and murine corneal endothelial cells (MCEnC) with normal and reduced levels of SLC4A11 (SLC4A11 KD pHCEnC) and Slc4a11 (Slc4a11-/- MCEnC), respectively. Validation of differentially expressed genes was performed using immunofluorescence staining of CHED corneal endothelium, as well as western blot and quantitative PCR analysis of SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC. Functional analyses were performed to investigate potential functional changes associated with the observed transcriptomic alterations. Results: CTA revealed inhibition of cell metabolism and ion transport function as well as mitochondrial dysfunction, leading to reduced adenosine triphosphate (ATP) production, in SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC. Co-localization of SNARE protein STX17 with mitochondria marker COX4 was observed in CHED corneal endothelium, as was activation of AMPK-p53/ULK1 in both SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC, providing additional evidence of mitochondrial dysfunction and mitophagy. Reduced Na+-dependent HCO3- transport activity and altered NH4Cl-induced membrane potential changes were observed in Slc4a11-/- MCEnC. Conclusions: Reduced steady-state ATP levels and subsequent activation of the AMPK-p53 pathway provide a link between the metabolic functional deficit and transcriptome alterations, as well as evidence of insufficient ATP to maintain the Na+/K+-ATPase corneal endothelial pump as the cause of the edema that characterizes SLC4A11-associated corneal endothelial dystrophies.

Diverse Clinical Presentations of C3 Dominant Glomerulonephritis.

C3 dominant immunofluorescence staining is present in a subset of patients with idiopathic immune complex membranoproliferative glomerulonephritis (iMPGN). It is increasingly recognized that iMPGN may be complement driven, as are cases of "typical" C3 glomerulopathy (C3G). In both iMPGN and C3G, a frequent membranoproliferative pattern of glomerular injury may indicate common pathogenic mechanisms via complement activation and endothelial cell damage. Dysregulation of the alternative complement pathway and mutations in certain regulatory factors are highly implicated in C3 glomerulopathy (which encompasses C3 glomerulonephritis, dense deposit disease, and cases of C3 dominant MPGN). We report three cases that demonstrate that an initial biopsy diagnosis of iMPGN does not exclude complement alterations similar to the ones observed in patients with a diagnosis of C3G. The first patient is a 39-year-old woman with iMPGN and C3 dominant staining, with persistently low C3 levels throughout her course. The second case is a 22-year-old woman with elevated anti-factor H antibodies and C3 dominant iMPGN findings on biopsy. The third case is a 25-year-old woman with C3 dominant iMPGN, dense deposit disease, and a crescentic glomerulonephritis on biopsy. We present the varied phenotypic variations of C3 dominant MPGN and review clinical course, complement profiles, genetic testing, treatment course, and peri-transplantation plans. Testing for complement involvement in iMPGN is important given emerging treatment options and transplant planning.

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.

Acute interstitial nephritis and drug-induced systemic lupus erythematosus due to chlorthalidone and amiodarone: A case report.

Drug-induced lupus erythematosus has features distinct from primary systemic lupus erythematosus. It can occur with a wide variety of agents that result in the generation of anti-histone or other types of antibodies. Systemic manifestations of drug-induced systemic lupus erythematosus may include renal dysfunction due to circulating immune complexes or due to other immune reactions to the culprit medication(s). Acute interstitial nephritis occurs due to DNA-drug or protein-drug complexes that trigger an allergic immune response. We report a patient who developed acute kidney injury, rash, and drug-induced systemic lupus diagnosed by serologies after starting chlorthalidone and amiodarone. A renal biopsy showed acute interstitial nephritis and not lupus-induced glomerulonephritis. It is important to note that systemic lupus erythematosus and acute interstitial nephritis can occur together, and this report highlights the role of the kidney biopsy in ascertaining the pathological diagnosis and outlining therapy in drug-induced lupus erythematosus.

Intravitreal bevacizumab-induced exacerbation of proteinuria in diabetic nephropathy, and amelioration by switching to ranibizumab.

Certain diabetic and hypertensive patients started on intravitreal vascular endothelial growth factor inhibition for diabetic retinopathy may experience worsening of hypertension and proteinuria. The etiology of this is the newly recognized absorption of intravitreally injected vascular endothelial growth factor inhibitors, and the susceptibility of patients with pre-existing renal disease to exacerbations depends on the degree of systemic absorption. There are eighteen reported cases of worsening hypertension, woresening proteinuria, worsening renal function, thrombotic microangiopathy, and glomerular disease noted after initiation of intravitreal vascular endothelial growth factor blockade. This nineteenth case demonstrates worsening hypertension and proteinuria with the start of bevacizumab. Both blood pressure and proteinuria parameters showed overall improvement with switching to the less absorbed and lower potency agent ranibizumab. There was a slight rise in serum creatinine after bevacizumab therapy, which stabilized at a new baseline, and the serum creatinine remained stable on ranibizumab. There were no other nephrotoxic exposures that explained the mild rise in serum creatinine. Because of improvement in renal function and proteinuria, a renal biopsy was deferred for the time. This case re-demonstrates the risk of worsening proteinuria with vascular endothelial growth factor inhibitors when given intravitreally in some patients. The demonstration of improvement in blood pressure and proteinuria with the use of lower potency agents like ranibizumab is novel and an important concept confirming observations from pharmacokinetic studies. The switch to ranibizumab offers a therapeutic option when proteinuria worsens with intravitreal vascular endothelial growth factor blockade, and the patient requires ongoing intravitreal therapy for treatment of diabetic retinopathy.

Worsening proteinuria and renal function after intravitreal vascular endothelial growth factor blockade for diabetic proliferative retinopathy.

Systemic vascular endothelial growth factor (VEGF) inhibitions can induce worsening hypertension, proteinuria and glomerular diseases of various types. These agents can also be used to treat ophthalmic diseases like proliferative diabetic retinopathy, diabetic macular edema, central retinal vein occlusion and age-related macular degeneration. Recently, pharmacokinetic studies confirmed that these agents are absorbed at levels that result in biologically significant suppression of intravascular VEGF levels. There have now been 23 other cases published that describe renal sequela of intravitreal VEGF blockade, and they unsurprisingly mirror known systemic toxicities of VEGF inhibitors. We present three cases where stable levels of proteinuria and chronic kidney disease worsened after initiation of these agents. Two of our three patients were biopsied. The first patient's biopsy showed diabetic nephropathy and focal and segmental glomerulosclerosis (FSGS) with collapsing features and acute interstitial nephritis (AIN). The second patient's biopsy showed AIN in a background of diabetic glomerulosclerosis. This is the second patient seen by our group, whose biopsy revealed segmental glomerulosclerosis with collapsing features in the setting of intravitreal VEGF blockade. Though FSGS with collapsing features and AIN are not the typical lesions seen with systemic VEGF blockade, they have been reported as rare case reports previously. In addition to reviewing known elements of intravitreal VEGF toxicity, the cases presented encompass renal pathology data supporting that intravitreal VEGF blockade can result in deleterious systemic and renal pathological disorders.

Slc4a11 disruption causes duct cell loss and impairs NaCl reabsorption in female mouse submandibular glands.

Slc4a11, a member of the Slc4 HCO3- transporter family, has a wide tissue distribution. In mouse salivary glands, the expression of Slc4a11 mRNA was more than eightfold greater than the other nine members of the Slc4 gene family. The Slc4a11 protein displayed a diffuse subcellular distribution in both the acinar and duct cells of mouse submandibular glands (SMG). Slc4a11 disruption induced a significant increase in the Na+ and Cl- concentrations of stimulated SMG saliva, whereas it did not affect the fluid secretion rate in response to either ß-adrenergic or cholinergic receptor stimulation. Heterologous expressed mouse Slc4a11 acted as a H+ /OH- transporter that was uncoupled of Na+ or Cl- movement, and this activity was blocked by ethyl-isopropyl amiloride (EIPA) but not 4,4'-Diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS). Slc4a11 disruption revealed that Slc4a11 does not play a major role in intracellular pH regulation in mouse salivary gland cells. In contrast, NaCl reabsorption was impaired in the SMG saliva of female compared to male Slc4a11 null mice, which correlated with the loss of duct cells and a decrease in expression of the duct-cell-specific transcription factor Ascl3. Together, our results suggest that Slc4a11 expression regulates the number of ducts cells in the mouse SMG and consequently NaCl reabsorption.

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.

ZEB1 insufficiency causes corneal endothelial cell state transition and altered cellular processing.

The zinc finger e-box binding homeobox 1 (ZEB1) transcription factor is a master regulator of the epithelial to mesenchymal transition (EMT), and of the reverse mesenchymal to epithelial transition (MET) processes. ZEB1 plays an integral role in mediating cell state transitions during cell lineage specification, wound healing and disease. EMT/MET are characterized by distinct changes in molecular and cellular phenotype that are generally context-independent. Posterior polymorphous corneal dystrophy (PPCD), associated with ZEB1 insufficiency, provides a new biological context in which to understand and evaluate the classic EMT/MET paradigm. PPCD is characterized by a cadherin-switch and transition to an epithelial-like transcriptomic and cellular phenotype, which we study in a cell-based model of PPCD generated using CRISPR-Cas9-mediated ZEB1 knockout in corneal endothelial cells (CEnCs). Transcriptomic and functional studies support the hypothesis that CEnC undergo a MET-like transition in PPCD, termed endothelial to epithelial transition (EnET), and lead to the conclusion that EnET may be considered a corollary to the classic EMT/MET paradigm.

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.

Renal Tubular Acidosis: H+/Base and Ammonia Transport Abnormalities and Clinical Syndromes.

Renal tubular acidosis (RTA) represents a group of diseases characterized by (1) a normal anion gap metabolic acidosis; (2) abnormalities in renal HCO3- absorption or new renal HCO3- generation; (3) changes in renal NH4+, Ca2+, K+, and H2O homeostasis; and (4) extrarenal manifestations that provide etiologic diagnostic clues. The focus of this review is to give a general overview of the pathogenesis of the various clinical syndromes causing RTA with a particular emphasis on type I (hypokalemic distal RTA) and type II (proximal) RTA while reviewing their pathogenesis from a physiological "bottom-up" approach. In addition, the factors involved in the generation of metabolic acidosis in both type I and II RTA are reviewed highlighting the importance of altered renal ammonia production/partitioning and new HCO3- generation. Our understanding of the underlying tubular transport and extrarenal abnormalities has significantly improved since the first recognition of RTA as a clinical entity because of significant advances in clinical acid-base chemistry, whole tubule and single-cell H+/base transport, and the molecular characterization of the various transporters and channels that are functionally affected in patients with RTA. Despite these advances, additional studies are needed to address the underlying mechanisms involved in hypokalemia, altered ammonia production/partitioning, hypercalciuria, nephrocalcinosis, cystic abnormalities, and CKD progression in these patients.

CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1.

Na+-coupled acid-base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na+-coupled acid-base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3.9 Å resolution by cryo electron microscopy. Our atomic model and functional mutagenesis revealed the ion accessibility pathway and the ion coordination site, the latter containing residues involved in human disease-causing mutations. We identified a small number of residues within the ion coordination site whose modification transformed NBCe1 into an anion exchanger. Our data suggest that symporters and exchangers utilize comparable transport machinery and that subtle differences in their substrate-binding regions have very significant effects on their transport mode.

Aminoacylase 3 Is a New Potential Marker and Therapeutic Target in Hepatocellular Carcinoma.

Ras proteins (HRas, KRas and NRas) are common oncogenes that require membrane association for activation. Previous approaches to block/inhibit Ras membrane association were unsuccessful for cancer treatment in human clinical studies. In the present study we utilized a new approach to decrease Ras membrane association in hepatocellular carcinoma (HCC) cell lines via inhibition of an enzyme aminoacylase 3 (AA3; EC 3.5.1.114). AA3 expression was significantly elevated in the livers of HCC patients and HCC cell lines. Treatment of HepG2 cells with AA3 inhibitors, and HepG2 and HuH7 with AA3 siRNA significantly decreased Ras membrane association and was toxic to these HCC cell lines. AA3 inhibitors also increased the levels of N-acetylfarnesylcysteine (NAFC) and N-acetylgeranylgeranylcysteine (NAGGC) in HepG2 and Huh7 cell lines. We hypothesized that AA3 deacetylates NAFC and NAGGC, and generated farnesylcysteine (FC) and geranylgeranylcysteine (GGC) that are used in HCC cells for the regeneration of farnesylpyrophosphate and geranylgeranylpyrophosphate providing the prenyl (farnesyl or geranylgeranyl) group for Ras prenylation required for Ras membrane association. This was confirmed experimentally where purified human AA3 was capable of efficiently deacetylating NAFC and NAGGC. Our findings suggest that AA3 inhibition may be an effective approach in the therapy of HCC and that elevated AA3 expression in HCC is potentially an important diagnostic marker.

SLC26A Gene Family Participate in pH Regulation during Enamel Maturation.

The bicarbonate transport activities of Slc26a1, Slc26a6 and Slc26a7 are essential to physiological processes in multiple organs. Although mutations of Slc26a1, Slc26a6 and Slc26a7 have not been linked to any human diseases, disruption of Slc26a1, Slc26a6 or Slc26a7 expression in animals causes severe dysregulation of acid-base balance and disorder of anion homeostasis. Amelogenesis, especially the enamel formation during maturation stage, requires complex pH regulation mechanisms based on ion transport. The disruption of stage-specific ion transporters frequently results in enamel pathosis in animals. Here we present evidence that Slc26a1, Slc26a6 and Slc26a7 are highly expressed in rodent incisor ameloblasts during maturation-stage tooth development. In maturation-stage ameloblasts, Slc26a1, Slc26a6 and Slc26a7 show a similar cellular distribution as the cystic fibrosis transmembrane conductance regulator (Cftr) to the apical region of cytoplasmic membrane, and the distribution of Slc26a7 is also seen in the cytoplasmic/subapical region, presumably on the lysosomal membrane. We have also examined Slc26a1 and Slc26a7 null mice, and although no overt abnormal enamel phenotypes were observed in Slc26a1-/- or Slc26a7-/- animals, absence of Slc26a1 or Slc26a7 results in up-regulation of Cftr, Ca2, Slc4a4, Slc4a9 and Slc26a9, all of which are involved in pH homeostasis, indicating that this might be a compensatory mechanism used by ameloblasts cells in the absence of Slc26 genes. Together, our data show that Slc26a1, Slc26a6 and Slc26a7 are novel participants in the extracellular transport of bicarbonate during enamel maturation, and that their functional roles may be achieved by forming interaction units with Cftr.

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.

Prevention of the disrupted enamel phenotype in Slc4a4-null mice using explant organ culture maintained in a living host kidney capsule.

Slc4a4-null mice are a model of proximal renal tubular acidosis (pRTA). Slc4a4 encodes the electrogenic sodium base transporter NBCe1 that is involved in transcellular base transport and pH regulation during amelogenesis. Patients with mutations in the SLC4A4 gene and Slc4a4-null mice present with dysplastic enamel, amongst other pathologies. Loss of NBCe1 function leads to local abnormalities in enamel matrix pH regulation. Loss of NBCe1 function also results in systemic acidemic blood pH. Whether local changes in enamel pH and/or a decrease in systemic pH are the cause of the abnormal enamel phenotype is currently unknown. In the present study we addressed this question by explanting fetal wild-type and Slc4a4-null mandibles into healthy host kidney capsules to study enamel formation in the absence of systemic acidemia. Mandibular E11.5 explants from NBCe1-/- mice, maintained in host kidney capsules for 70 days, resulted in teeth with enamel and dentin with morphological and mineralization properties similar to cultured NBCe1+/+ mandibles grown under identical conditions. Ameloblasts express a number of proteins involved in dynamic changes in H+/base transport during amelogenesis. Despite the capacity of ameloblasts to dynamically modulate the local pH of the enamel matrix, at least in the NBCe1-/- mice, the systemic pH also appears to contribute to the enamel phenotype. Extrapolating these data to humans, our findings suggest that in patients with NBCe1 mutations, correction of the systemic metabolic acidosis at a sufficiently early time point may lead to amelioration of enamel abnormalities.