Reduced surface pH and upregulated AE2 anion exchange in SLC26A3-deleted polarized intestinal epithelial cells.

Loss of function mutations in the SLC26A3 gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question, SLC26A3 was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pHi and apical and basolateral Cl-/HCO3- exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl-/HCO3- exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pHi was not significantly different from that of WT cells, but basolateral Cl-/HCO3- exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl-/HCO3- exchange rate and apical surface pH, but sustained a normal steady-state pHi due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide.NEW & NOTEWORTHY SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl-/HCO3- exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.

Role of ecto-5'-nucleotidase in bladder function.

Purinergic signaling plays an important role in regulating bladder contractility and voiding. Abnormal purinergic signaling is associated with lower urinary tract symptoms (LUTS). Ecto-5'-nucleotidase (NT5E) catalyzes dephosphorylation of extracellular AMP to adenosine, which in turn promotes adenosine-A2b receptor signaling to relax bladder smooth muscle (BSM). The functional importance of this mechanism was investigated using Nt5e knockout (Nt5eKO) mice. Increased voiding frequency of small voids revealed by voiding spot assay was corroborated by urodynamic studies showing shortened voiding intervals and decreased bladder compliance. Myography indicated reduced contractility of Nt5eKO BSM. These data support a role for NT5E in regulating bladder function through modulation of BSM contraction and relaxation. However, the abnormal bladder phenotype of Nt5eKO mice is much milder than we previously reported in A2b receptor knockout (A2bKO) mice, suggesting compensatory response(s) in Nt5eKO mouse bladder. To better understand this compensatory mechanism, we analyzed changes in purinergic and other receptors controlling BSM contraction and relaxation in the Nt5eKO bladder. We found that the relative abundance of muscarinic CHRM3 (cholinergic receptor muscarinic 3), purinergic P2X1, and A2b receptors was unchanged, whereas P2Y12 receptor was significantly downregulated, suggesting a negative feedback response to elevated ADP signaling. Further studies of additional ecto-nucleotidases indicated significant upregulation of the nonspecific urothelial alkaline phosphatase ALPL, which might mitigate the degree of voiding dysfunction by compensating for Nt5e deletion. These data suggest a mechanistic complexity of the purinergic signaling network in bladder and imply a paracrine mechanism in which urothelium-released ATP and its rapidly produced metabolites coordinately regulate BSM contraction and relaxation.

Concurrent impact of de novo mutations on cranial and cortical development in nonsyndromic craniosynostosis.

OBJECTIVE: Nonsyndromic craniosynostosis (nsCS), characterized by premature cranial suture fusion, is considered a primary skull disorder in which impact on neurodevelopment, if present, results from the mechanical hindrance of brain growth. Despite surgical repair of the cranial defect, neurocognitive deficits persist in nearly half of affected children. Therefore, the authors performed a functional genomics analysis of nsCS to determine when, where, and in what cell types nsCS-associated genes converge during development. METHODS: The authors integrated whole-exome sequencing data from 291 nsCS proband-parent trios with 29,803 single-cell transcriptomes of the prenatal and postnatal neurocranial complex to inform when, where, and in what cell types nsCS-mutated genes might exert their pathophysiological effects. RESULTS: The authors found that nsCS-mutated genes converged in cranial osteoprogenitors and pial fibroblasts and their transcriptional networks that regulate both skull ossification and cerebral neurogenesis. Nonsyndromic CS-mutated genes also converged in inhibitory neurons and gene coexpression modules that overlapped with autism and other developmental disorders. Ligand-receptor cell-cell communication analysis uncovered crosstalk between suture osteoblasts and neurons via the nsCS-associated BMP, FGF, and noncanonical WNT signaling pathways. CONCLUSIONS: These data implicate a concurrent impact of nsCS-associated de novo mutations on cranial morphogenesis and cortical development via cell- and non-cell-autonomous mechanisms in a developmental nexus of fetal osteoblasts, pial fibroblasts, and neurons. These results suggest that neurodevelopmental outcomes in nsCS patients may be driven more by mutational status than surgical technique.

A novel SMARCC1 BAFopathy implicates neural progenitor epigenetic dysregulation in human hydrocephalus.

Hydrocephalus, characterized by cerebral ventriculomegaly, is the most common disorder requiring brain surgery in children. Recent studies have implicated SMARCC1, a component of the BRG1-associated factor (BAF) chromatin remodelling complex, as a candidate congenital hydrocephalus gene. However, SMARCC1 variants have not been systematically examined in a large patient cohort or conclusively linked with a human syndrome. Moreover, congenital hydrocephalus-associated SMARCC1 variants have not been functionally validated or mechanistically studied in vivo. Here, we aimed to assess the prevalence of SMARCC1 variants in an expanded patient cohort, describe associated clinical and radiographic phenotypes, and assess the impact of Smarcc1 depletion in a novel Xenopus tropicalis model of congenital hydrocephalus. To do this, we performed a genetic association study using whole-exome sequencing from a cohort consisting of 2697 total ventriculomegalic trios, including patients with neurosurgically-treated congenital hydrocephalus, that total 8091 exomes collected over 7 years (2016-23). A comparison control cohort consisted of 1798 exomes from unaffected siblings of patients with autism spectrum disorder and their unaffected parents were sourced from the Simons Simplex Collection. Enrichment and impact on protein structure were assessed in identified variants. Effects on the human fetal brain transcriptome were examined with RNA-sequencing and Smarcc1 knockdowns were generated in Xenopus and studied using optical coherence tomography imaging, in situ hybridization and immunofluorescence. SMARCC1 surpassed genome-wide significance thresholds, yielding six rare, protein-altering de novo variants localized to highly conserved residues in key functional domains. Patients exhibited hydrocephalus with aqueductal stenosis; corpus callosum abnormalities, developmental delay, and cardiac defects were also common. Xenopus knockdowns recapitulated both aqueductal stenosis and cardiac defects and were rescued by wild-type but not patient-specific variant SMARCC1. Hydrocephalic SMARCC1-variant human fetal brain and Smarcc1-variant Xenopus brain exhibited a similarly altered expression of key genes linked to midgestational neurogenesis, including the transcription factors NEUROD2 and MAB21L2. These results suggest de novo variants in SMARCC1 cause a novel human BAFopathy we term 'SMARCC1-associated developmental dysgenesis syndrome', characterized by variable presence of cerebral ventriculomegaly, aqueductal stenosis, developmental delay and a variety of structural brain or cardiac defects. These data underscore the importance of SMARCC1 and the BAF chromatin remodelling complex for human brain morphogenesis and provide evidence for a 'neural stem cell' paradigm of congenital hydrocephalus pathogenesis. These results highlight utility of trio-based whole-exome sequencing for identifying pathogenic variants in sporadic congenital structural brain disorders and suggest whole-exome sequencing may be a valuable adjunct in clinical management of congenital hydrocephalus patients.

Challenges to dialysis treatment during the COVID-19 pandemic: a qualitative study of patients' and experts' perspectives.

Background: The global COVID-19 pandemic transformed healthcare services in ways that have impacted individual physical and psychological health. The substantial health challenges routinely faced by dialysis-dependent patients with advanced kidney disease have increased considerably during the ongoing COVID-19 pandemic but remain inadequately investigated. We therefore decided to analyze and compare the perspectives of dialysis patients on their own needs and challenges during the COVID-19 pandemic with those of their professional healthcare providers through interviews with both groups. Methods: Qualitative study of seven in-center hemodialysis patients, seven peritoneal dialysis patients, seven dialysis nurses, and seven physicians at the Medical University of Vienna between March 2020 and February 2021, involving content analysis of semi-structured interviews supported by a natural language processing technique. Results: Among the main themes emerging from interviews with patients were: (1) concerns about being a 'high-risk patient'; (2) little fear of COVID-19 as a patient on hemodialysis; (3) questions about home dialysis as a better choice than in-center dialysis. Among the main themes brought up by physicians and nurses were: (1) anxiety, sadness, and loneliness of peritoneal dialysis patients; (2) negative impact of changes in clinical routine on patients' well-being; (3) telehealth as a new modality of care. Conclusion: Preventive measures against COVID-19 (e.g., use of facemasks, distancing, isolation), the introduction of telemedicine, and an increase in home dialysis have led to communication barriers and reduced face-to-face and direct physical contact between healthcare providers and patients. Physicians did not perceive the full extent of patients' psychological burdens. Selection/modification of dialysis modality should include analysis of the patient's support network and proactive discussion between dialysis patients and their healthcare providers about implications of the ongoing COVID-19 epidemic. Modification of clinical routine care to increase frequency of psychological evaluation should be considered in anticipation of future surges of COVID-19 or currently unforeseen pandemics.

Human genetics and molecular genomics of Chiari malformation type 1.

Chiari malformation type 1 (CM1) is the most common structural brain disorder involving the craniocervical junction, characterized by caudal displacement of the cerebellar tonsils below the foramen magnum into the spinal canal. Despite the heterogeneity of CM1, its poorly understood patho-etiology has led to a 'one-size-fits-all' surgical approach, with predictably high rates of morbidity and treatment failure. In this review we present multiplex CM1 families, associated Mendelian syndromes, and candidate genes from recent whole exome sequencing (WES) and other genetic studies that suggest a significant genetic contribution from inherited and de novo germline variants impacting transcription regulation, craniovertebral osteogenesis, and embryonic developmental signaling. We suggest that more extensive WES may identify clinically relevant, genetically defined CM1 subtypes distinguished by unique neuroradiographic and neurophysiological endophenotypes.

Blockade of the mineralocorticoid receptor improves markers of human endothelial cell dysfunction and hematological indices in a mouse model of sickle cell disease.

Increased endothelin-1 (ET-1) levels in patients with sickle cell disease (SCD) and transgenic mouse models of SCD contribute to disordered hematological, vascular, and inflammatory responses. Mineralocorticoid receptor (MR) activation by aldosterone, a critical component of the Renin-Angiotensin-Aldosterone-System, modulates inflammation and vascular reactivity, partly through increased ET-1 expression. However, the role of MR in SCD remains unclear. We hypothesized that MR blockade in transgenic SCD mice would reduce ET-1 levels, improve hematological parameters, and reduce inflammation. Berkeley SCD (BERK) mice, a model of severe SCD, were randomized to either sickle standard chow or chow containing the MR antagonist (MRA), eplerenone (156 mg/Kg), for 14 days. We found that MRA treatment reduced ET-1 plasma levels (p = .04), improved red cell density gradient profile (D50 ; p < .002), and increased mean corpuscular volume in both erythrocytes (p < .02) and reticulocytes (p < .024). MRA treatment also reduced the activity of the erythroid intermediate-conductance Ca2+ -activated K+ channel - KCa 3.1 (Gardos channel, KCNN4), reduced cardiac levels of mRNAs encoding ET-1, Tumor Necrosis Factor Receptor-1, and protein disulfide isomerase (PDI) (p < .01), and decreased plasma PDI and myeloperoxidase activity. Aldosterone (10-8 M for 24 h in vitro) also increased PDI mRNA levels (p < .01) and activity (p < .003) in EA.hy926 human endothelial cells, in a manner blocked by pre-incubation with the MRA canrenoic acid (1 µM; p < .001). Our results suggest a novel role for MR activation in SCD that may exacerbate SCD pathophysiology and clinical complications.

Computational drug repositioning of clopidogrel as a novel therapeutic option for focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a glomerular lesion often associated with nephrotic syndrome. It is also associated with a high risk of progression to end-stage kidney disease. Current treatment of FSGS is limited to systemic corticosteroids or calcineurin inhibition, along with inhibitors of the renin-angiotensin-aldosterone system. FSGS is heterogeneous in etiology, and novel therapies targeting specific, dysregulated molecular pathways represent a major unmet medical need. We have generated a network-based molecular model of FSGS pathophysiology using previously established systems biology workflows to allow computational evaluation of compounds for their predicted interference with molecular processes contributing to FSGS. We identified the anti-platelet drug clopidogrel as a therapeutic option to counterbalance dysregulated FSGS pathways. This prediction of our computational screen was validated by testing clopidogrel in the adriamycin FSGS mouse model. Clopidogrel improved key FSGS outcome parameters and significantly reduced urinary albumin to creatinine ratio (P < 0.01) and weight loss (P < 0.01), and ameliorated histopathological damage (P < 0.05). Clopidogrel is used to treat several cardiovascular diseases linked to chronic kidney disease. Clopidogrel's favorable safety profile and its efficacy in the adriamycin mouse FSGS model thus recommend it as an attractive drug repositioning candidate for clinical trial in FSGS.

A novel SMARCC1 -mutant BAFopathy implicates epigenetic dysregulation of neural progenitors in hydrocephalus.

Importance: Hydrocephalus, characterized by cerebral ventriculomegaly, is the most common disorder requiring brain surgery. A few familial forms of congenital hydrocephalus (CH) have been identified, but the cause of most sporadic cases of CH remains elusive. Recent studies have implicated SMARCC1 , a component of the B RG1- a ssociated factor (BAF) chromatin remodeling complex, as a candidate CH gene. However, SMARCC1 variants have not been systematically examined in a large patient cohort or conclusively linked with a human syndrome. Moreover, CH-associated SMARCC1 variants have not been functionally validated or mechanistically studied in vivo . Objectives: The aims of this study are to (i) assess the extent to which rare, damaging de novo mutations (DNMs) in SMARCC1 are associated with cerebral ventriculomegaly; (ii) describe the clinical and radiographic phenotypes of SMARCC1 -mutated patients; and (iii) assess the pathogenicity and mechanisms of CH-associated SMARCC1 mutations in vivo . Design setting and participants: A genetic association study was conducted using whole-exome sequencing from a cohort consisting of 2,697 ventriculomegalic trios, including patients with neurosurgically-treated CH, totaling 8,091 exomes collected over 5 years (2016-2021). Data were analyzed in 2023. A comparison control cohort consisted of 1,798 exomes from unaffected siblings of patients with autism spectrum disorder and their unaffected parents sourced from the Simons simplex consortium. Main outcomes and measures: Gene variants were identified and filtered using stringent, validated criteria. Enrichment tests assessed gene-level variant burden. In silico biophysical modeling estimated the likelihood and extent of the variant impact on protein structure. The effect of a CH-associated SMARCC1 mutation on the human fetal brain transcriptome was assessed by analyzing RNA-sequencing data. Smarcc1 knockdowns and a patient-specific Smarcc1 variant were tested in Xenopus and studied using optical coherence tomography imaging, in situ hybridization, and immunofluorescence microscopy. Results: SMARCC1 surpassed genome-wide significance thresholds in DNM enrichment tests. Six rare protein-altering DNMs, including four loss-of-function mutations and one recurrent canonical splice site mutation (c.1571+1G>A) were detected in unrelated patients. DNMs localized to the highly conserved DNA-interacting SWIRM, Myb-DNA binding, Glu-rich, and Chromo domains of SMARCC1 . Patients exhibited developmental delay (DD), aqueductal stenosis, and other structural brain and heart defects. G0 and G1 Smarcc1 Xenopus mutants exhibited aqueductal stenosis and cardiac defects and were rescued by human wild-type SMARCC1 but not a patient-specific SMARCC1 mutant. Hydrocephalic SMARCC1 -mutant human fetal brain and Smarcc1 -mutant Xenopus brain exhibited a similarly altered expression of key genes linked to midgestational neurogenesis, including the transcription factors NEUROD2 and MAB21L2 . Conclusions: SMARCC1 is a bona fide CH risk gene. DNMs in SMARCC1 cause a novel human BAFopathy we term " S MARCC1- a ssociated D evelopmental D ysgenesis S yndrome (SaDDS)", characterized by cerebral ventriculomegaly, aqueductal stenosis, DD, and a variety of structural brain or cardiac defects. These data underscore the importance of SMARCC1 and the BAF chromatin remodeling complex for human brain morphogenesis and provide evidence for a "neural stem cell" paradigm of human CH pathogenesis. These results highlight the utility of trio-based WES for identifying risk genes for congenital structural brain disorders and suggest WES may be a valuable adjunct in the clinical management of CH patients. KEY POINTS: Question: What is the role of SMARCC1 , a core component of the B RG1- a ssociated factor (BAF) chromatin remodeling complex, in brain morphogenesis and congenital hydrocephalus (CH)? Findings: SMARCC1 harbored an exome-wide significant burden of rare, protein-damaging de novo mutations (DNMs) (p = 5.83 × 10 -9 ) in the largest ascertained cohort to date of patients with cerebral ventriculomegaly, including treated CH (2,697 parent-proband trios). SMARCC1 contained four loss-of-function DNMs and two identical canonical splice site DNMs in a total of six unrelated patients. Patients exhibited developmental delay, aqueductal stenosis, and other structural brain and cardiac defects. Xenopus Smarcc1 mutants recapitulated core human phenotypes and were rescued by the expression of human wild-type but not patient-mutant SMARCC1 . Hydrocephalic SMARCC1 -mutant human brain and Smarcc1 -mutant Xenopus brain exhibited similar alterationsin the expression of key transcription factors that regulate neural progenitor cell proliferation. Meaning: SMARCC1 is essential for human brain morphogenesis and is a bona fide CH risk gene. SMARCC1 mutations cause a novel human BAFopathy we term " S MARCC1- a ssociated D evelopmental D ysgenesis S yndrome (SaDDS)". These data implicate epigenetic dysregulation of fetal neural progenitors in the pathogenesis of hydrocephalus, with diagnostic and prognostic implications for patients and caregivers.

Multiomic analyses implicate a neurodevelopmental program in the pathogenesis of cerebral arachnoid cysts.

Cerebral arachnoid cysts (ACs) are one of the most common and poorly understood types of developmental brain lesion. To begin to elucidate AC pathogenesis, we performed an integrated analysis of 617 patient-parent (trio) exomes, 152,898 human brain and mouse meningeal single-cell RNA sequencing transcriptomes and natural language processing data of patient medical records. We found that damaging de novo variants (DNVs) were highly enriched in patients with ACs compared with healthy individuals (P = 1.57 × 10-33). Seven genes harbored an exome-wide significant DNV burden. AC-associated genes were enriched for chromatin modifiers and converged in midgestational transcription networks essential for neural and meningeal development. Unsupervised clustering of patient phenotypes identified four AC subtypes and clinical severity correlated with the presence of a damaging DNV. These data provide insights into the coordinated regulation of brain and meningeal development and implicate epigenomic dysregulation due to DNVs in AC pathogenesis. Our results provide a preliminary indication that, in the appropriate clinical context, ACs may be considered radiographic harbingers of neurodevelopmental pathology warranting genetic testing and neurobehavioral follow-up. These data highlight the utility of a systems-level, multiomics approach to elucidate sporadic structural brain disease.

The choroid plexus links innate immunity to CSF dysregulation in hydrocephalus.

The choroid plexus (ChP) is the blood-cerebrospinal fluid (CSF) barrier and the primary source of CSF. Acquired hydrocephalus, caused by brain infection or hemorrhage, lacks drug treatments due to obscure pathobiology. Our integrated, multi-omic investigation of post-infectious hydrocephalus (PIH) and post-hemorrhagic hydrocephalus (PHH) models revealed that lipopolysaccharide and blood breakdown products trigger highly similar TLR4-dependent immune responses at the ChP-CSF interface. The resulting CSF "cytokine storm", elicited from peripherally derived and border-associated ChP macrophages, causes increased CSF production from ChP epithelial cells via phospho-activation of the TNF-receptor-associated kinase SPAK, which serves as a regulatory scaffold of a multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents PIH and PHH by antagonizing SPAK-dependent CSF hypersecretion. These results reveal the ChP as a dynamic, cellularly heterogeneous tissue with highly regulated immune-secretory capacity, expand our understanding of ChP immune-epithelial cell cross talk, and reframe PIH and PHH as related neuroimmune disorders vulnerable to small molecule pharmacotherapy.

A neural stem cell paradigm of pediatric hydrocephalus.

Pediatric hydrocephalus, the leading reason for brain surgery in children, is characterized by enlargement of the cerebral ventricles classically attributed to cerebrospinal fluid (CSF) overaccumulation. Neurosurgical shunting to reduce CSF volume is the default treatment that intends to reinstate normal CSF homeostasis, yet neurodevelopmental disability often persists in hydrocephalic children despite optimal surgical management. Here, we discuss recent human genetic and animal model studies that are shifting the view of pediatric hydrocephalus from an impaired fluid plumbing model to a new paradigm of dysregulated neural stem cell (NSC) fate. NSCs are neuroprogenitor cells that comprise the germinal neuroepithelium lining the prenatal brain ventricles. We propose that heterogenous defects in the development of these cells converge to disrupt cerebrocortical morphogenesis, leading to abnormal brain-CSF biomechanical interactions that facilitate passive pooling of CSF and secondary ventricular distention. A significant subset of pediatric hydrocephalus may thus in fact be due to a developmental brain malformation leading to secondary enlargement of the ventricles rather than a primary defect of CSF circulation. If hydrocephalus is indeed a neuroradiographic presentation of an inborn brain defect, it suggests the need to focus on optimizing neurodevelopment, rather than CSF diversion, as the primary treatment strategy for these children.

OXGR1 is a candidate disease gene for human calcium oxalate nephrolithiasis.

PURPOSE: Nephrolithiasis (NL) affects 1 in 11 individuals worldwide, leading to significant patient morbidity. NL is associated with nephrocalcinosis (NC), a risk factor for chronic kidney disease. Causative genetic variants are detected in 11% to 28% of NL and/or NC, suggesting that additional NL/NC-associated genetic loci await discovery. Therefore, we employed genomic approaches to discover novel genetic forms of NL/NC. METHODS: Exome sequencing and directed sequencing of the OXGR1 locus were performed in a worldwide NL/NC cohort. Putatively deleterious, rare OXGR1 variants were functionally characterized. RESULTS: Exome sequencing revealed a heterozygous OXGR1 missense variant (c.371T>G, p.L124R) cosegregating with calcium oxalate NL and/or NC disease in an autosomal dominant inheritance pattern within a multigenerational family with 5 affected individuals. OXGR1 encodes 2-oxoglutarate (α-ketoglutarate [AKG]) receptor 1 in the distal nephron. In response to its ligand AKG, OXGR1 stimulates the chloride-bicarbonate exchanger, pendrin, which also regulates transepithelial calcium transport in cortical connecting tubules. Strong amino acid conservation in orthologs and paralogs, severe in silico prediction scores, and extreme rarity in exome population databases suggested that the variant was deleterious. Interrogation of the OXGR1 locus in 1107 additional NL/NC families identified 5 additional deleterious dominant variants in 5 families with calcium oxalate NL/NC. Rare, potentially deleterious OXGR1 variants were enriched in patients with NL/NC compared with Exome Aggregation Consortium controls (χ2 = 7.117, P = .0076). Wild-type OXGR1-expressing Xenopus oocytes exhibited AKG-responsive Ca2+ uptake. Of 5 NL/NC-associated missense variants, 5 revealed impaired AKG-dependent Ca2+ uptake, demonstrating loss of function. CONCLUSION: Rare, dominant loss-of-function OXGR1 variants are associated with recurrent calcium oxalate NL/NC disease.

Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes.

Two heterozygous missense variants (G1 and G2) of Apolipoprotein L1 (APOL1) found in individuals of recent African ancestry can attenuate the severity of infection by some forms of Trypanosoma brucei. However, these two variants within a broader African haplotype also increase the risk of kidney disease in Americans of African descent. Although overexpression of either variant G1 or G2 causes multiple pathogenic changes in cultured cells and transgenic mouse models, the mechanism(s) promoting kidney disease remain unclear. Human serum APOL1 kills trypanosomes through its cation channel activity, and cation channel activity of recombinant APOL1 has been reconstituted in lipid bilayers and proteoliposomes. Although APOL1 overexpression increases whole cell cation currents in HEK-293 cells, the ion channel activity of APOL1 has not been assessed in glomerular podocytes, the major site of APOL1-associated kidney diseases. We characterize APOL1-associated whole cell and on-cell cation currents in HEK-293 T-Rex cells and demonstrate partial inhibition of currents by anti-APOL antibodies. We detect in primary human podocytes a similar cation current inducible by interferon-γ (IFNγ) and sensitive to inhibition by anti-APOL antibody as well as by a fragment of T. brucei Serum Resistance-Associated protein (SRA). CRISPR knockout of APOL1 in human primary podocytes abrogates the IFNγ-induced, antibody-sensitive current. Our novel characterization in HEK-293 cells of heterologous APOL1-associated cation conductance inhibited by anti-APOL antibody and our documentation in primary human glomerular podocytes of endogenous IFNγ-stimulated, APOL1-mediated, SRA and anti-APOL-sensitive ion channel activity together support APOL1-mediated channel activity as a therapeutic target for treatment of APOL1-associated kidney diseases.

Magnesium homeostasis in deoxygenated sickle erythrocytes is modulated by endothelin-1 via Na+ /Mg2+ exchange.

Painful crises in sickle cell disease (SCD) are associated with increased plasma cytokines levels, including endothelin-1 (ET-1). Reduced red cell magnesium content, mediated in part by increased Na+ /Mg2+ exchanger (NME) activity, contributes to erythrocyte K+ loss, dehydration and sickling in SCD. However, the relationship between ET-1 and the NME in SCD has remained unexamined. We observed increased NME activity in sickle red cells incubated in the presence of 500 nM ET-1. Deoxygenation of sickle red cells, in contrast, led to decreased red cell NME activity and cellular dehydration that was reversed by the NME inhibitor, imipramine. Increased NME activity in sickle red cells was significantly blocked by pre-incubation with 100 nM BQ788, a selective blocker of ET-1 type B receptors. These results suggest an important role for ET-1 and for cellular magnesium homeostasis in SCD. Consistent with these results, we observed increased NME activity in sickle red cells of three mouse models of sickle cell disease greater than that in red cells of C57BL/J6 mice. In vivo treatment of BERK sickle transgenic mice with ET-1 receptor antagonists reduced red cell NME activity. Our results suggest that ET-1 receptor blockade may be a promising therapeutic approach to control erythrocyte volume and magnesium homeostasis in SCD and may thus attenuate or retard the associated chronic inflammatory and vascular complications of SCD.

Combined Treatment with KV Channel Inhibitor 4-Aminopyridine and either γ-Cystathionine Lyase Inhibitor ß-Cyanoalanine or Epinephrine Restores Blood Pressure, and Improves Survival in the Wistar Rat Model of Anaphylactic Shock.

The mechanism of anaphylactic shock (AS) remains incompletely understood. The potassium channel blocker 4-aminopyridine (4-AP), the inhibitors of cystathionine γ-lyase (ICSE), dl-propargylglycine (DPG) or ß-cyanoalanine (BCA), and the nitric oxide (NO) synthase produce vasoconstriction and could be an alternative for the treatment of AS. The aim of this study was to demonstrate the ability of L-NAME, ICSE alone or in combination with 4-AP to restore blood pressure (BP) and improve survival in ovalbumin (OVA) rats AS. Experimental groups included non-sensitized Wistar rats (n = 6); AS (n = 6); AS (n = 10 per group) treated i.v. with 4-AP (AS+4-AP), epinephrine (AS+EPI), AS+DPG, AS+BCA, or with L-NAME (AS+L-NAME); or AS treated with drug combinations 4-AP+DPG, 4-AP+BCA, 4-AP+L-NAME, or 4-AP+EPI. AS was induced by i.v. OVA (1 mg). Treatments were administered i.v. one minute after AS induction. Mean arterial BP (MAP), heart rate (HR), and survival were monitored for 60 min. Plasma levels of histamine, prostaglandin E2 (PGE2) and F2 (PGF2α), leukotriene B4 and C4, angiotensin II, vasopressin, oxidative stress markers, pH, HCO3, PaO2, PaCO2, and K+ were measured. OVA induced severe hypotension and all AS rats died. Moreover, 4-AP, 4-AP+EPI, or 4-AP+BCA normalized both MAP and HR and increased survival. All sensitized rats treated with 4-AP alone or with 4-AP+BCA survived. The time-integrated MAP "area under the curve" was significantly higher after combined 4-AP treatment with ICSE. Metabolic acidosis was not rescued and NO, ICSE, and Kv inhibitors differentially alter oxidative stress and plasma levels of anaphylactic mediators. The AS-induced reduction of serum angiotensin II levels was prevented by 4-AP treatment alone or in combination with other drugs. Further, 4-AP treatment combined with EPI or with BCA also increased serum PGF2α, whereas only the 4-AP+EPI combination increased serum LTB4. Serum vasopressin and angiotensin II levels were increased by 4-AP treatment alone or in combination with other drugs. Moreover, 4-AP alone and in combination with inhibition of cystathionine γ-lyase or EPI normalizes BP, increases serum vasoconstrictor levels, and improves survival in the Wistar rat model of AS. These findings suggest possible investigative treatment pathways for research into epinephrine-refractory anaphylactic shock in patients.

ADAR regulates APOL1 via A-to-I RNA editing by inhibition of MDA5 activation in a paradoxical biological circuit.

APOL1 risk variants are associated with increased risk of kidney disease in patients of African ancestry, but not all individuals with the APOL1 high-risk genotype develop kidney disease. As APOL1 gene expression correlates closely with the degree of kidney cell injury in both cell and animal models, the mechanisms regulating APOL1 expression may be critical determinants of risk allele penetrance. The APOL1 messenger RNA includes Alu elements at the 3' untranslated region that can form a double-stranded RNA structure (Alu-dsRNA) susceptible to posttranscriptional adenosine deaminase acting on RNA (ADAR)-mediated adenosine-to-inosine (A-to-I) editing, potentially impacting gene expression. We studied the effects of ADAR expression and A-to-I editing on APOL1 levels in podocytes, human kidney tissue, and a transgenic APOL1 mouse model. In interferon-γ (IFN-γ)-stimulated human podocytes, ADAR down-regulates APOL1 by preventing melanoma differentiation-associated protein 5 (MDA5) recognition of dsRNA and the subsequent type I interferon (IFN-I) response. Knockdown experiments showed that recognition of APOL1 messenger RNA itself is an important contributor to the MDA5-driven IFN-I response. Mathematical modeling suggests that the IFN-ADAR-APOL1 network functions as an incoherent feed-forward loop, a biological circuit capable of generating fast, transient responses to stimuli. Glomeruli from human kidney biopsies exhibited widespread editing of APOL1 Alu-dsRNA, while the transgenic mouse model closely replicated the edited sites in humans. APOL1 expression in mice was inversely correlated with Adar1 expression under IFN-γ stimuli, supporting the idea that ADAR regulates APOL1 levels in vivo. ADAR-mediated A-to-I editing is an important regulator of APOL1 expression that could impact both penetrance and severity of APOL1-associated kidney disease.

The erythroid K-Cl cotransport inhibitor [(dihydroindenyl)oxy]acetic acid blocks erythroid Ca2+-activated K+ channel KCNN4.

Red cell volume is a major determinant of HbS concentration in sickle cell disease. Cellular deoxy-HbS concentration determines the delay time, the interval between HbS deoxygenation and deoxy-HbS polymerization. Major membrane transporter protein determinants of sickle red cell volume include the SLC12/KCC K-Cl cotransporters KCC3/SLC12A6 and KCC1/SLC12A4, and the KCNN4/KCa3.1 Ca2+-activated K+ channel (Gardos channel). Among standard inhibitors of KCC-mediated K-Cl cotransport, only [(dihydroindenyl)oxy]acetic acid (DIOA) has been reported to lack inhibitory activity against the related bumetanide-sensitive erythroid Na-K-2Cl cotransporter NKCC1/SLC12A2. DIOA has been often used to inhibit K-Cl cotransport when studying the expression and regulation of other K+ transporters and K+ channels. We report here that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also abrogate activity of the KCNN4/KCa3.1 Gardos channel in human and mouse red cells and in human sickle red cells. DIOA inhibition of A23187-stimulated erythroid K+ uptake (Gardos channel activity) was chloride-independent and persisted in mouse red cells genetically devoid of the principal K-Cl cotransporters KCC3 and KCC1. DIOA also inhibited YODA1-stimulated, chloride-independent erythroid K+ uptake. In contrast, DIOA exhibited no inhibitory effect on K+ influx into A23187-treated red cells of Kcnn4-/- mice. DIOA inhibition of human KCa3.1 was validated (IC50 42 µM) by whole cell patch clamp in HEK-293 cells. RosettaLigand docking experiments identified a potential binding site for DIOA in the fenestration region of human KCa3.1. We conclude that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also block the KCNN4/KCa3.1 Gardos channel in normal and sickle red cells.

Activation of 2-oxoglutarate receptor 1 (OXGR1) by α-ketoglutarate (αKG) does not detectably stimulate Pendrin-mediated anion exchange in Xenopus oocytes.

SLC26A4/Pendrin is the major electroneutral Cl- /HCO3- exchanger of the apical membrane of the Type B intercalated cell (IC) of the connecting segment (CNT) and cortical collecting duct (CCD). Pendrin mediates both base secretion in response to systemic base load and Cl- reabsorption in response to systemic volume depletion, manifested as decreased nephron salt and water delivery to the distal nephron. Pendrin-mediated Cl- /HCO3- exchange in the apical membrane is upregulated through stimulation of the ß-IC apical membrane G protein-coupled receptor, 2-oxoglutarate receptor 1 (OXGR1/GPR99), by its ligand α-ketoglutarate (αKG). αKG is both filtered by the glomerulus and lumenally secreted by proximal tubule apical membrane organic anion transporters (OATs). OXGR1-mediated regulation of Pendrin by αKG has been documented in transgenic mice and in isolated perfused CCD. However, aspects of the OXGR1 signaling pathway have remained little investigated since its original discovery in lymphocytes. Moreover, no ex vivo cellular system has been reported in which to study the OXGR1 signaling pathway of Type B-IC, a cell type refractory to survival in culture in its differentiated state. As Xenopus oocytes express robust heterologous Pendrin activity, we investigated OXGR1 regulation of Pendrin in oocytes. Despite functional expression of OXGR1 in oocytes, co-expression of Pendrin and OXGR1 failed to exhibit αKG-sensitive stimulation of Pendrin-mediated Cl- /anion exchange under a wide range of conditions. We conclude that Xenopus oocytes lack one or more essential molecular components or physical conditions required for OXGR1 to regulate Pendrin activity.