Histologic and Clinical Factors Associated with Kidney Outcomes in IgA Vasculitis Nephritis.

BACKGROUND: Nephritis is a common manifestation of IgA vasculitis and is morphologically indistinguishable from IgA nephropathy. While MEST-C scores are predictive of kidney outcomes in IgA nephropathy, their value in IgA vasculitis nephritis has not been investigated in large multiethnic cohorts. METHODS: Biopsies from 262 children and 99 adults with IgA vasculitis nephritis ( N =361) from 23 centers in North America, Europe, and Asia were independently scored by three pathologists. MEST-C scores were assessed for correlation with eGFR/proteinuria at biopsy. Because most patients ( N =309, 86%) received immunosuppression, risk factors for outcomes were evaluated in this group using latent class mixed models to identify classes of eGFR trajectories over a median follow-up of 2.7 years (interquartile range, 1.2-5.1). Clinical and histologic parameters associated with each class were determined using logistic regression. RESULTS: M, E, T, and C scores were correlated with either eGFR or proteinuria at biopsy. Two classes were identified by latent class mixed model, one with initial improvement in eGFR followed by a late decline (class 1, N =91) and another with stable eGFR (class 2, N =218). Class 1 was associated with a higher risk of an established kidney outcome (time to ≥30% decline in eGFR or kidney failure; hazard ratio, 5.84; 95% confidence interval, 2.37 to 14.4). Among MEST-C scores, only E1 was associated with class 1 by multivariable analysis. Other factors associated with class 1 were age 18 years and younger, male sex, lower eGFR at biopsy, and extrarenal noncutaneous disease. Fibrous crescents without active changes were associated with class 2. CONCLUSIONS: Kidney outcome in patients with biopsied IgA vasculitis nephritis treated with immunosuppression was determined by clinical risk factors and endocapillary hypercellularity (E1) and fibrous crescents, which are features that are not part of the International Study of Diseases of Children classification.

Medication patterns and dosing guidance in pediatric patients supported with intermittent hemodialysis or continuous kidney replacement therapy.

BACKGROUND: Hemodialysis is a life-saving technology used during periods of acute or chronic kidney failure to remove toxins, and maintain fluid, electrolyte and metabolic balance. While this technology plays an important role for pediatric patients with kidney dysfunction, it can alter the pharmacokinetic behavior of medications placing patients at risk for suboptimal dosing and drug toxicity. The ability to directly translate pharmacokinetic alterations into dosing recommendations has thus far been limited and dosing guidance specific to pediatric hemodialysis patients is rare. Despite differences in dialysis prescription and patient populations, intermittent (iHD) and continuous kidney replacement therapy (CKRT) patients are often pooled together. In order to develop evidence-based dosing guidelines, it is important to first prioritize drugs for study in each modality. METHODS: Here we aim to identify priority drugs in two hemodialysis modalities, through: 1) Identification of hospitalized, pediatric patients who received CKRT or intermittent hemodialysis (iHD) using a machine learning-based predictive model based on medications; 2) Identification of medication administration patterns in these patient cohorts; and 3) Identification of the most commonly prescribed drugs that lack published dosing guidance. RESULTS: Notable differences were found in the pattern of medications and drug dosing guidance between iHD and CKRT patients. Antibiotics, diuretics and sedatives were more common in CKRT patients. Out of the 50 most commonly administered medications in the two modalities, only 34% and 28% had dosing guidance present for iHD and CKRT, respectively. CONCLUSIONS: Our results add to the understanding of the differences between iHD and CKRT patient populations by identifying commonly used medications that lack dosing guidance for each hemodialysis modality, helping to pinpoint priority medications for further study. Overall, this study provides an overview of the current limitations in medication use in this at-risk population, and provides a framework for future studies by identifying commonly used medications in pediatric CKRT and iHD patients.

Genome-wide association analyses define pathogenic signaling pathways and prioritize drug targets for IgA nephropathy.

IgA nephropathy (IgAN) is a progressive form of kidney disease defined by glomerular deposition of IgA. Here we performed a genome-wide association study of 10,146 kidney-biopsy-diagnosed IgAN cases and 28,751 controls across 17 international cohorts. We defined 30 genome-wide significant risk loci explaining 11% of disease risk. A total of 16 loci were new, including TNFSF4/TNFSF18, REL, CD28, PF4V1, LY86, LYN, ANXA3, TNFSF8/TNFSF15, REEP3, ZMIZ1, OVOL1/RELA, ETS1, IGH, IRF8, TNFRSF13B and FCAR. The risk loci were enriched in gene orthologs causing abnormal IgA levels when genetically manipulated in mice. We also observed a positive genetic correlation between IgAN and serum IgA levels. High polygenic score for IgAN was associated with earlier onset of kidney failure. In a comprehensive functional annotation analysis of candidate causal genes, we observed convergence of biological candidates on a common set of inflammatory signaling pathways and cytokine ligand-receptor pairs, prioritizing potential new drug targets.

Acute kidney injury requiring kidney replacement therapy in childhood lupus nephritis: a cohort study of the Pediatric Nephrology Research Consortium and Childhood Arthritis and Rheumatology Research Alliance.

BACKGROUND: Acute kidney injury (AKI) is common in lupus nephritis (LN) and a risk factor for development of chronic kidney disease. In adults with LN, AKI severity correlates with the incidence of kidney failure and patient survival. Data on AKI outcomes in children with LN, particularly those requiring kidney replacement therapy (KRT), are limited. METHODS: A multicenter, retrospective cohort study was performed in children diagnosed between 2010 and 2019 with LN and AKI stage 3 treated with dialysis (AKI stage 3D). Descriptive statistics were used to characterize demographics, clinical data, and kidney biopsy findings; treatment data for LN were not included. Logistic regression was used to examine the association of these variables with kidney failure. RESULTS: Fifty-nine patients (mean age 14.3 years, 84.7% female) were identified. The most common KRT indications were fluid overload (86.4%) and elevated blood urea nitrogen/creatinine (74.6%). Mean follow-up duration was 3.9 ± 2.9 years. AKI recovery without progression to kidney failure occurred in 37.3% of patients. AKI recovery with later progression to kidney failure occurred in 25.4% of patients, and there was no kidney recovery from AKI in 35.6% of patients. Older age, severe (> 50%) tubular atrophy and interstitial fibrosis, and National Institutes of Health (NIH) chronicity index score > 4 on kidney biopsy were associated with kidney failure. CONCLUSIONS: Children with LN and AKI stage 3D have a high long-term risk of kidney failure. Severe tubular atrophy and interstitial fibrosis at the time of AKI, but not AKI duration, are predictive of kidney disease progression. A higher resolution version of the Graphical abstract is available as Supplementary information.

Medication based machine learning to identify subpopulations of pediatric hemodialysis patients in an electronic health record database.

Electronic health records (EHRs) have given rise to large and complex databases of medical information that have the potential to become powerful tools for clinical research. However, differences in coding systems and the detail and accuracy of the information within EHRs can vary across institutions. This makes it challenging to identify subpopulations of patients and limits the widespread use of multi-institutional databases. In this study, we leveraged machine learning to identify patterns in medication usage among hospitalized pediatric patients receiving renal replacement therapy and created a predictive model that successfully differentiated between intermittent (iHD) and continuous renal replacement therapy (CRRT) hemodialysis patients. We trained six machine learning algorithms (logistical regression, Naïve Bayes, k-nearest neighbor, support vector machine, random forest, and gradient boosted trees) using patient records from a multi-center database (n = 533) and prescribed medication ingredients (n = 228) as features to discriminate between the two hemodialysis types. Predictive skill was assessed using a 5-fold cross-validation, and the algorithms showed a range of performance from 0.7 balanced accuracy (logistical regression) to 0.86 (random forest). The two best performing models were further tested using an independent single-center dataset and achieved 84-87% balanced accuracy. This model overcomes issues inherent within large databases and will allow us to utilize and combine historical records, significantly increasing population size and diversity within both iHD and CRRT populations for future clinical studies. Our work demonstrates the utility of using medications alone to accurately differentiate subpopulations of patients in large datasets, allowing codes to be transferred between different coding systems. This framework has the potential to be used to distinguish other subpopulations of patients where discriminatory ICD codes are not available, permitting more detailed insights and new lines of research.

IgA vasculitis with nephritis: update of pathogenesis with clinical implications.

IgA vasculitis with nephritis (IgAVN) shares many pathogenetic features with IgA nephropathy (IgAN). The purpose of this review is to describe our current understanding of the pathogenesis of pediatric IgAVN, particularly as it relates to the four-hit hypothesis for IgAN. These individual steps, i.e., hits, in the pathogenesis of IgAN are (1) elevated production of IgA1 glycoforms with some O-glycans deficient in galactose (galactose-deficient IgA1; Gd-IgA1), (2) generation of circulating IgG autoantibodies specific for Gd-IgA1, (3) formation of pathogenic circulating Gd-IgA1-containing immune complexes, and (4) kidney deposition of the Gd-IgA1-IgG immune complexes from the circulation and induction of glomerular injury. Evidence supporting the four-hit hypothesis in the pathogenesis of pediatric IgAVN is detailed. The genetics, pediatric outcomes, and kidney histopathologic features and the impact of these findings on future treatment and potential biomarkers are discussed. In summary, the evidence points to the critical roles of Gd-IgA1-IgG immune complexes and complement activation in the pathogenesis of IgAVN. Future studies are needed to characterize the features of the immune and autoimmune responses that enable progression of IgA vasculitis to IgAVN.

Safety and efficacy of sucroferric oxyhydroxide in pediatric patients with chronic kidney disease.

BACKGROUND: Pediatric patients with advanced chronic kidney disease (CKD) are often prescribed oral phosphate binders (PBs) for the management of hyperphosphatemia. However, available PBs have limitations, including unfavorable tolerability and safety. METHODS: This phase 3, multicenter, randomized, open-label study investigated safety and efficacy of sucroferric oxyhydroxide (SFOH) in pediatric and adolescent subjects with CKD and hyperphosphatemia. Subjects were randomized to SFOH or calcium acetate (CaAc) for a 10-week dose titration (stage 1), followed by a 24-week safety extension (stage 2). Primary efficacy endpoint was change in serum phosphorus from baseline to the end of stage 1 in the SFOH group. Safety endpoints included treatment-emergent adverse events (TEAEs). RESULTS: Eighty-five subjects (2-18 years) were randomized and treated (SFOH, n = 66; CaAc, n = 19). Serum phosphorus reduction from baseline to the end of stage 1 in the overall SFOH group (least squares [LS] mean ± standard error [SE]) was - 0.488 ± 0.186 mg/dL; p = 0.011 (post hoc analysis). Significant reductions in serum phosphorus were observed in subjects aged ≥ 12 to ≤ 18 years (LS mean ± SE - 0.460 ± 0.195 mg/dL; p = 0.024) and subjects with serum phosphorus above age-related normal ranges at baseline (LS mean ± SE - 0.942 ± 0.246 mg/dL; p = 0.005). Similar proportions of subjects reported ≥ 1 TEAE in the SFOH (75.8%) and CaAc (73.7%) groups. Withdrawal due to TEAEs was more common with CaAc (31.6%) than with SFOH (18.2%). CONCLUSIONS: SFOH effectively managed serum phosphorus in pediatric patients with a low pill burden and a safety profile consistent with that reported in adult patients.

Publisher Correction: Whole Transcriptome Analysis of Renal Intercalated Cells Predicts Lipopolysaccharide Mediated Inhibition of Retinoid X Receptor alpha Function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human Papillomavirus Vaccination in Male and Female Adolescents Before and After Kidney Transplantation: A Pediatric Nephrology Research Consortium Study.

Background: Kidney transplant (KT) recipients have higher incidence of malignancies, including Human Papillomavirus (HPV)-associated cancers. Thus, HPV vaccines may have an important role in preventing HPV-related disease in this population; however, immunogenicity and safety data are lacking. Objective: To examine the immunological response and tolerability to HPV vaccination in pediatric KT recipients compared to future KT candidates. Methods: The quadrivalent HPV vaccine was administered to girls and boys age 9-18 recruited from seven centers part of the Pediatric Nephrology Research Consortium. Subjects were recruited for three groups: (1) CKD: chronic kidney disease stages 3, 4, and 5 not on dialysis; (2) Dialysis; (3) KT recipients. The outcome consisted of antibody concentrations against HPV 6, 11, 16, and 18. Geometric mean titers (GMTs) and seroconversion rates were compared. Vaccine tolerability was assessed. Results: Sixty-five participants were recruited: 18 in the CKD, 18 in the dialysis, and 29 into the KT groups. KT patients had significantly lower GMTs after vaccination for all serotypes. The percentages of subjects who reached seroconversion were overall lower for the KT group, reaching statistical significance for HPV 6, 11, and 18. Comparing immunosuppressed subjects (anyone taking immunosuppression medications, whether KT recipient or not) with the non-immunosuppressed participants, the former had significantly lower GMTs for all the HPV serotypes and lower seroconversion rates for HPV 6, 11, and 18. KT females had higher GMTs and seroconversion rates for certain serotypes. There were no adverse events in either group. Conclusions: HPV vaccine was well-tolerated in this population. Pediatric KT recipients had in general lower GMTs and seroconversion rates compared to their peers with CKD or on dialysis. Immunosuppression played a role in the lack of seroconversion. Our results emphasize the importance of advocating for HPV vaccination prior to KT and acknowledge its safety post transplantation. Future studies are needed to investigate the effect of a supplemental dose of HPV vaccine in KT recipients who do not seroconvert and to evaluate the long-term persistence of antibodies post-KT.

Whole Transcriptome Analysis of Renal Intercalated Cells Predicts Lipopolysaccharide Mediated Inhibition of Retinoid X Receptor alpha Function.

The renal collecting duct consists of intercalated cells (ICs) and principal cells (PCs). We have previously demonstrated that collecting ducts have a role in the innate immune defense of the kidney. Transcriptomics is an important tool used to enhance systems-level understanding of cell biology. However, transcriptomics performed on whole kidneys provides limited insight of collecting duct cell gene expression, because these cells comprise a small fraction of total kidney cells. Recently we generated reporter mouse models to enrich collecting duct specific PC and ICs and reported targeted gene expression of anti-microbial peptide genes. Here we report transcriptomics on enriched ICs and PCs and performed a pilot study sequencing four single ICs. We identified 3,645 genes with increased relative expression in ICs compared to non-ICs. In comparison to non-PCs, 2,088 genes had higher relative expression in PCs. IC associated genes included the innate interleukin 1 receptor, type 1 and the antimicrobial peptide(AMP) adrenomedullin. The top predicted canonical pathway for enriched ICs was lipopolysaccharide/Interleukin 1 mediated inhibition of Retinoid X Receptor alpha function and decreased Retinoid X Receptor expression was confirmed to occur 1-hour post experimental murine UTI in ICs but not in non-ICs.

Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct.

The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanism by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells that consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H+-ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cre+tdT+ mice to fluorescently label ICs and AQP2-cre+tdT+ mice to fluorescently label PCs, and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure antimicrobial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC) challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge, this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.

Vitamin D in incident nephrotic syndrome: a Midwest Pediatric Nephrology Consortium study.

BACKGROUND: Cross-sectional studies of children with prevalent nephrotic syndrome (NS) have shown 25-vitamin D (25(OH)D) deficiency rates of 20-100 %. Information on 25(OH)D status in incident patients or following remission is limited. This study aimed to assess 25(OH)D status of incident idiopathic NS children at presentation and longitudinally with short-term observation. METHODS: Multicenter longitudinal study of children (2-18 years old) from 14 centers across the Midwest Pediatric Nephrology Consortium with incident idiopathic NS. 25(OH)D levels were assessed at diagnosis and 3 months later. RESULTS: Sixty-one children, median age 5 (3, 11) years, completed baseline visit and 51 completed second visit labs. All 61 (100 %) had 25(OH)D < 20 ng/ml at diagnosis. Twenty-seven (53 %) had 25(OH)D < 20 ng/ml at follow-up. Fourteen (28 %) children were steroid resistant. Univariate analysis showed that children prescribed vitamin D supplements were less likely to have 25(OH)D deficiency at follow-up (OR 0.2, 95 % CI 0.04, 0.6). Steroid response, age, and season did not predict 25(OH)D deficiency. Multivariable linear regression modeling showed higher 25(OH)D levels at follow-up by 13.2 ng/ml (SE 4.6, p < 0.01) in children supplemented with vitamin D. CONCLUSIONS: In this incident idiopathic NS cohort, all children at diagnosis had 25(OH)D deficiency and the majority continued to have a deficiency at 2-4 months. Supplemental vitamin D decreased the odds of 25(OH)D deficiency at follow-up, supporting a role for supplementation in incident NS.

Reducing αENaC expression in the kidney connecting tubule induces pseudohypoaldosteronism type 1 symptoms during K+ loading.

Genetic inactivation of the epithelial Na(+) channel α-subunit (αENaC) in the renal collecting duct (CD) does not interfere with Na(+) and K(+) homeostasis in mice. However, inactivation in the CD and a part of the connecting tubule (CNT) induces autosomal recessive pseudohypoaldosteronism type 1 (PHA-1) symptoms in subjects already on a standard diet. In the present study, we further examined the importance of αENaC in the CNT. Knockout mice with αENaC deleted primarily in a part of the CNT (CNT-KO) were generated using Scnn1a(lox/lox) mice and Atp6v1b1::Cre mice. With a standard diet, plasma Na(+) concentration ([Na(+)]) and [K(+)], and urine Na(+) and K(+) output were unaffected. Seven days of Na(+) restriction (0.01% Na(+)) led to a higher urine Na(+) output only on days 3-5, and after 7 days plasma [Na(+)] and [K(+)] were unaffected. In contrast, the CNT-KO mice were highly susceptible to a 2-day 5% K(+) diet and showed lower food intake and relative body weight, lower plasma [Na(+)], higher fractional excretion (FE) of Na(+), higher plasma [K(+)], and lower FE of K(+). The higher FE of Na(+) coincided with lower abundance and phosphorylation of the Na(+)-Cl(-) cotransporter. In conclusion, reducing ENaC expression in the CNT induces clear PHA-1 symptoms during high dietary K(+) loading.

Renal replacement therapy in infants and children with hepatorenal syndrome awaiting liver transplantation: a case-control study.

Limited data on short- and long-term outcomes of renal replacement therapy (RRT) in pediatric liver transplantation (LT) patients exist. We evaluated risk factors for RRT in pediatric LT recipients with hepatorenal syndrome (HRS) and described the outcomes. We performed a single-center, case-control study of LT recipients who required RRT for HRS from 1999 to 2011. Three controls who did not receive RRT were matched with each case on the basis of age, diagnosis, and LT date. We identified 8 recipients among 133 recipients of 152 LT cases [6%, 95% confidence interval = 2%-10%; mean age = 7.7 years, range = 0.5-19.8 years) who required RRT before LT for HRS. Four patients were <1 year old and weighed 5.6 to 6.6 kg. Biliary atresia was the most common LT indication. Cases had higher Model for (Pediatric) End-Stage Liver Disease scores at listing (26 versus 16, P = 0.01) and lower glomerular filtration rates (GFRs; 15 versus 102 mL/minute/1.73 m(2) , P < 0.001) at RRT initiation or LT. Ascites, gastrointestinal bleeding, and infections occurred more commonly among cases: (100% versus 54%, P = 0.03; 100% versus 46%, P = 0.01; and 88% versus 33%, P = 0.01, respectively). Cases also experienced toxic vancomycin troughs more frequently (38% versus 0%, P = 0.01) and received RRT for a median of 21 days (range = 3-355 days). The case mortality rate was 37.5% (3/8 at 1, 26, and 346 days after LT) and 0% for controls. The 4 infants required 0 to 3 dialysis catheter replacements during RRT. Cases and controls had similar median follow-ups [3.2 years (range = 1.5-7.6 years) versus 4.9 years (range = 0.2-11 years), P = 0.29]. After LT, they also had similar GFRs (83 versus 99 mL/minute/1.73 m(2) at 1 month, P = 0.19; 80 versus 107 mL/minute/1.73 m2 at 1 year, P > 0.99; and 97 versus 114 mL/minute/1.73 m2 at the most recent follow-up, P = 0.09). The case survival rates were 75% and 63% at 1 month and 1 year, respectively; 4 cases required antihypertensives and diuretics 1 month after LT, but at the last follow-up, only 1 case required antihypertensive therapy, and none required diuretics. In conclusion, pediatric patients with HRS, including infants, benefit from RRT. Although HRS decreases survival, patients with HRS who undergo LT generally recover renal function within 1 month that persists during long-term follow-up.

SLC26A4 targeted to the endolymphatic sac rescues hearing and balance in Slc26a4 mutant mice.

Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4 (Δ/Δ) mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4 (Δ/Δ) line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4.

Overexpression of pendrin in intercalated cells produces chloride-sensitive hypertension.

Inherited and acquired disorders that enhance the activity of transporters mediating renal tubular Na(+) reabsorption are well established causes of hypertension. It is unclear, however, whether primary activation of an Na(+)-independent chloride transporter in the kidney can also play a pathogenic role in this disease. Here, mice overexpressing the chloride transporter pendrin in intercalated cells of the distal nephron (Tg(B1-hPDS) mice) displayed increased renal absorption of chloride. Compared with normal mice, these transgenic mice exhibited a delayed increase in urinary NaCl and ultimately, developed hypertension when exposed to a high-salt diet. Administering the same sodium intake as NaHCO3 instead of NaCl did not significantly alter BP, indicating that the hypertension in the transgenic mice was chloride-sensitive. Moreover, excessive chloride absorption by pendrin drove parallel absorption of sodium through the epithelial sodium channel ENaC and the sodium-driven chloride/bicarbonate exchanger (Ndcbe), despite an appropriate downregulation of these sodium transporters in response to the expanded vascular volume and hypertension. In summary, chloride transport in the distal nephron can play a primary role in driving NaCl transport in this part of the kidney, and a primary abnormality in renal chloride transport can provoke arterial hypertension. Thus, we conclude that the chloride/bicarbonate exchanger pendrin plays a major role in controlling net NaCl absorption, thereby influencing BP under conditions of high salt intake.

Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities.

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K(+) and H(+) excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin-RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K(+) and pH homeostasis.

Denys-Drash syndrome with neonatal renal failure in monozygotic twins due to c.1097G>A mutation in the WT1 gene.

Denys-Drash syndrome, characterized by nephrosis, dysgenetic gonads and a predisposition to Wilms tumor, is due to germline mutations in the WT1 gene. We report the pathologic findings on monozygotic twins, both of whom presented with male pseudohermaphroditism, nephrotic syndrome, and progressed to renal failure and death within the first month of life. Sequence analysis of WT1 demonstrated a G-to-A substitution in exon 8 of the gene (c.1097G > A), resulting in an arginine-to-histidine (R366H) substitution in the second zinc finger domain. To the best of our knowledge, this is only the second set of monozygotic twins with Denys-Drash syndrome reported to date.

Deletion of hensin/DMBT1 blocks conversion of beta- to alpha-intercalated cells and induces distal renal tubular acidosis.

Acid-base transport in the renal collecting tubule is mediated by two canonical cell types: the ß-intercalated cell secretes HCO(3) by an apical Cl:HCO(3) named pendrin and a basolateral vacuolar (V)-ATPase. Acid secretion is mediated by the α-intercalated cell, which has an apical V-ATPase and a basolateral Cl:HCO(3) exchanger (kAE1). We previously suggested that the ß-cell converts to the α-cell in response to acid feeding, a process that depended on the secretion and deposition of an extracellular matrix protein termed hensin (DMBT1). Here, we show that deletion of hensin from intercalated cells results in the absence of typical α-intercalated cells and the consequent development of complete distal renal tubular acidosis (dRTA). Essentially all of the intercalated cells in the cortex of the mutant mice are canonical ß-type cells, with apical pendrin and basolateral or diffuse/bipolar V-ATPase. In the medulla, however, a previously undescribed cell type has been uncovered, which resembles the cortical ß-intercalated cell in ultrastructure, but does not express pendrin. Polymerization and deposition of hensin (in response to acidosis) requires the activation of ß1 integrin, and deletion of this gene from the intercalated cell caused a phenotype that was identical to the deletion of hensin itself, supporting its critical role in hensin function. Because previous studies suggested that the conversion of ß- to α-intercalated cells is a manifestation of terminal differentiation, the present results demonstrate that this differentiation proceeds from HCO(3) secreting to acid secreting phenotypes, a process that requires deposition of hensin in the ECM.

Role of the Rhesus glycoprotein, Rh B glycoprotein, in renal ammonia excretion.

Rh B glycoprotein (Rhbg) is a member of the Rh glycoprotein family of ammonia transporters. In the current study, we examine Rhbg's role in basal and acidosis-stimulated acid-base homeostasis. Metabolic acidosis induced by HCl administration increased Rhbg expression in both the cortex and outer medulla. To test the functional significance of increased Rhbg expression, we used a Cre-loxP approach to generate mice with intercalated cell-specific Rhbg knockout (IC-Rhbg-KO). On normal diet, intercalated cell-specific Rhbg deletion did not alter urine ammonia excretion, pH, or titratable acid excretion significantly, but it did decrease glutamine synthetase expression in the outer medulla significantly. After metabolic acidosis was induced, urinary ammonia excretion was significantly less in IC-Rhbg-KO than in control (C) mice on days 2-4 of acid loading, but not on day 5. Urine pH and titratable acid excretion and dietary acid intake did not differ significantly between acid-loaded IC-Rhcg-KO and C mice. In IC-Rhbg-KO mice, acid loading increased connecting segment (CNT) cell and outer medullary collecting duct principal cell Rhbg expression. In both C and IC-Rhbg-KO mice, acid loading decreased glutamine synthetase in both the cortex and outer medulla; the decrease on day 3 was similar in IC-Rhbg-KO and C mice, but on day 5 it was significantly greater in IC-Rhbg-KO than in C mice. We conclude 1) intercalated cell Rhbg contributes to acidosis-stimulated renal ammonia excretion, 2) Rhbg in CNT and principal cells may contribute to renal ammonia excretion, and 3) decreased glutamine synthetase expression may enable normal rates of ammonia excretion under both basal conditions and on day 5 of acid loading in IC-Rhbg-KO mice.