An essential role for the Zn2+ transporter ZIP7 in B cell development.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

Clinical, biochemical, and pathophysiological analysis of SLC34A1 mutations.

Mutations in SLC34A1, encoding the proximal tubular sodium-phosphate transporter NaPi-IIa, may cause a range of clinical phenotypes including infantile hypercalcemia, a proximal renal Fanconi syndrome, which are typically autosomal recessive, and hypophosphatemic nephrolithiasis, which may be an autosomal dominant trait. Here, we report two patients with mixed clinical phenotypes, both with metabolic acidosis, hyperphosphaturia, and renal stones. Patient A had a single heterozygous pathogenic missense mutation (p.I456N) in SLC34A1, consistent with the autosomal dominant pattern of renal stone disease in this family. Patient B, with an autosomal recessive pattern of disease, was compound heterozygous for SLC34A1 variants; a missense variant (p.R512C) together with a relatively common in-frame deletion p.V91A97del7 (91del7). Xenopus oocyte and renal (HKC-8) cell line transfection studies of the variants revealed limited cell surface localization, consistent with trafficking defects. Co-expression of wild-type and I456N and 91del7 appeared to cause intracellular retention in HKC-8, whereas the R512C mutant had a less dominant effect. Expression in Xenopus oocytes failed to demonstrate a significant dominant negative effect for I456N and R512C; however, a negative impact of 91del7 on [32 P]phosphate transport was found. In conclusion, we have investigated pathogenic alleles of SLC34A1 which contribute to both autosomal dominant and autosomal recessive renal stone disease.

The NF-κB1 is a key regulator of acute but not chronic renal injury.

The NF-κB family of transcription factors is important for many cellular functions, in particular initiation and propagation of inflammatory and immune responses. However, recent data has suggested that different subunits of the NF-κB family can suppress the inflammatory response. NF-κB1, from the locus nfκb1, can inhibit transcription, acting as a brake to the recognised pro-inflammatory activity of other NF-κB subunits. We tested the function of NF-κB1 in an acute (nephrotoxic serum (NTS) nephritis) and a chronic (unilateral ureteric obstruction (UUO)) model of renal injury using NF-κB1 (nfκb1-/-) knockout mice. Deficiency in NF-κB1 increased the severity of glomerular injury in NTS-induced nephritis and was associated with greater proteinuria and persistent pro-inflammatory gene expression. Induction of disease in bone marrow chimeric mice demonstrated that the absence of NF-κB1 in either bone marrow or glomerular cells increased the severity of injury. Early after UUO (day 3) there was more severe histological injury in the nfκb1-/- mice but by day 10, disease severity was equivalent in wild type and nfκb1-/- mice. In conclusion, NF-κB1 modifies acute inflammatory renal injury but does not influence chronic fibrotic injury.

Ectopically expressed Slc34a2a sense-antisense transcripts cause a cerebellar phenotype in zebrafish embryos depending on RNA complementarity and Dicer.

Natural antisense transcripts (NATs) are complementary to protein coding genes and potentially regulate their expression. Despite widespread occurrence of NATs in the genomes of higher eukaryotes, their biological role and mechanism of action is poorly understood. Zebrafish embryos offer a unique model system to study sense-antisense transcript interplay at whole organism level. Here, we investigate putative antisense transcript-mediated mechanisms by ectopically co-expressing the complementary transcripts during early zebrafish development. In zebrafish the gene Slc34a2a (Na-phosphate transporter) is bi-directionally transcribed, the NAT predominantly during early development up to 48 hours after fertilization. Declining levels of the NAT, Slc34a2a(as), coincide with an increase of the sense transcript. At that time, sense and antisense transcripts co-localize in the endoderm at near equal amounts. Ectopic expression of the sense transcript during embryogenesis leads to specific failure to develop a cerebellum. The defect is RNA-mediated and dependent on sense-antisense complementarity. Overexpression of a Slc34a2a paralogue (Slc34a2b) or the NAT itself had no phenotypic consequences. Knockdown of Dicer rescued the brain defect suggesting that RNA interference is required to mediate the phenotype. Our results corroborate previous reports of Slc34a2a-related endo-siRNAs in two days old zebrafish embryos and emphasize the importance of coordinated expression of sense-antisense transcripts. Our findings suggest that RNAi is involved in gene regulation by certain natural antisense RNAs.

Molecular determinants of transport function in zebrafish Slc34a Na-phosphate transporters.

The epithelial Na+-coupled phosphate cotransporter family Slc34a (NaPi-II) is well conserved in vertebrates and plays an essential role in maintaining whole body levels of inorganic phosphate (Pi). A three-dimensional model of the transport protein has recently been proposed with defined substrate coordination sites. Zebrafish express two NaPi-II isoforms with high sequence identity but a 10-fold different apparent Km for Pi ([Formula: see text]). We took advantage of the two zebrafish isoforms to investigate the contribution of specific amino acids to Pi coordination and transport. Mutations were introduced to gradually transform the low-affinity isoform into a high-affinity transporter. The constructs were expressed in Xenopus laevis oocytes and functionally characterized. Becaue the cotransport of Pi and Na involves multiple steps that could all influence [Formula: see text], we performed a detailed functional analysis to characterize the impact of the mutations on particular steps of the transport cycle. We used varying concentrations of the substrates Pi and its slightly larger analog, arsenate, as well as the cosubstrate, Na+ Moreover, electrogenic kinetics were performed to assess intramolecular movements of the transporter. All of the mutations were found to affect multiple transport steps, which suggested that the altered amino acids induced subtle structural changes rather than coordinating Pi directly. The likely positions of the critical residues were mapped to the model of human Slc34a, and their localization in relation to the proposed substrate binding pockets concurs well with the observed functional data.

Complement activation in progressive renal disease.

Chronic kidney disease (CKD) is common and the cause of significant morbidity and mortality. The replacement of functioning nephrons by fibrosis is characteristic of progressive disease. The pathways that lead to fibrosis are not fully understood, although chronic non-resolving inflammation in the kidney is likely to drive the fibrotic response that occurs. In patients with progressive CKD there is histological evidence of inflammation in the interstitium and strategies that reduce inflammation reduce renal injury in pre-clinical models of CKD. The complement system is an integral part of the innate immune system but also augments adaptive immune responses. Complement activation is known to occur in many diverse renal diseases, including glomerulonephritis, thrombotic microangiopathies and transplant rejection. In this review we discuss current evidence that complement activation contributes to progression of CKD, how complement could cause renal inflammation and whether complement inhibition would slow progression of renal disease.

Pivotal role of CD4+ T cells in renal fibrosis following ureteric obstruction.

Tubulointerstitial fibrosis is a common consequence of a diverse range of kidney diseases that lead to end-stage renal failure. The degree of fibrosis is related to leukocyte infiltration. Here, we determined the role of different T cell populations on renal fibrosis in the well-characterized mouse model of unilateral ureteric obstruction. Depletion of CD4(+) T cells in wild-type mice with a monoclonal antibody significantly reduced the amount of interstitial expansion and collagen deposition after 2 weeks of obstruction. Reconstitution of lymphopenic RAG knockout mice with purified CD4(+) but not CD8(+) T cells, prior to ureteric obstruction, resulted in a significant increase in interstitial expansion and collagen deposition. Wild-type mice had significantly greater interstitial expansion and collagen deposition compared with lymphopenic RAG(-/-) mice, following ureteric obstruction; however, macrophage infiltration was equivalent in all groups. Thus, our results suggest that renal injury with subsequent fibrosis is likely to be a multifactorial process, with different arms of the immune system involved at different stages. In this ureteric obstruction model, we found a critical role for CD4(+) T cells in kidney fibrosis. These cells could be a potential target of therapeutic intervention to prevent excessive fibrosis and loss of function due to renal injury.

Characterization of H+ and HCO3- transporters in CFPAC-1 human pancreatic duct cells.

AIM: To characterize H+ and HCO3- transporters in polarized CFPAC-1 human pancreatic duct cells, which were derived from a cystic fibrosis patient with the DeltaF508 CFTR mutation. METHODS: CFPAC-1 cells were seeded at high density onto permeable supports and grown to confluence. The cells were loaded with the pH-sensitive fluorescent dye BCECF, and mounted into a perfusion chamber, which allowed the simultaneous perfusion of the basolateral and apical membranes. Transmembrane base flux was calculated from the changes in intracellular pH and the buffering capacity of the cells. RESULTS: Our results showed differential permeability to HCO3-/CO2 at the apical and basolateral membranes of CFPAC-1 cells. Na+/ HCO3- co-transporters (NBCs) and Cl-/ HCO3- exchangers (AEs) were present on the basolateral membrane, and Na+/H+ exchangers (NHEs) on both the apical and basolateral membranes of the cells. Basolateral HCO3- uptake was sensitive to variations of extracellular K+ concentration, the membrane permeable carbonic anhydrase (CA) inhibitors acetazolamide (100 micromol/L) and ethoxyzolamide (100 micromol/L), and was partially inhibited by H2-DIDS (600 micromol/L). The membrane-impermeable CA inhibitor 1-N-(4-sulfamoylphenylethyl)-2,4,6-trimethylpyridine perchlorate did not have any effect on HCO3- uptake. The basolateral AE had a much higher activity than that in the apical membrane, whereas there was no such difference with the NHE under resting conditions. Also, 10 micromol/L forskolin did not significantly influence Cl-/ HCO3- exchange on the apical and basolateral membranes. The administration of 250 micromol/L H2-DIDS significantly inhibited the basolateral AE. Amiloride (300 micromol/L) completely inhibited NHEs on both membranes of the cells. RT-PCR revealed the expression of pNBC1, AE2, and NHE1 mRNA. CONCLUSION: These data suggest that apart from the lack of CFTR and apical Cl-/ HCO3- exchanger activity, CFPAC-1 cells express similar H+ and HCO3- transporters to those observed in native animal tissue.