A novel mouse model for familial hypocalciuric hypercalcemia (FHH1) reveals PTH-dependent and independent CaSR defects

The Calcium-sensing receptor (CaSR) senses extracellular calcium, regulates parathyroid hormone (PTH) secretion, and has additional functions in various organs related to systemic and local calcium and mineral homeostasis. Familial hypocalciuric hypercalcemia type I (FHH1) is caused by heterozygous loss-of-function mutations in the CaSR gene, and is characterized by the combination of hypercalcemia, hypocalciuria, normal to elevated PTH, and facultatively hypermagnesemia and mild bone mineralization defects. To date, only heterozygous Casr null mice have been available as model for FHH1. Here we present a novel mouse FHH1 model identified in a large ENU-screen that carries an c.2579 T > A (p.Ile859Asn) variant in the Casr gene (Casr$^{BCH002}$ mice). In order to dissect direct effects of the genetic variant from PTH-dependent effects, we crossed Casr$^{BCH002}$ mice with PTH deficient mice. Heterozygous Casr$^{BCH002}$ mice were fertile, had normal growth and body weight, were hypercalcemic and hypermagnesemic with inappropriately normal PTH levels and urinary calcium excretion replicating some features of FHH1. Hypercalcemia and hypermagnesemia were independent from PTH and correlated with higher expression of claudin 16 and 19 in kidneys. Likewise, reduced expression of the renal TRPM6 channel in Casr$^{BCH002}$ mice was not dependent on PTH. In bone, mutations in Casr rescued the bone phenotype observed in Pth null mice by increasing osteoclast numbers and improving the columnar pattern of chondrocytes in the growth zone. In summary, Casr$^{BCH002}$ mice represent a new model to study FHH1 and our results indicate that only a part of the phenotype is driven by PTH

Erythropoietin stimulates fibroblast growth factor 23 (FGF23) in mice and men

Fibroblast growth factor 23 (FGF23) is a major endocrine regulator of phosphate and 1,25 (OH)2 vitamin D3 metabolism and is mainly produced by osteocytes. Its production is upregulated by a variety of factors including 1,25 (OH)2 vitamin D3, high dietary phosphate intake, and parathyroid hormone (PTH). Recently, iron deficiency and hypoxia have been suggested as additional regulators of FGF23 and a role of erythropoietin (EPO) was shown. However, the regulation of FGF23 by EPO and the impact on phosphate and 1,25(OH)2 vitamin D3 are not completely understood. Here, we demonstrate that acute administration of recombinant human EPO (rhEPO) to healthy humans increases the C-terminal fragment of FGF23 (C-terminal FGF23) but not intact FGF23 (iFGF23). In mice, rhEPO stimulates acutely (24 h) C-terminal FGF23 but iFGF23 only after 4 days without effects on PTH and plasma phosphate. 1,25 (OH)2 D3 levels and αklotho expression in the kidney decrease after 4 days. rhEPO induced FGF23 mRNA in bone marrow but not in bone, with increased staining of FGF23 in CD71+ erythroid precursors in bone marrow. Chronic elevation of EPO in transgenic mice increases iFGF23. Finally, acute injections of recombinant FGF23 reduced renal EPO mRNA expression. Our data demonstrate stimulation of FGF23 levels in mice which impacts mostly on 1,25 (OH)2 vitamin D3 levels and metabolism. In humans, EPO is mostly associated with the C-terminal fragment of FGF23; in mice, EPO has a time-dependent effect on both FGF23 forms. EPO and FGF23 may form a feedback loop controlling and linking erythropoiesis and mineral metabolism

The (pro)renin receptor (ATP6ap2) facilitates receptor-mediated endocytosis and lysosomal function in the renal proximal tubule

The ATP6ap2 (Pro)renin receptor protein associates with H(+)-ATPases which regulate organellar, cellular, and systemic acid-base homeostasis. In the kidney, ATP6ap2 colocalizes with H(+)-ATPases in various cell types including the cells of the proximal tubule. There, H(+)-ATPases are involved in receptor-mediated endocytosis of low molecular weight proteins via the megalin/cubilin receptors. To study ATP6ap2 function in the proximal tubule, we used an inducible shRNA Atp6ap2 knockdown rat model (Kd) and an inducible kidney-specific Atp6ap2 knockout mouse model. Both animal lines showed higher proteinuria with elevated albumin, vitamin D binding protein, and procathepsin B in urine. Endocytosis of an injected fluid-phase marker (FITC- dextran, 10 kDa) was normal whereas processing of recombinant transferrin, a marker for receptor-mediated endocytosis, to lysosomes was delayed. While megalin and cubilin expression was unchanged, abundance of several subunits of the H(+)-ATPase involved in receptor-mediated endocytosis was reduced. Lysosomal integrity and H(+)-ATPase function are associated with mTOR signaling. In ATP6ap2, KO mice mTOR and phospho-mTOR appeared normal but increased abundance of the LC3-B subunit of the autophagosome was observed suggesting a more generalized impairment of lysosomal function in the absence of ATP6ap2. Hence, our data suggests a role for ATP6ap2 for proximal tubule function in the kidney with a defect in receptor-mediated endocytosis in mice and rats

Acidosis and Deafness in Patients with Recessive Mutations in FOXI1

Maintenance of the composition of inner ear fluid and regulation of electrolytes and acid-base homeostasis in the collecting duct system of the kidney require an overlapping set of membrane transport proteins regulated by the forkhead transcription factor FOXI1. In two unrelated consanguineous families, we identified three patients with novel homozygous missense mutations in FOXI1 (p.L146F and p.R213P) predicted to affect the highly conserved DNA binding domain. Patients presented with early-onset sensorineural deafness and distal renal tubular acidosis. In cultured cells, the mutations reduced the DNA binding affinity of FOXI1, which hence, failed to adequately activate genes crucial for normal inner ear function and acidbase regulation in the kidney. A substantial proportion of patients with a clinical diagnosis of inherited distal renal tubular acidosis has no identified causative mutations in currently known disease genes. Our data suggest that recessive mutations in FOXI1 can explain the disease in a subset of these patients

Colocalization of the (Pro)renin receptor/Atp6ap2 with H+-AT pases in mouse kidney but prorenin does not acutely regulate intercalated cell H+-ATPase activity

The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and nonproteolytically activate prorenin. Additionally, (P)RR is associated with H+-ATPases and alternative functions in H+-ATPase regulation as well as inWnt signalling have been reported. Kidneys express very high levels of H+-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is co

Silver nanoparticles promote the emergence of heterogeneic human neutrophil sub-populations

Neutrophil surveillance is central to nanoparticle clearance. Silver nanoparticles (AgNP) have numerous uses, however conflicting evidence exists as to their impact on neutrophils and whether they trigger damaging inflammation. Neutrophil’s importance in innate defence and regulating immune networks mean it’s essential we understand AgNP’s impact on neutrophil function. Human neutrophil viability following AgNP or Ag Bulk treatment was analysed by flow cytometry and AnV/PI staining. Whilst AgNP exposure did not increase the total number of apoptotic neutrophils, the number of late apoptotic neutrophils was increased, suggesting AgNP increase transit through apoptosis. Mature (CD16bright/CD62Lbright), immature (CD16dim/CD62Lbright) and apoptotic (CD16dim/CD62Ldim) neutrophil populations were evident within isolated neutrophil preparations. AgNP exposure significantly reduced CD62L staining of CD16bright/CD62Lbright neutrophils, and increased CD16 staining of CD16dim/CD62Lbright populations, suggesting AgNPs trigger neutrophil activation and maturation, respectively. AgNP exposure dramatically increased IL-8, yet not classical pro-inflammatory cytokine release, suggesting AgNP triggers neutrophil activation, without pro-inflammation or damaging, necrotic cell death. For the first time, we show AgNPs differentially affect distinct sub-populations of circulating human neutrophils; activating mature neutrophils with the emergence of CD16bright/CD62Ldim neutrophils. This may stimulate particle clearance without harmful inflammation, challenging previous assumptions that silver nanomaterials induce neutrophil toxicity and damaging inflammatory responses

Adaptive response of the murine collecting duct to alkali loading

Fine-tuning of salt and acid-base homeostasis is achieved in the renal collecting duct through the action of intercalated and principal cells. Their activity is tightly regulated adapting to changes in systemic acid-base, fluid, or electrolyte status. The relative number of acid or bicarbonate secretory intercalated cells changes in response to acid or alkali loading. Several factors that may induce collecting duct plasticity in response to acid loading have been identified including cell proliferation, Growth Differentiation Factor 15 (Gdf15), hensin (DMBT1), and SDF1 (or CXCL12). Also, the transcription factors Foxi1 and CP2L1, or the Notch2-Jag1 signaling pathway, may play a role. However, little is known about the mechanisms mediating the adaptive response of the collecting duct to alkali loading. Here, we examined in mouse kidney the response of these factors to alkali loading. Mice were left untreated or received NaHCO$_{3}$ or NaCl over 7 days. Cell proliferation in vivo was monitored by Ki67 labeling or BrdU incorporation and expression of cell markers, and regulatory factors were examined. Foxi1 and GDF15 were upregulated and CP2L1 downregulated during alkali loading. Ki67 staining and BrdU incorporation were frequent in AQP2-positive cells in the NaCl and NaHCO$_{3}$ groups, but no evidence was found for increased Ki67 or BrdU staining in bicarbonate-secretory cells consistent with a model that AQP2 positive precursor cells may differentiate into intercalated cells. Thus, alkali loading alters the cellular profile of the collecting duct, which may involve cell proliferation and changes in the network of molecules determining the plasticity of the collecting duct

The (pro)renin receptor (ATP6ap2) facilitates receptor-mediated endocytosis and lysosomal function in the renal proximal tubule

The ATP6ap2 (Pro)renin receptor protein associates with H+-ATPases which regulate organellar, cellular, and systemic acid-base homeostasis. In the kidney, ATP6ap2 colocalizes with H+-ATPases in various cell types including the cells of the proximal tubule. There, H+-ATPases are involved in receptor-mediated endocytosis of low molecular weight proteins via the megalin/cubilin receptors. To study ATP6ap2 function in the proximal tubule, we used an inducible shRNA Atp6ap2 knockdown rat model (Kd) and an inducible kidney-specific Atp6ap2 knockout mouse model. Both animal lines showed higher proteinuria with elevated albumin, vitamin D binding protein, and procathepsin B in urine. Endocytosis of an injected fluid-phase marker (FITC- dextran, 10 kDa) was normal whereas processing of recombinant transferrin, a marker for receptor-mediated endocytosis, to lysosomes was delayed. While megalin and cubilin expression was unchanged, abundance of several subunits of the H+-ATPase involved in receptor-mediated endocytosis was reduced. Lysosomal integrity and H+-ATPase function are associated with mTOR signaling. In ATP6ap2, KO mice mTOR and phospho-mTOR appeared normal but increased abundance of the LC3-B subunit of the autophagosome was observed suggesting a more generalized impairment of lysosomal function in the absence of ATP6ap2. Hence, our data suggests a role for ATP6ap2 for proximal tubule function in the kidney with a defect in receptor-mediated endocytosis in mice and rats. Keywords: Endocytosis; H+-ATPase; Low molecular weight proteins; Lysosome; Proximal tubul

Expression of NaPi-IIb in rodent and human kidney and upregulation in a model of chronic kidney disease

Na$^{+}$-coupled phosphate cotransporters from the SLC34 and SLC20 families of solute carriers mediate transepithelial transport of inorganic phosphate (Pi). NaPi-IIa/Slc34a1, NaPi-IIc/Slc34a3, and Pit-2/Slc20a2 are all expressed at the apical membrane of renal proximal tubules and therefore contribute to renal Pi reabsorption. Unlike NaPi-IIa and NaPi-IIc, which are rather kidney-specific, NaPi-IIb/Slc34a2 is expressed in several epithelial tissues, including the intestine, lung, testis, and mammary glands. Recently, the expression of NaPi-IIb was also reported in kidneys from rats fed on high Pi. Here, we systematically quantified the mRNA expression of SLC34 and SLC20 cotransporters in kidneys from mice, rats, and humans. In all three species, NaPi-IIa mRNA was by far the most abundant renal transcript. Low and comparable mRNA levels of the other four transporters, including NaPi-IIb, were detected in kidneys from rodents and humans. In mice, the renal expression of NaPi-IIa transcripts was restricted to the cortex, whereas NaPi-IIb mRNA was observed in medullary segments. Consistently, NaPi-IIb protein colocalized with uromodulin at the luminal membrane of thick ascending limbs of the loop of Henle segments. The abundance of NaPi-IIb transcripts in kidneys from mice was neither affected by dietary Pi, the absence of renal NaPi-IIc, nor the depletion of intestinal NaPi-IIb. In contrast, it was highly upregulated in a model of oxalate-induced kidney disease where all other SLC34 phosphate transporters were downregulated. Thus, NaPi-IIb may contribute to renal phosphate reabsorption, and its upregulation in kidney disease might promote hyperphosphatemia

Elevated FGF23 and disordered renal mineral handling with reduced bone mineralization in chronically erythropoietin over-expressing transgenic mice

Fibroblast Growth Factor 23 (FGF23) is a phosphaturic factor causing increased renal phosphate excretion as well as suppression of 1,25 (OH)$_{2}$-vitamin D$_{3.}$ Highly elevated FGF23 can promote development of rickets and osteomalacia. We and others previously reported that acute application of erythropoietin (EPO) stimulates FGF23 production. Considering that EPO is clinically used as chronic treatment against anemia, we used here the Tg6 mouse model that constitutively overexpresses human EPO in an oxygen-independent manner, to examine the consequences of long-term EPO therapy on mineral and bone metabolism. Six to eight weeks old female Tg6 mice showed elevated intact and C-terminal fragment of FGF23 but normal plasma levels of PTH, calcitriol, calcium and phosphate. Renal function showed moderate alterations with higher urea and creatinine clearance and mild albuminuria. Renal phosphate excretion was normal whereas mild hypercalciuria was found. Renal expression of the key proteins TRPV5 and calbindin D28k involved in active calcium reabsorption was reduced in Tg6 mice. Plasma levels of the bone turnover marker osteocalcin were comparable between groups. However, urinary excretion of deoxypyridinoline (DPD) was lower in Tg6 mice. MicroCT analysis showed reduced total, cortical, and trabecular bone mineral density in femora from Tg6 mice. Our data reveal that chronic elevation of EPO is associated with high FGF23 levels and disturbed mineral homeostasis resulting in reduced bone mineral density. These observations imply the need to study the impact of therapeutically applied EPO on bone mineralization in patients, especially those suffering from chronic kidney disease