Functional tests to guide management in an adult with loss of function of type-1 angiotensin II receptor

BACKGROUND: Genetic loss of function of AGT (angiotensinogen), REN (renin), ACE (angiotensin-converting enzyme), or AGTR1 (type-1 angiotensin II receptor) leads to renal tubular dysgenesis (RTD). This syndrome is almost invariably lethal. Most surviving patients reach stage 5 chronic kidney disease at a young age. METHODS: Here, we report a 28-year-old male with a homozygous truncating mutation in AGTR1 (p.Arg216*), who survived the perinatal period with a mildly impaired kidney function. In contrast to classic RTD, kidney biopsy showed proximal tubules that were mostly normal. During the subsequent three decades, we observed evidence of both tubular dysfunction (hyperkalemia, metabolic acidosis, salt-wasting and a urinary concentrating defect) and glomerular dysfunction (reduced glomerular filtration rate, currently ~30 mL/min/1.73 m(2), accompanied by proteinuria). To investigate the recurrent and severe hyperkalemia, we performed a patient-tailored functional test and showed that high doses of fludrocortisone induced renal potassium excretion by 155%. Furthermore, fludrocortisone lowered renal sodium excretion by 39%, which would have a mitigating effect on salt-wasting. In addition, urinary pH decreased in response to fludrocortisone. Opposite effects on urinary potassium and pH occurred with administration of amiloride, further supporting the notion that a collecting duct is present and able to react to fludrocortisone. CONCLUSIONS: This report provides living proof that even truncating loss-of-function mutations in AGTR1 are compatible with life and relatively good GFR and provides evidence for the prescription of fludrocortisone to treat hyperkalemia and salt-wasting in such patients. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00467-021-05018-7

2009 Homer W. Smith Award: Minerals in motion: from new ion transporters to new concepts.

Contains fulltext : 89674.pdf (publisher's version ) (Open Access)The kidneys play a critical role in maintaining the systemic balance of Mg(2+) and Ca(2+) cations. The reabsorptive capacity of these divalent cations adapt to changes in their plasma concentrations. Active reabsorption of Mg(2+) and Ca(2+) takes place in the distal convoluted and connecting tubules, respectively, and is initiated by cellular transport through selective transient receptor potential (TRP) channels located along the luminal membrane and modulated by hormonal stimuli. Recent characterization of underlying molecular defects in renal Mg(2+) handling illuminate complex transport processes in the kidney and their contribution to the overall mineral balance. Likewise, studies of Ca(2+) transport proteins in null mice disclose molecular mechanisms maintaining normal plasma Ca(2+) levels and the hypercalciuria-related adaptations important in the prevention of kidney stones. Current knowledge of Mg(2+) and Ca(2+) transport is summarized here as comprehensive cellular models of the distal nephron.1 augustus 201

Enhanced passive Ca²⁺ reabsorption and reduced Mg²⁺ channel abundance explains thiazide-induced hypocalciuria and hypomagnesemia

Thiazide diuretics enhance renal Na⁺ excretion by blocking the Na⁺-Cl⁻ cotransporter (NCC), and mutations in NCC result in Gitelman syndrome. The mechanisms underlying the accompanying hypocalciuria and hypomagnesemia remain debated. Here, we show that enhanced passive Ca²⁺ transport in the proximal tubule rather than active Ca²⁺ transport in distal convolution explains thiazide-induced hypocalciuria. First, micropuncture experiments in mice demonstrated increased reabsorption of Na⁺ and Ca²⁺ in the proximal tubule during chronic hydrochlorothiazide (HCTZ) treatment, whereas Ca²⁺ reabsorption in distal convolution appeared unaffected. Second, HCTZ administration still induced hypocalciuria in transient receptor potential channel subfamily V, member 5–knockout (Trpv5-knockout) mice, in which active distal Ca²⁺ reabsorption is abolished due to inactivation of the epithelial Ca²⁺ channel Trpv5. Third, HCTZ upregulated the Na⁺/H⁺ exchanger, responsible for the majority of Na⁺ and, consequently, Ca²⁺ reabsorption in the proximal tubule, while the expression of proteins involved in active Ca²⁺ transport was unaltered. Fourth, experiments addressing the time-dependent effect of a single dose of HCTZ showed that the development of hypocalciuria parallels a compensatory increase in Na⁺ reabsorption secondary to an initial natriuresis. Hypomagnesemia developed during chronic HCTZ administration and in NCC-knockout mice, an animal model of Gitelman syndrome, accompanied by downregulation of the epithelial Mg²⁺ channel transient receptor potential channel subfamily M, member 6 (Trpm6). Thus, Trpm6 downregulation may represent a general mechanism involved in the pathogenesis of hypomagnesemia accompanying NCC inhibition or inactivation

Functionomics of novel mutations in NCC and their relevance in development of Gitelman syndrome

<p>Gitelman syndrome is a genetic disease characterized by low blood pressure and salt wasting. In most cases, Gitelman syndrome results from loss-of-function mutations in the solute carrier family 12 (SLC12A3) gene, which encodes the thiazide-sensitive sodium chloride co-transporter (NCC). At present, more than 250 distinct loss-of-function mutations have been identified in patients with Gitelman syndrome. Functional analysis has been limited by the use of only Xenopus laevis oocytes as a model system. The aim of the present study is to understand the functional consequences of these NCC mutations in mammalian cell line systems. Recently, our group published a new technique to isolate primary cells using Complex Object Parametric Analyzer and Sorter (COPAS). We have pioneered with this COPAS sorting platform to isolate and culture distal convoluted tubules (DCT) based on parvalbumin-eGFP transgenic mice model. These primary cultures exhibit several characteristics of the original epithelium including thiazide-sensitive transepithelial NaCl transport. By generating crossbred of parvalbumin-eGFP mice with NCC knockout we aim to obtain DCT primary cell line without endogenous expression of wild type NCC, which in turn will be substituted by ectopic expression of mutated NCC. Generation of this model will create an excellent platform for further functional analysis of mutant NCC proteins. Currently, out of several patients with Gitelman syndrome symptoms eight new mutations were selected. The majority of these mutations exhibited a disturbed phosphorylation and glycosylation pattern as well as diminished expression of NCC. However, functional consequences of these mutations remain to be elucidated. Overall, functional screening of many NCC mutations in DCT cultures are now possible and will also allow to unravel a complex system of NCC regulatory mechanisms.</p

1,25-Dihydroxyvitamin D₃-independent stimulatory effect of estrogen on the expression of ECaCl in the kidney

Estrogen deficiency results in a negative Ca2+ balance and bone loss in postmenopausal women. In addition to bone, the intestine and kidney are potential sites for estrogen action and are involved in Ca2+ handling and regulation. The epithelial Ca2+ channel ECaCl (or TRPV5) is the entry channel involved in active Ca2+ transport. Ca2+ entry is followed by cytosolic diffusion, facilitated by calbindin-D28K and/or calbindin-D9k, and active extrusion across the basolateral membrane by the Na+/Ca2+-exchanger (NCX1) and plasma membrane Ca2+-ATPase (PMCA1b). In this transcellular Ca2+ transport, ECaCl probably represents the final regulatory target for hormonal control. The aim of this study was to determine whether 17β-estradiol (17β-E2) is involved in Ca2+ reabsorption via regulation of the expression of ECaCl. The ovariectomized rat model was used to investigate the regulation of ECaCl, at the mRNA and protein levels, by 17β-E2 replacement therapy. Using real-time quantitative PCR and immunohistochemical analyses, this study demonstrated that 17β-E2 treatment at pharmacologic doses increased renal mRNA levels of ECaCl, calbindin-D28K, NCX1, and PMCA1b and increased the protein abundance of ECaCl. Furthermore, the involvement of 1,25-dihydroxyvitamin D3 in the effects of 17β-E2 was examined in 25-hydroxyvitamin D3-1α-hydroxylase-knockout mice. Renal mRNA expression of calbindin-D9K, calbindin-D28K, NCX1, and PMCA1b was not significantly altered after 17β-E2 treatment. In contrast, ECaCl mRNA and protein levels were both significantly upregulated. Moreover, 17β-E2 treatment partially restored serum Ca2+ levels, from 1.63 ± 0.06 to 2.03 ± 0.12 mM. In conclusion, this study suggests that 17β-E2 is positively involved in renal Ca2+ reabsorption via the upregulation of ECaCl, an effect independent of 1,25-dihydroxyvitamin D3.</p

Regulation of the epithelial Ca2+ channels in small intestine as studied by quantitative mRNA detection

The epithelial Ca2+ channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca2+ absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the effect of 17β-estradiol (17β-E2), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and dietary Ca2+ on the expression of the duodenal Ca2+ transport proteins was investigated in vivo and analyzed using realtime quantitative PCR. Supplementation with 17β-E2 increased duodenal gene expression of TRPV5 and TRPV6 but also calbindin-D9K and plasma membrane Ca2+-ATPase (PMCA1b) in ovariectomized rats. 25-Hydroxyvitamin D3-1α hydroxylase (1α-OHase) knockout mice are characterized by hyperparathyroidism, rickets, hypocalcemia, and undetectable levels of 1,25(OH)2D3 and were used to study the 1,25(OH)2D3-dependency of the stimulatory effects of 17β-E2. Treatment with 17β-E2 upregulated mRNA levels of duodenal TRPV6 in these 1α-OHase knockout mice, which was accompanied by increased serum Ca2+ concentrations from 1.69 ± 0.10 to 2.03 ± 0.12 mM (P < 0.05). In addition, high dietary Ca2+ intake normalized serum Ca2+ in these mice and upregulated expression of genes encoding the duodenal Ca2+ transport proteins except for PMCA1b. Supplementation with 1,25(OH)2D3 resulted in increased expression of TRPV6, calbindin-D9K, and PMCA1b and normalization of serum Ca2+. Expression levels of duodenal TRPV5 mRNA are below detection limits in these 1α-OHase knockout mice, but supplementation with 1,25(OH)2D3 upregulated the expression to significant levels. In conclusion, TRPV5 and TRPV6 are regulated by 17β-E2 and 1,25(OH)2D3, whereas dietary Ca2+ is positively involved in the regulation of TRPV6 only

Parathyroid Hormone Activates TRPV5 via PKA-Dependent Phosphorylation

Low extracellular calcium (Ca2+) promotes release of parathyroid hormone (PTH), which acts on multiple organs to maintain overall Ca2+ balance. In the distal part of the nephron, PTH stimulates active Ca2+ reabsorption via the adenylyl cyclase–cAMP–protein kinase A (PKA) pathway, but the molecular target of this pathway is unknown. The transient receptor potential vanilloid 5 (TRPV5) channel constitutes the luminal gate for Ca2+ entry in the distal convoluted tubule and has several putative PKA phosphorylation sites. Here, we investigated the effect of PTH-induced cAMP signaling on TRPV5 activity. Using fluorescence resonance energy transfer, we studied cAMP and Ca2+ dynamics during PTH stimulation of HEK293 cells that coexpressed the PTH receptor and TRPV5. PTH increased cAMP levels, followed by a rise in TRPV5-mediated Ca2+ influx. PTH (1 to 31) and forskolin, which activate the cAMP pathway, mimicked the stimulation of TRPV5 activity. Remarkably, TRPV5 activation was limited to conditions of strong intracellular Ca2+ buffering. Cell surface biotinylation studies demonstrated that forskolin did not affect TRPV5 expression on the cell surface, suggesting that it alters the single-channel activity of a fixed number of TRPV5 channels. Application of the PKA catalytic subunit, which phosphorylated TRPV5, directly increased TRPV5 channel open probability. Alanine substitution of threonine-709 abolished both in vitro phosphorylation and PTH-mediated stimulation of TRPV5. In summary, PTH activates the cAMP-PKA signaling cascade, which rapidly phosphorylates threonine-709 of TRPV5, increasing the channel's open probability and promoting Ca2+ reabsorption in the distal nephron