Autosomal dominant hypocalcemia with mild type 5 Bartter syndrome

Type 5 Bartter syndrome has been recently defined as a Bartter syndrome due to the most activating mutations of the calcium-sensing receptor (CaSR). It has been attributed to the inhibition exerted by CaSR activity on sodium transport in the thick ascending limb of the loop of Henle (TALH). Two monozygotic twin sisters (T1 and T2) with autosomal dominant hypocalcemia (ADH) due to a nonconservative activating CaSR mutation in the extracellular domain (K29E) were studied. They developed a Bartter-like syndrome characterized by a mild phenotype: hypokalemia occurred only at the age of 22 years; it was corrected with small doses of oral potassium in one twin, while the other twin needed no potassium supplements to maintain borderline levels of plasma potassium; alkalosis was absent; plasma renin and aldosterone production were not markedly activated. Furthermore, the natriuretic response to furosemide, a inhibitor of sodium reabsorption in the TALH, was conserved in both twins. The K29E mutation was previously reported as one of the most activating mutations of the CaSR gene leading to a very marked increase in CaSR sensitivity to calcium ions. These findings confirm that Bartter syndrome is typically associated with ADH provided that the underlying mutation of CaSR is able to produce a conspicuous gain of function. However, the phenotype of type 5 Bartter syndrome may manifest with variable severity, not directly related with the in vitro potency of the CaSR activating mutation.     

A case of gain-of-function mutation in calcium-sensing receptor: supplemental hydration is required for renal protection

AIMS: The calcium-sensing receptor (CaSR) regulates the extracellular calcium level, mainly by controlling parathyroid hormon secretion and renal calcium reabsorption. In gain-of-function CaSR mutations, the genetic abnormalities increase CaSR activity leading to the development of such clinical manifestations as hypercalciuric hypocalcemia and hypoparathyroidism. We report a Japanese case of CaSR gain-of-function mutation and represent a therapeutic intervention based on the functional characteristics of CaSR in renal tubule. METHODS AND RESULTS (CASE): DNA sequence analysis revealed a heterozygous G to T mutation identified in a 12-year-old Japanese girl presenting with sporadic onset of hypercalciuric hypocalcemia and hypoparathyroidism. The mutation is located in the N-terminal extracellular domain of the CaSR gene, one of the most important parts for the three-dimensional construction of the receptor, resulting in the substitution of phenylalanine for cysteine at amino acid 131 (C131F) in exon 3. Based on the diagnosis of the gain-of-function mutation in the CaSR, oral hydrochlorothiazide administration and supplemental hydration were started in addition to calcium supplementation. The combination therapy of thiazide and supplemental hydration markedly reduced both renal calcium excretion and urinary calcium concentration from 0.4-0.7 to less than 0.1 mg/mg (urinary calcium/creatinine ratio) and from 10-15 to 3-5 mg/dl (urinary calcium concentration), respectively. This therapy stopped the progression of renal calcification during the follow-up period. CONCLUSION: Supplemental hydration should be considered essential for the following reasons: (1) calcium supplementation activates the CaSR in the kidney and suppresses renal urinary concentrating ability, (2) the thiazide has a diuretic effect, (3) as calcium supplementation increases renal calcium excretion, the supplemental hydration decreases urinary calcium concentration by increasing urinary volume, thereby diminishing the risk of intratubular crystallization of calcium ion.     

Autosomal dominant hypocalcemia caused by a novel mutation in the loop 2 region of the human calcium receptor extracellular domain.

We report a novel missense mutation N124K in the extracellular calcium receptor (CaR) identified in two related subjects with the phenotypic features of autosomal dominant hypocalcemia (ADH). Expression of the N124K mutant receptor created by site-directed mutagenesis and transfected into HEK-293 cells was comparable with that of the wild-type (WT) receptor and two other mutant receptors N118K and L125P identified in subjects with ADH. Functional characterization by the extracellular Ca2+ ion ([Ca2+]0)-stimulated phosphoinositide (PI) hydrolysis in transfected HEK-293 cells showed that the N124K mutant receptor was left-shifted in Ca2+ sensitivity. This biochemical gain-of-function is comparable with that seen in other missense mutations of the CaR identified in subjects with ADH. We tested a series of missense substitutions (R, Q, E, and G) in addition to K for N124 and found that only the N124K mutation and to a much lesser extent N124R caused a left shift in Ca2+ sensitivity. Thus, a specific substitution, not merely a mutation of the N124 residue, is required for receptor activation. The N124K mutation is one of eight naturally occurring mutations in subjects with ADH identified in a short segment A116-C129 of the CaR extracellular domain (ECD). We present a hypothesis to explain receptor activation by mutations in this region based on the recently described three-dimensional structure of the related metabotropic glutamate type 1 receptor (mGluR1).     

Pathogenesis of hypokalemia in autosomal dominant hypocalcemia type 1

Background

Autosomal dominant hypocalcemia type 1 (ADH1) is a relatively rare endocrine disorder characterized by hypocalcemia and inadequate parathyroid hormone secretion. ADH is caused by activating mutations in the calcium-sensing receptor (CaSR) gene, CASR. CaSR plays a crucial role in calcium and magnesium homeostasis in the kidney. ADH may be accompanied by hypokalemia and metabolic alkalosis when it is classified as type V Bartter syndrome. However, the mechanism underlying hypokalemia in this disease is unclear.

Methods

We investigated a 33-year-old woman with hypocalcemia and hypoparathyroidism since childhood, whose mother also had hypocalcemia and hypoparathyroidism, but with no clinical symptoms. Blood examinations showed hypokalemia and metabolic alkalosis in the patient, but not her mother. We conducted mutation analysis and diuretic tests to clarify the patient’s and her mother’s diagnosis and to investigate the onset mechanism of hypokalemia in ADH1. We also determined the localization of CaSR in the kidney by immunohistochemistry.

Results

We detected a known gain-of-function mutation in CASR in both the patient and her mother. Diuretic tests revealed a response to furosemide and no reaction to thiazide in the patient, although the mother responded well to both diuretics. CaSR co-localized with the Na⁺–Cl^? cotransporter (NCCT) on distal tubular epithelial cells.

Conclusions

These results indicate that the NCCT in the distal convoluted tubule was secondarily affected in this patient. We conclude that the main pathogenesis of secondary hypokalemia in ADH1 in this patient was secondary NCCT dysfunction.

     

Acid-base, calcium, potassium and aldosterone metabolism in renal tubular acidosis.

Classic renal tubular acidosis is characterized by a primary defect in establishment of a large hydrogen ion gradient across the distal renal tubule. Thus the development of hyperchlorenic metabolic acidosis follows. In addition, hypokalemia results from renal potassium wasting secondary hyperaldosteronism from sodium wasting and contraction of the extracellular fluid. The presenting signs and symptoms are growth retardation, fatigue, periodic paralysis, polyuria, polydipsia, vomiting and constipation as well as nephrocalcinosis and nephrolithiasis. It is suggested that effective treatment with alkali therapy requires markedly higher doses than formerly recommended, and may related to a higher rate of endogenous acid production from (1) intermediary metabolism of sulfur amino acids and organic acids, (2) impaired tubular reabsorption of bicarbonate and (3) hydrogen ion release from hydroxyapatite formation. It is also suggested that acidosis may interfere with vitamin D metabolism and thus play an important role in the pathoetiology of the growth failure in children with this disorder.     

Paracellin-1 is critical for magnesium and calcium reabsorption in the human thick ascending limb of Henle

BACKGROUND: A new protein, named paracellin 1 (PCLN-1), expressed in human thick ascending limb (TAL) tight junctions, possibly plays a critical role in the control of magnesium and calcium reabsorption, since mutations of PCLN-1 are present in the hypomagnesemia hypercalciuria syndrome (HHS). However, no functional experiments have demonstrated that TAL magnesium and calcium reabsorption were actually impaired in patients with HHS. METHODS: Genetic studies were performed in the kindred of two unrelated patients with HHS. Renal magnesium and calcium reabsorption in TAL were analyzed in one homozygous affected patient of each family, one patient with extrarenal hypomagnesemia (ERH), and two control subjects (CSs). RESULTS: We found two yet undescribed mutations of PCLN-1 (Gly 162 Val, Ala 139 Val). In patients with HHS, renal magnesium and calcium reabsorptions were impaired as expected; NaCl renal conservation during NaCl deprivation and NaCl tubular reabsorption in diluting segment were intact. Furosemide infusion in CS markedly increased NaCl, Mg, and Ca urinary excretion rates. In HHS patients, furosemide similarly increased NaCl excretion, but failed to increase Mg and Ca excretion. Acute MgCl(2) infusion in CS and ERH patient provoked a dramatic increase in urinary calcium excretion without change in NaCl excretion. When combined with MgCl(2) infusion, furosemide infusion remained able to induce normal natriuretic response, but was unable to increase urinary magnesium and calcium excretion further. In HHS patients, calciuric response to MgCl(2) infusion was blunted. CONCLUSION: This study is the first to our knowledge to demonstrate that homozygous mutations of PCLN-1 result in a selective defect in paracellular Mg and Ca reabsorption in the TAL, with intact NaCl reabsorption ability at this site. In addition, the study supports a selective physiological effect of basolateral Mg(2+) and Ca(2+) concentration on TAL divalent cation paracellular permeability, that is, PCLN-1 activity.     

Gentamicin-induced Bartter-like syndrome

Gentamicin is well known to be associated with nephrotoxicity, including acute renal failure and renal tubular dysfunction. A Bartter-like syndrome has also been described as a toxic manifestation of gentamicin therapy in adults, but this nephrotoxic syndrome has not been well characterized in children. In this report we describe the clinical course of four patients with gentamicin-associated Bartter-like syndrome. These patients ranged in age from 4 months to 17 years; they all demonstrated evidence of renal tubulopathy, primarily affecting the distal nephron. Hypocalcemia, hypomagnesemia, alkalosis, and hypokalemia were the main manifestations in these patients. After discontinuation of gentamicin, recovery of the renal tubular functions and resolution of the electrolyte abnormalities were complete in all patients. Received December 10, 1996; received in revised form May 5, 1997; accepted May 14, 1997     

Calcilytic Ameliorates Abnormalities of Mutant Calcium‐Sensing Receptor (CaSR) Knock‐In Mice Mimicking Autosomal Dominant Hypocalcemia (ADH)

Activating mutations of calcium‐sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D₃ or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT‐305/MK‐5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT‐305/MK‐5442 suppressed the hypersensitivity to extracellular Ca²⁺ of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock‐in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT‐305/MK‐5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1‐34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock‐in mice exhibited low bone turnover due to the deficiency of PTH, and JTT‐305/MK‐5442 as well as PTH(1‐34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a novel therapeutic agent for ADH. © 2015 American Society for Bone and Mineral Research.     

Distal nephron function in Bartter's syndrome: abnormal conductance to chloride in the cortical collecting tubule?

Five patients with the clinical patterns of Bartter's syndrome underwent a series of clearance studies in order to characterize the underlying tubule defect. Free water generation during maximal water diuresis (CH2O), expressed as percentage of the distal delivery (CH2O + CCl), was lower in the patients (72.5 +/- 3.2%) than in controls (84.4 +/- 5.5, p < 0.0001). During maximal water diuresis and furosemide administration (40 mg i.v. as bolus), NaCl reabsorption along the diluting nephron segments could be separated into 2 components, that occurring in the loop of Henle (DRNaHL) and that occurring in tubule segments beyond the macula densa (DRNaDT): DRNaHL was normal, while DRNaDT was reduced (3.1 +/- 0.8 vs. 6.2 +/- 2.5 ml/min in controls, p < 0.015). Thus, according to this furosemide protocol, our patients had normal solute reabsorption in the loop of Henle but reduced NaCl reabsorption in tubule segments beyond the macula densa. During 0.9% saline infusion (2 liters in 2 h, after stimulation of distal Na reabsorption with fludrocortisone) fractional excretion (FE) of K showed a linear rise with the increase of FECl-FEK, however, was much higher in the patients than in controls for every FECl level. In contrast, the infusion of Na2SO4, after fludrocortisone administration, induced similar FEK increases in patients and in controls. Thus, in these patients Na reabsorption in the distal nephron (possibly the cortical collecting tubule) was associated with the generation of a higher than normal electric potential gradient in the presence of Cl but not of another poorly reabsorbable anion, such as SO4(2-). These observations indicate that, in our patients, Henle's loop function is normal, while the collecting tubule function is abnormal. We suggest that NaCl wasting and enhanced tubular secretion of H+ and K in our patients might result from an abnormally low conductance to Cl in distal nephron site(s) where Na reabsorption is electrogenic, possibly the cortical collecting tubule. A larger than normal transtubular electric gradient would be generated by Na reabsorption, causing: (1) a direct stimulation of tubular secretion of K and H+ (leading to hypokalemia and alkalosis) and (2) inhibition of the reabsorption of Na ('trapped' into the tubular lumen by electric forces), with consequent extracellular volume contraction, hyperreninemia and hyperaldosteronism.     

Renal tubular sodium and water metabolism in brain tumour patients submitted to craniotomy

SummaryPurpose To evaluate the effect of Brain Tomour (BT) and Neurosurgery (NS) on the renal handling of H₂O and Na, and the clinical importance of SIADH in this setting.Methods Fourteen patients with BT pre-op for NS and 6 controls (C) pre-op for general surgery, were assessed in a controlled prospective trial.All patients were normovolaemic, with normal renal function. They received 400 mg of lithium carbonate (Li) 8 hours before each of two test periods (I and II) and a standard water load only before period II. Clearances studies were performed pre-op (period I) and 24 hours post-op (period II).Results Serum Na was normal at all times. Despite normovolaemia, a 1% decrement in serum osmolality and the water load, ADH dramaticaly increased from time I to II mainly in the BT group (36.2±9.4 vs 7.1±0.6 pmol/L, p=0.02). FENa, FELi and FEUricA were significantly more elevated in the BT group pre and post-op (at time II respectively 4.6±1.6 vs 1.1±0.3%; 29.3±4.9 vs 22.6±5.5; 26.0±8.1 vs 11.3±2.2, p=0.03). Proximal and distal H₂O re-absorption and distal fractional Na re-absorption were identical in both groups pre and post-operatively.Conclusions 1-BT and NS always induce a SIADH. 2-There was a primary Na loss at the proximal tubule level not explained by ADH increment, that did not significantly changed H₂O handling. 3-To prevent hyponatraemia, hypotonic I.V. fluids should be avoided, but more importantly saline must be provided to this potentially salt-wasting condition.Abbreviations Al Aldosterone - ADH Anti diuretic hormone - UNa Urine sodium (mEq/l) - SNa Serum sodium (mEq/l) - CrCl Creatinine clearance - LiCl Lithium clearance - H₂OCl Free water clearance - UricACl Uric acid clearance - NaCl Sodium clearance - FENa Fractional excretion of sodium (NaCl/CrCl) - FEUricA Fractional excretion of uric acid (UricACl/CrCl) - FELi Fractional excretion of Lithium (LiCl/CrCl) - FNa Filtered load of sodium (CrCl^*SNa/1000 mEq/min) - DistNa Distal renal tubular delivery of sodium (LiCl^*SNa/1000 mEq/min) - NaPR Proximal renal tubular reabsorption of sodium (DistNa-(UNa^*Urine Volume)) - NaDR Distal renal tubular sodium reabsorption (DistNa-(UNa^*Urine volume)) - H₂OPR Proximal renal tubular water reabsorption (CrCl-LiCr)ml/min - H₂ODR Distal renal tubular water reabsorption (Licl-Urine volume)ml/min - NaPFR Renal tubular proximal sodium fractional reabsorption (NaPR/FNa^*100)% - NaDFR Renal tubular distal sodium fractional reabsorption (NaDR/DistNa^*100)%     

Pseudoaldosteronism with increased serum cortisol associated with pneumonia, hypouricemia, hypocalcemia, and hypophosphatemia

We describe here the interesting case of a 73-year-old hypertensive man with pseudoaldosteronism. He had been taking glycyrrhizin at a dose of 75 mg/day for 12 years because of mild liver damage, but had never experienced any previous symptoms associated with hypokalemia. He was referred to our hospital because of hypokalemic tetraparesis and rhabdomyolysis. At that time, we noted mineralocorticoid excess characterized by hypokalemia due to urinary K loss, exacerbation of hypertension due to increased tubular Na reabsorption, metabolic alkalosis, and suppression of both plasma renin activity and plasma aldosterone concentration. His urinary free cortisol excretion rate and the urinary ratio of free cortisol to free cortisone were markedly elevated. Thus we diagnosed pseudoaldosteronism that was related to the long-term use of glycyrrhizin. When he developed pseudoaldosteronism, he also contracted pneumonia, and exhibited elevated levels of serum cortisol and creatinine clearance (CCr) as well as hypouricemia, hypocalcemia, and hypophosphatemia. All normalized after the recovery from pneumonia and the administration of spironolactone. The extracellular volume expansion associated with increased tubular Na reabsorption by the aldosterone-sensitive distal nephron and the resulting increase in CCr caused an inhibition of proximal tubular reabsorption of uric acid, Ca, and inorganic phosphate, leading to their renal loss and therefore hypouricemia, hypocalcemia, and hypophosphatemia, respectively. In this patient, the increased circulating cortisol associated with the stress of inflammation caused by pneumonia triggered the development of pseudoaldosteronism.     

Unmasking of primary hyperaldosteronism by renal transplantation

BACKGROUND: Primary hyperaldosteronism is an uncommon cause of hypertension in the general population. Given the mechanism of action of aldosterone clinical manifestations may not occur in the setting of end stage renal disease. However, if a successful renal transplant is performed clinical manifestations may occur. METHODS: We present a case of a patient with a preexisting adrenal adenoma who only presented with clinical signs of hyperaldosteronism after renal transplantation. Patients' work-up included plasma aldosterone, plasma renin activity, serum cortisol, and estimation of trans tubular potassium gradient. RESULTS: The patient's serum aldosterone was markedly elevated with a relatively suppressed plasma renin activity. Trans tubular potassium gradient was high in the presence of hypokalemia. CONCLUSION: Previously silent hyperaldosteronism may be unmasked by a successful renal transplant.     

Inherited renal tubulopathies associated with metabolic alkalosis: effects on blood pressure

Inherited tubular disorders associated with metabolic alkalosis are caused by several gene mutations encoding different tubular transporters responsible for NaCl renal handling. Body volume and renin-angiotensin-aldosterone system status are determined by NaCl reabsorption in the distal nephron. Two common hallmarks in affected individuals: hypokalemia and normal / high blood pressure, support the differential diagnosis. Bartter's syndrome, characterized by hypokalemia and normal blood pressure, is a heterogenic disease caused by the loss of function of SLC12A1 (type 1), KCNJ1 (type 2), CLCNKB (type 3), or BSND genes (type 4). As a result, patients present with renal salt wasting and hypercalciuria. Gitelman's syndrome is caused by the loss of funcion of the SLC12A3 gene and may resemble Bartter's syndrome, though is associated with the very low urinary calcium. Liddle's syndrome, also with similar phenotype but with hypertension, is produced by the gain of function of the SNCC1B or SNCC1G genes, and must be distinguished from other entities of inherited hypertension such as Apparently Mineralocorticoid Excess, of glucocorticoid remediable hypertension.     

Pendrin regulation in mouse kidney primarily is chloride-dependent

Recent studies indicate that pendrin, an apical Cl-/HCO3- exchanger, mediates chloride reabsorption in the connecting tubule and the cortical collecting duct and therefore is involved in extracellular fluid volume regulation. The purpose of this study was to test whether pendrin is regulated in vivo primarily by factors that are associated with changes in renal chloride transport, by aldosterone, or by the combination of both determinants. For achievement of this goal, pendrin protein abundance was studied by semiquantitative immunoblotting in different mouse models with altered aldosterone secretion or tubular chloride transport, including NaCl loading, hydrochlorothiazide administration, NaCl co-transporter knockout mice, and mice with Liddle's mutation. The parallel regulation of the aldosterone-regulated epithelial sodium channel (ENaC) was examined as a control for biologic effects of aldosterone. Major changes in pendrin protein expression were found in experimental models that are associated with altered renal chloride transport, whereas no significant changes were detected in pendrin protein abundance in models with altered aldosterone secretion. Moreover, in response to hydrochlorothiazide administration, pendrin was downregulated despite a marked secondary hyperaldosteronism. In contrast, alpha-ENaC was markedly upregulated, and the molecular weight of a large fraction of gamma-ENaC subunits was shifted from 85 to 70 kD, consistent with previous results from rat models with elevated plasma aldosterone levels. These results suggest that factors that are associated with changes in distal chloride delivery govern pendrin expression in the connecting tubule and cortical collecting duct.     

A novel mutation of the thiazide-sensitive sodium chloride cotransporter gene in a Japanese family with Gitelman syndrome

Gitelman syndrome is an inherited renal disorder characterized by impaired NaCl reabsorption in the distal convoluted tubule leading to hypokalemia, hypomagnesemia and normocalcemic hypocalciuria. It has been shown that this syndrome results from mutations in the gene encoding the thiazide-sensitive sodium chloride cotransporter (TSC). We performed the mutational analysis in the TSC gene of a 30-year-old Japanese woman with Gitelman syndrome and found two mutations at adjacent spots in both alleles. One was a frame shift mutation which generated stop codon at position 671, the other was a single nucleotide mutation, which resulted in an aminoacid substitution at position 672, Met to Ile. Her 52-year-old mother and two daughters had neither hypokalemia nor hypomagnesemia. However, her mother and her 8-year-old daughter had the Met672Ile mutation as heterozygotes. Her 4-year-old daughter had the same frame shift mutation as her mother, a heterozygotic mutation. These results suggest that Gitelman syndrome requires 2 compound heterozygotic mutations and the coexistence of the large deletion in the C-terminal domain with Met672Ile substitution of the TSC could impair the transporter activity underling the hypokalemia and hypomagnesemia in this patient.     

TRPV5: an ingeniously controlled calcium channel

Body Ca(2+) homeostasis is tightly controlled and slight disturbances in renal Ca(2+) reabsorption can lead to excessive urine Ca(2+) excretion and promote kidney stone formation. The epithelial Ca(2+) channel TRPV5 constitutes the rate-limiting step of active Ca(2+) reabsorption in the kidney. Elucidation of the molecular pathways controlling TRPV5 function provides important information for our understanding of renal Ca(2+) handling, since active Ca(2+) reabsorption fine-tunes the final amount of Ca(2+) excreted into the urine. Over the last years, the molecular regulation of TRPV5 has been dismantled in detail. Various calciotropic hormones, known to alter renal Ca(2+) reabsorption, affect the expression of TRPV5. Others stimulate the trafficking of TRPV5 to the plasma membrane, while a number of associated proteins and ions control channel activity at the plasma membrane. Dynamic cell surface presence of TRPV5 is largely mediated by endosomal recycling processes allowing internalized channels to reappear at the plasma membrane. We present recently identified factors shown to modulate TRPV5 activity by diverse mechanisms to ultimately control renal Ca(2+) handling. The selected factors include klotho, tissue kallikrein, pH, Ca(2+), Mg(2+), PIP(2) and WNK4. This review covers the distinctive properties and regulation of the highly Ca(2+)-selective TRPV5 channel and highlights the implications for our understanding of the process of Ca(2+) reabsorption.     

Aluminum is a weak agonist for the calcium-sensing receptor

BACKGROUND: Aluminum (Al3+) has diverse biological effects mediated through activation of a putative extracellular cation-sensing receptor. A recently identified calcium-sensing receptor (CaSR), which has been identified in target tissues for Al3+, may transduce some of the biological effects of Al3+. METHODS: To test this possibility, we transfected human embryonic kidney 293 (HEK 293) cells with a cDNA encoding the rat CaSR and evaluated CaSR expression by Western blot analysis and function by measurement of intracellular calcium ([Ca2+]i) levels and inositol monophosphate (IP1) generation following stimulation with Al3+ and a panel of CaSR agonists. RESULTS: The CaSR protein was detected by immunoblot analysis in cells transfected with the CaSR cDNA but not in nontransfected HEK 293 cells. In addition, [Ca2+]i levels and IP1 generation were enhanced in a dose-dependent fashion by additions of the CaSR agonists calcium (Ca2+), magnesium (Mg2+), gadolinium (Gd3+), and neomycin only in cells transfected with CaSR. To determine if Al3+ activated CaSR, we stimulated cells transfected with rat CaSR with 10 microM to 1 mM concentrations of Al3+. Concentrations of Al3+ in the range of 10 microM to 100 microM had no effect on [Ca2+]i levels or IP1 generation. In contrast, 1 mM Al3+ induced small but significant increases in both parameters. Whereas Gd3+ antagonized calcium-mediated activation of CaSR, pretreatment with Al3+ failed to block subsequent activation of rat CaSR by Ca2+, suggesting a distinct mechanism of Al3+ action. CONCLUSION: Al3+ is not a potent agonist for CaSR. Because Al3+ affects a variety of target tissues at micromolar concentrations, it seems unlikely that CaSR mediates these cellular actions of Al3+.     

Cyclosporin A tubular effects contribute to nephrotoxicity: role for Ca2+ and Mg2+ ions.

BACKGROUND: Cyclosporin A (CsA) nephrotoxicity has been attributed primarily to renal haemodynamic alterations caused by afferent arteriolar vasoconstriction. However, CsA nephropathy is also characterized by CsA-induced pre-glomerular disturbances and interstitial injury that may occur independently of haemodynamic changes. Given the high lipophilic activity of CsA, we hypothesized that direct tubular injury is likely to contribute to nephrotoxicity. METHODS: To investigate tubular toxicity of CsA, increasing concentrations of CsA (1, 2.5, 10, 25, 50 and 100 micro g/ml) and its vehicle (cremophor) were added to isolated rat proximal tubules (PT). Cell injury was assessed by lactate dehydrogenase (LDH) release. The role of Ca(2+) ions in tubular toxicity and the effect of calcium channel blockers on CsA toxicity were evaluated by measuring intracellular calcium using the fluorescent dye Fura-2 AM. The role of Mg(2+) ions was assessed using high extracellular Mg(2+) medium (2 mM). RESULTS: Whereas cremophor alone was not toxic to PT, CsA caused PT injury but only at the highest concentration (100 micro g/ml). After 90 min incubation, LDH was 22.5% in control PT and 41.9% in PT treated with 100 micro g/ml CsA (P < 0.001, n = 11). There was a transient increase in intracellular calcium ([Ca(2+)](i)) after CsA administration. A low calcium medium (100 nM) prevented CsA injury to renal tubules. However, verapamil, but not nifedipine, enhanced cell damage. Only nifedipine completely prevented [Ca(2+)](i) increases following CsA. Finally, a high Mg(2+) medium attenuated CsA-induced injury. CONCLUSION: We found that high CsA concentrations caused Ca(2+)- and Mg(2+)-dependent PT injury. Thus, low extracellular Ca(2+) and high Mg(2+) media attenuated CsA-induced tubular injury. Verapamil, but not nifedipine, enhanced CsA tubular toxicity. Therefore, CsA-induced tubular injury may contribute to CsA nephrotoxicity independently of haemodynamic disturbances.     

The effect of calcitriol on fasting urine calcium loss and renal tubular reabsorption of calcium in patients with mild renal failure--actions of a permissive hormone

AIM: Renal production of 1,25-dihydroxycholecalciferol is attenuated in early renal failure. Renal tubular reabsorption of calcium is diminished in moderate renal failure and we wished to see if this were true in the early stages and whether supplementary calcitriol would bring about correction. We were interested in the idea of 1,25-dihydroxycholecalciferol being a permissive agent, operating indirectly. METHODS: We measured calcium-related variables, including calculated ultrafiltrable serum calcium, before and after calcitriol 0.5 microg daily for six days in 34 subjects with stable mild renal failure. RESULTS: The mean serum creatinine was 0.21 (+/- 0.08) mmol/l. The mean serum Ca++ was normal (1.18 mmol/l) but nine patients had values outside the normal range and in six cases, with low-normal serum Ca++ levels, there was a diminished tubular reabsorption. In five cases, basal serum Ca++ was mildly elevated. The coefficient of variation for serum Ca++ was 4.4%. PTH (1-84) levels were mildly elevated and 1,25-dihydroxycholecalciferol levels low-normal. The urine Ca/Cr, representing net bone resorption, was elevated in six cases. After calcitriol, the mean serum Ca++ level rose slightly and the coefficient of variation decreased to 3.6%. Changes in Ca++ whether upward or downward were accounted for by minor alterations in tubular reabsorption and a tendency to less net bone resorption. The initial Ca++ predicted (negatively) the magnitude of the correction. Neither the prevailing PTH nor the 1,25-dihydroxycholecalciferol levels explained any of the observed changes. CONCLUSION: In early renal failure, there may be impaired regulation of serum Ca++. Despite elevated PTH, mild hypocalcemia may exist in the presence of increased net bone resorption relative to GFR. Hypocalcemia was accounted for by reduced renal tubular reabsorption of calcium which corrected after calcitriol. Net bone resorption tended to fall after calcitriol. Mild hypercalcemia, when present, was corrected by a reduction in tubular reabsorption. Calcitriol did not have a simple unidirectional effect but instead contributed to efficiency of the homeostatic mechanisms controlling the serum Ca++ set-point.