Giacomo Colussi

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.

Correction of Hypokalemia with Antialdosterone Therapy in Gitelman’s Syndrome

Six adult patients (4 females and 2 males, age range 26-57 years) with Gitelmans syndrome (GS) were treated with spironolactone 200-300 mg/day (n = 5) and/or amiloride 10-30 mg/day (n = 3) for 1-18 months. The patients had hypokalemia, hyperreninemia, chloride-resistant metabolic alkalosis, renal hypomagnesemia (n = 5), and hypocalciuria (n = 5). Free water clearance studies during maximal water diuresis and furosemide administration were suggestive of a solute reabsorptive defect beyond the loop of Henle. Antialdosterone therapy induced a significant increase of PK (from 2.6 +/- 0.4 to 3.4 +/- 0.4 mM; p < 0.0001) and a decrease of CK (from 21.4 +/- 13.2 to 10.6 +/- 4.8 ml/min, p < 0.02) and FEK (from 21.0 +/- 13.6 to 13.4 +/- 5.7%; p < 0.03); PMg increased from 1.38 +/- 0.38 to 1.64 +/- 0.21 mg/dl (p < 0.03) with a parallel fall of CMg (from 5.5 +/- 2.3 to 2.9 +/- 1.5 ml/min; p < 0.02) and FEMg (from 5.7 +/- 2.6 to 2.9 +/- 0.6%; p < 0.05); arterial blood pH and HCO3- did not change (P = plasma, C = clearance, FE = fractional excretion). The creatinine clearance fell (from 90.5 +/- 16.8 to 65.8 +/- 20.9 ml/min; p < 0.05), and Prenin rose (from 16.6 +/- 8.9 to 35.3 +/- 25.3 ng/ml/h; p < 0.02, as did Paldo (from 26.1 +/- 12.3 to 109 +/- 82.6 ng/dl; p < 0.01), indicating extracellular fluid volume contraction; however no significant clinical symptoms of hypovolemia ensued.(ABSTRACT TRUNCATED AT 250 WORDS)

Pseudohyperkalemia in Extreme Leukocytosis

Spurious elevation of blood K levels is a well known occurrence in patients with extreme leukocytosis. A common explanation is the in vitro release of K from leukocytes undergoing lysis during the clotting process. Since in clinical practice blood electrolytes are now being evaluated in plasma or whole heparinized blood rather than in serum, this source of error should almost have disappeared. Another mechanism may be prolonged storage of blood at room temperature or in the cold before performing the test, most likely since unphysiological conditions and/or shortage of metabolic fuels may impair Na/K-ATPase activity in leukocytes, ensuing in K release from these cells. For these reasons, it is commonly advised that patients with extreme leukocytosis should have K levels determined on plasma samples that are separated promptly from the cellular elements. We have recently observed a case of pseudohyperkalemia in a patient with chronic lymphocytic leukemia which was unrelated to both of these mechanisms, and was instead related to a common mode of drawing blood, i.e. with vacuum tubes.

Chronic hypercalcaemia from inactivating mutations of vitamin D 24-hydroxylase (CYP24A1): implications for mineral metabolism changes in chronic renal failure

BACKGROUND Loss-of-function mutations of vitamin D-24 hydroxylase have recently been recognized as a cause of hypercalcaemia and nephrocalcinosis/nephrolithiasis in infants and adults. True prevalence and natural history of this condition are still to be defined. METHODS We describe two adult patients with homozygous mutations and six related heterozygous carriers. Mineral and hormonal data in these patients were compared with that in 27 patients with stage 2-3 chronic kidney disease and 39 healthy adult kidney donors. RESULTS Probands had recurrent nephrolithiasis, chronic hypercalcaemia with depressed parathyroid hormone (PTH) and increased 1,25(OH)(2)D levels; carriers had nephrolithiasis (two of six), hypercalciuria (two of six) and high or normal-high 1,25(OH)(2)D (four of four). Corticosteroids did not reduce plasma and urine calcium levels, but ketoconazole did, indicating that 1,25(OH)(2)D production is not maximally depressed despite coexisting hypercalcaemia, high 1,25(OH)(2)D and depressed PTH, and that 1,25(OH)(2)D degradation through vitamin D-24 hydroxylase is a regulator of plasma 1,25(OH)(2)D levels. Both probands had vascular calcifications and high bone mineral content. One developed stage 3b renal failure: in this patient 1,25(OH)(2)D decreased within normal limits as glomerular filtration rate (GFR) fell and PTH rose to high-normal values, yet hypercalcaemia persisted and the ratio of 1,25(OH)(2)D to GFR remained higher than normal for any degree of GFR. CONCLUSIONS This natural model indicates that vitamin D-24 hydroxylase is a key physiologic regulator of calcitriol and plasma calcium levels, and that balanced reduction of 1,25(OH)(2)D and GFR is instrumental for the maintenance of physiologic calcium levels and balance in chronic kidney diseases.

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).

The genetic and clinical spectrum of a large cohort of patients with distal renal tubular acidosis

Primary distal renal tubular acidosis is a rare genetic disease. Mutations in SLC4A1, ATP6V0A4, and ATP6V1B1 genes have been described as the cause of the disease, transmitted as either an autosomal dominant or recessive trait. Particular clinical features, such as sensorineural hearing loss, have been mainly described in association with mutations in one gene instead of the others. Nevertheless, the diagnosis of distal renal tubular acidosis is essentially based on clinical and laboratory findings, and the series of patients described so far are usually represented by small cohorts. Therefore, a strict genotype-phenotype correlation is still lacking, and questions about whether clinical and laboratory data should direct the genetic analysis remain open. Here, we applied next-generation sequencing in 89 patients with a clinical diagnosis of distal renal tubular acidosis, analyzing the prevalence of genetic defects in SLC4A1, ATP6V0A4, and ATP6V1B1 genes and the clinical phenotype. A genetic cause was determined in 71.9% of cases. In our group of sporadic cases, clinical features, including sensorineural hearing loss, are not specific indicators of the causal underlying gene. Mutations in the ATP6V0A4 gene are quite as frequent as mutations in ATP6V1B1 in patients with recessive disease. Chronic kidney disease was frequent in patients with a long history of the disease. Thus, our results suggest that when distal renal tubular acidosis is suspected, complete genetic testing could be considered, irrespective of the clinical phenotype of the patient.

Distal Nephron Function in Familial Hypokalemia - Hypomagnesemia (Gitelman's Syndrome)

Dr. Giacomo Colussi, Divisione di Nefrologia e Dialisi, Ospedale Niguarda-Ca’ Granda, Piazza Ospedale Maggiore, 3, I20162 Milan (Italy) Dear Sir, In the November 1992 issue of Nephron , Zarraga Larrondo et al. [1] described a patient with the so-called familial hypokalemia-hy-pomagnesemia, or Gitelman’s syndrome [2], also known as ‘hypocalciuric variant’ of Bartter’s syndrome [3, 4]. They contend that fractional distal solute reabsorption (evaluated as the Ch2o/[CH2o + CCJ ratio during maximally diluted diuresis) was reduced, as expected (indicating abnormal distal nephron function) in the patient when urine dilution was achieved by hypotonic saline infusion, but was instead normal when urine was diluted by an oral water load. These apparently divergent results on distal nephron function are rather difficult to explain, and indeed efforts made by the authors to conciliate them appear rather unconvincing. However, if one looks at their data, it appears that all their efforts might have been unnecessary. In tables 1 and 2 of their paper, C1⁄8o and Cα values are given as 8.09 and 4.15 ml/min, respectively (this latter value was obtained by correcting the reported CCι value of 3.46 ml/dl GF by the corresponding GFR of 120 ml/ min). It is easy to calculate that the Ch2o/(Ch2o + Cα) ratio corresponds to 66.1% (a much lower value than the 80-92 reference range), and not to 88.3%, as reported in table 1 [1]. Accordingly, since CNa is also reported (2.25 ml/dl GF, i.e. 2.7 ml/min), one can calculate a C1⁄8o + CNa value of 10.79 ml/min (and not of 10.31 ml/min as reported in table 1), with a C7⁄8o/(C7⁄8o + CNa) ratio of 75%, again an abnormally low value. Thus, it appears that some computation mistake might have occurred, leading to an erroneous evaluation of the fractional distal solute reabsorption. Indeed, no ‘avid reabsorption of chloride’ can be said to exist in this patient, whereas abnormal distal nephron function could instead be detected not only during hypotonic saline infusion, but also during oral water load. In the same paper [1], the authors compared a series of parameters in their patient with reference values from a paper of ours [5]; unfortunately, some of these reference values were taken incorrectly. Among others, the reference value for Cα after furosemide is not 15.2 ± 1.6 (table 2) but 20.9 ± 6.5 ml/dl GF, CNa after furosemide (table 2) was not given in our paper, and the reference value for C1⁄8o after furosemide is not 15 ± 3 ml/min (table 1). This latter value in our paper [5] represents ‘total’ C7⁄8o generated by the distal nephron, i.e. the sum of C1⁄8o before furosemide and free water back-diffusion (which is the difference between urine flow rate after and before furosemide). Values for free water generation after furosemide administration were not given in our paper [5], and would correspond to 6.2 ± 2.5 ml/min [4]. Thus, it would appear

Aldosterone influences serum magnesium in Gitelman syndrome.

Dependent variable: log (magnesium concentration). Dear Sir, Gitelman syndrome is a genetic tubular disorder caused by mutations in the Na-Cl thiazide-sensitive cotransporter (TSC) gene, expressed in the distal convoluted tubule [1]. The cardinal features of this syndrome are hypokalemia, metabolic alkalosis, hypocalciuria, and hypomagnesemia. However, a few patients with normal serum magnesium levels were reported [2, 3]. In such a syndrome, the cause of hypomagnesemia is still unknown [4, 5]. Many different factors can stimulate net magnesium transport in the distal convoluted tubule, such as volume depletion, metabolic alkalosis, vasopressin, and aldosterone [4–6]. In 30 patients with Gitelman syndrome (mean age 20.3, range 7–42 years), we evaluated the relationship between serum magnesium and the most important biochemical parameters. All of the patients had hypokalemia (serum potassium 3.1 B 0.4 mmol/l), metabolic alkalosis (plasma HCO3 31.2 B 3.4 mmol/l), hyperreninemia (plasma renin 17.6 B 10.8 ng/ml/h), low urinary calcium creatinine molar ratio (0.08 B 0.09), increased fractional excretion of magnesium (5.7 B 2.5%), and normotension. The mean serum magnesium concentration was 0.60 B 0.13 mmol/l; 3 patients had normal serum levels of magnesium (10.75 mmol/l). The mean serum aldosterone level was 284 B 149 pg/ml. All of the patients had a normal glomerular filtration rate (GFR; creatinine clearance 102 B 14 ml/min/1.73/m2). Consistent mutations in the TSC gene [1, 3, personal commun.] were detected in all patients. A negligible correlation (Spearman’s coefficients between –0.09 and 0.14) was found between magnesium and age, serum electrolytes, plasma renin, pH, and bicarbonates. To evaluate simultaneously the effects of aldosterone and GFR on serum magnesium, a multiple regression analysis was performed. The magnesium concentration was log transformed to improve the normality of magnesium distribution; the regression model showed that, independently, both aldosterone and GFR were positively associated with the magnesium concentration (table 1). In this group of patients, aldosterone and creatinine clearance together accounted for about one third (r2 = 0.30) of the total variability of magnesium in Gitelman syndrome. These data are in accordance with the hypothesis that aldosterone can stimulate magnesium reabsorption directly or by potentiating the effect of vasopressin in distal convoluted tubules [4, 6] and can also partly explain the variability of serum magnesium in Gitelman syndrome patients. References

Calcium Oxalate (CaOx) Urine Supersaturation in Calcium Stone Formers (CSF): Hypercalciuria versus Hyperoxaluria

Calcium oxalate is the most frequent constituent of urinary calculi in idiopathic stone disease. Urinary excretion of both calcium (Ca) and oxalate (Ox) are recognised as risk factors1; however, there is still disagreement on the relative role played by each of the two in the genesis of urine supersaturation for CaOx. On the one hand, it has been shown that stone recurrence is related to oxalate excretion but not to calcium excretion2. On the other hand, it is widely recognized that reduction in Ca excretion (either by diet or with drugs) is highly effective in reducing the stone recurrence rate3,4, even though Ox excretion may increase. This is because intestinal calcium absorption and Ca excretion are frequently increased in CSF and the decline in urinary calcium is typically more prominent than the modest increase in urinary oxalate3. The aim of our study was to evaluate CaOx urine supersaturation in relation to either Ca or Ox excretion in a large group of idiopathic CSF in an attempt to determine if either of the two has a more relevant clinical importance.

Phosphate and Calcium Control in Short Frequent Hemodialysis with the NxStage System One Cycler: Mass Balance Studies and Comparison with Standard Thrice-Weekly Bicarbonate Dialysis

Background: Short frequent dialysis with NxStage System One cycler (NSO) has become increasingly popular as home hemodialysis prescription. Short dialysis sessions with NSO might not allow adequate phosphate (P) removal. Methods: Single-session and weekly balances of P and calcium (Ca) were compared in 14 patients treated with NSO (6 sessions/week) and in 14 patients on standard bicarbonate dialysis (BHD). Results: NSO and BHD showed similar plasma P fall, with end-dialysis plasma P slightly lower in BHD (2.2 ± 0.5 vs. 2.7 ± 0.8 mg/dL, p < 0.02). Single-session P removal was lower in NSO, but weekly removal was higher (3,488 ± 1,181 mg vs. 2,634 ± 878, p < 0.003). Plasma Ca increase was lower in NSO, with similar PTH fall. Ca balance varied according to start plasma Ca, dialysate to blood Ca gradient and net ultrafiltration. Conclusions: short, frequent home hemodialysis with NSO, on a 6/week-based prescription, allows higher weekly P removal than BHD. With the dialysate Ca concentration in use (6 mg/dL), total plasma Ca and iCa concentration increase is lower in NSO.