Laboratory changes during adrenocorticotropic hormone therapy associated with renal calcified lesions.

BACKGROUND: Renal calcified lesions are known as one of the complications during adrenocorticotropic hormone (ACTH) therapy for intractable epilepsy. However, laboratory changes during the therapy or laboratory features of high-risk cases with renal calcified lesions are yet to be clarified. METHODS: In this study, 43 patients with West syndrome aged ≤2 years were included. We retrospectively reviewed age and body mass index at the beginning of ACTH therapy, as well as the amount of fluid intake, daily urinary volume, and laboratory data during therapy. In addition, we studied the urinary sediment of the cases with renal calcified lesions diagnosed by computed tomography. RESULTS: After initiating ACTH treatment, urinary calcium (Ca)/creatinine ratio and urinary pH increased within 2 weeks. Urinary crystals and renal tubular epithelial cells (RTECs) in urinary sediment were frequently found in most cases. Urinary Ca levels, proteinuria or frequency of urinary crystals, and number of RTECs in the urinary sediment were significantly higher in patients with epithelial casts (ECs) or hematuria than in patients without these findings. Among the seven patients who underwent abdominal CT, ECs or hematuria were found only in those with renal calcified lesions. These findings suggested that patients with ECs or hematuria were more likely to have calcified lesions. CONCLUSIONS: The risk of renal calcified lesions increased after 2 weeks of ACTH treatment. Abnormal findings in urinary sediments might be an early sign of renal calcification during ACTH therapy.

Kidney volume, kidney function, and ambulatory blood pressure in children born extremely preterm with and without nephrocalcinosis.

BACKGROUND: Reduced kidney volume (KV) following prematurity is a proxy for reduced nephron number and is associated with the development of hypertension and end-stage renal disease in adults. We investigated whether extreme prematurity affects KV, function, and blood pressure in school-aged children and if nephrocalcinosis (NC) developed during the neonatal period had additional effects. METHODS: We investigated 60 children at a mean age of 7.7 years: 20 born extremely preterm (EPT < 28 weeks gestational age with NC (NC+)), 20 born EPT without NC (NC-), and 19 born as full-term infants (control). We measured KV by ultrasound, collected blood and urine samples to evaluate renal function, and measured office and 24-h ambulatory blood pressure (ABPM). RESULTS: Children born EPT had significantly smaller kidneys (EPT (NC+ NC-) vs control (estimated difference, 11.8 (CI - 21.51 to - 2.09 ml), p = 0.018) and lower but normal cystatin C-based glomerular filtration rate compared with control (estimated difference, - 10.11 (CI - 0.69 to - 19.5), p = 0.035). KV and function were not different between NC+ and NC- groups. Change in KV in relation to BSA (KV/BSA) from the neonatal period to school age showed significantly more EPT children with neonatal NC having a negative evolution of KV (p = 0.01). Blood pressure was normal and not different between the 3 groups. Fifty percent of EPT had a less than 10% day-to-night decline in ABPM. CONCLUSIONS: Kidney growth and volume is affected by EPT birth with NC being a potential aggravating factor. Circadian blood pressure regulation seems abnormal in EPT-born children.

Mice with a Brd4 Mutation Represent a New Model of Nephrocalcinosis.

Nephrolithiasis (NL) and nephrocalcinosis (NC), which comprise renal calcification of the collecting system and parenchyma, respectively, have a multifactorial etiology with environmental and genetic determinants and affect ∼10% of adults by age 70 years. Studies of families with hereditary NL and NC have identified >30 causative genes that have increased our understanding of extracellular calcium homeostasis and renal tubular transport of calcium. However, these account for <20% of the likely genes that are involved, and to identify novel genes for renal calcification disorders, we investigated 1745 12-month-old progeny from a male mouse that had been treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for radiological renal opacities. This identified a male mouse with renal calcification that was inherited as an autosomal dominant trait with >80% penetrance in 152 progeny. The calcification consisted of calcium phosphate deposits in the renal papillae and was associated with the presence of the urinary macromolecules osteopontin and Tamm-Horsfall protein, which are features found in Randall's plaques of patients with NC. Genome-wide mapping located the disease locus to a ∼30 Mbp region on chromosome 17A3.3-B3 and whole-exome sequence analysis identified a heterozygous mutation, resulting in a missense substitution (Met149Thr, M149T), in the bromodomain-containing protein 4 (BRD4). The mutant heterozygous (Brd4+/M149T ) mice, when compared with wild-type (Brd4+/+ ) mice, were normocalcemic and normophosphatemic, with normal urinary excretions of calcium and phosphate, and had normal bone turnover markers. BRD4 plays a critical role in histone modification and gene transcription, and cDNA expression profiling, using kidneys from Brd4+/M149T and Brd4+/+ mice, revealed differential expression of genes involved in vitamin D metabolism, cell differentiation, and apoptosis. Kidneys from Brd4+/M149T mice also had increased apoptosis at sites of calcification within the renal papillae. Thus, our studies have established a mouse model, due to a Brd4 Met149Thr mutation, for inherited NC. © 2019 American Society for Bone and Mineral Research.

Generation of a Primary Hyperoxaluria Type 1 Disease Model Via CRISPR/Cas9 System in Rats.

BACKGROUND: Primary hyperoxaluria type 1 (PH1) is an inherited disease caused by mutations in alanine-glyoxylate aminotransferase (AGXT). It is characterized by abnormal metabolism of glyoxylic acid in the liver leading to endogenous oxalate overproduction and deposition of oxalate in multiple organs, mainly the kidney. Patients of PH1 often suffer from recurrent urinary tract stones, and finally renal failure. There is no effective treatment other than combined liver-kidney transplantation. METHODS: Microinjection was administered to PH1 rats. Urine samples were collected for urine analysis. Kidney tissues were for Western blotting, quantitative PCR, AGT assays and histological evaluation. RESULTS: In this study, we generated a novel PH1 disease model through CRISPR/Cas9 mediated disruption of mitochondrial localized Agxt gene isoform in rats. Agxt-deficient rats excreted more oxalate in the urine than WT animals. Meanwhile, mutant rats exhibited crystalluria and showed a slight dilatation of renal tubules with mild fibrosis in the kidney. When supplied with 0.4% ethylene glycol (EG) in drinking water, mutant rats excreted greater abundance of oxalate and developed severe nephrocalcinosis in contrast to WT animals. Significantly elevated expression of inflammation- and fibrosisrelated genes was also detected in mutants. CONCLUSION: These data suggest that Agxt-deficiency in mitochondria impairs glyoxylic acid metabolism and leads to PH1 in rats. This rat strain would not only be a useful model for the study of the pathogenesis and pathology of PH1 but also a valuable tool for the development and evaluation of innovative drugs and therapeutics.

The Long Pentraxin PTX3 Is an Endogenous Inhibitor of Hyperoxaluria-Related Nephrocalcinosis and Chronic Kidney Disease.

The long pentraxin 3 (PTX3) exerts a variety of regulatory functions in acute and chronic tissue inflammation. In particular, PTX3 acts as an opsonin for a variety of pathogens and endogenous particles. We hypothesized that PTX3 would exhibit opsonin-like functions toward calcium oxalate crystals, too, and inhibit crystal growth. This process is fundamental in kidney stone disease as well as in hyperoxaluria-related nephrocalcinosis, the paradigmatic cause of chronic kidney disease (CKD) in children with primary hyperoxaluria type I due to genetic defects in oxalate metabolism. Direct effects of PTX3 on calcium oxalate crystals were investigated in chemico by adding recombinant PTX3 to supersaturated calcium and oxalate solutions. PTX3, but not isomolar concentrations of albumin, dose-dependently inhibited crystal growth. In vivo, the PTX3 protein was undetectable in tubular epithelial cells and urine of wild-type mice under physiological conditions. However, its levels increased within 3 weeks of feeding an oxalate-rich diet, an exposure inducing hyperoxaluria-related nephrocalcinosis and CKD in selected mouse strains (male and female C57BL/6N and male Balb/c mice) but not in others (male and female 129SV and CD-1, male and female Balb/c mice). Genetic ablation of ptx3 in nephrocalcinosis un-susceptible B6;129 mice was sufficient to raise the oxalate nephropathy phenotype observed in susceptible strains. We conclude that PTX3 is an endogenous inhibitor of calcium oxalate crystal growth. This mechanism limits hyperoxaluria-related nephrocalcinosis, e.g., in primary or secondary hyperoxaluria, and potentially also in the more prevalent kidney stone disease.

Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods.

BACKGROUND: Molecular and cellular pathophysiological events occurring in the majority of rare kidney diseases remain to be elucidated. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive disorder caused by mutations in either CLDN16 or CLDN19 genes. This disease is characterized by massive urinary wasting of magnesium and calcium, osmosis deregulation and polyuria. Patients with p.G20D homozygous mutation in CLDN19 gene exhibit different progression to kidney failure suggesting that beyond the pathogenic mutation itself, other molecular events are favoring disease progression. Due to the fact that biopsy is not clinically indicated in these patients, urinary exosome-like vesicles (uEVs) can be envisioned as a valuable non-invasive source of information of events occurring in the kidney. Exosome research has increased notably to identify novel disease biomarkers but there is no consensus standardized protocols for uEVs isolation in patients with polyuria. For this reason, this work was aimed to evaluate and refine different uEVs isolation methods based on differential centrifugation, the gold standard method. RESULTS: Characterization by NTA, cryo-TEM and immunoblotting techniques identified the most appropriate protocol to obtain the highest yield and purest uEVs enriched fraction possible from urine control samples and FHHNC patients. Moreover, we tested five different RNA extraction methods and evaluated the miRNA expression pattern by qRT-PCR. CONCLUSIONS: In summary, we have standardized the conditions to proceed with the identification of differentially expressed miRNAs in uEVs of FHHNC patients, or other renal diseases characterized by polyuria.

Short-Term Effect of High-Dose Pantoprazol on Serum and Urinary Magnesium Levels.

BACKGROUND: Proton pump inhibitor (PPI) induced hypomagnesemia is a completely unexplained issue and cases are still being reported. Long-term use is the main factor, but there are a few articles stating that it may also emerge with short-term use. We aimed to evaluate the changes of serum and urine magnesium levels during shortterm high dose pantoprazol treatment. METHODS: The serum and 24-hour urine magnesium levels of 58 patients were evaluated during the course of 2 days. Of 58 patients, 25 were allowed oral intake on the 3rd day of hospitalization and thus, 24-hour urine for 3 days was collected from 33 patients. RESULTS: There were no significant differences in the mean levels of serum magnesium and the median levels of urine magnesium. When the magnesium levels were evaluated by age over and under 60 years, the baseline serum magnesium level was significantly higher than the 1st level in patients aged ≥ 60 years (p = 0.029). The 3rd day serum magnesium level was significantly higher than the baseline and 1st day levels in those aged < 60 years (p = 0.049). CONCLUSIONS: We showed that plasma levels and urinary excretion of magnesium did not change significantly during high-dose pantoprazol treatment. It can be hypothesized that magnesium levels are not affected by PPIs in short-term usage. Age and other contributing factors may have more impact on PPI induced hypomagnesemia. Patients aged over 60 years might be handled carefully under proton pump inhibitors treatment.

Type 1 primary hyperoxaluria: A case report and focus on bone impairment of systemic oxalosis.

Primary hyperoxaluria is a rare genetic disorder characterized by oxalate overproduction, leading to kidney failure due to nephrocalcinosis, and is eventually responsible for systemic oxalosis. Bone impairment, secondary to oxalate deposits, is one of the many complications that may occur. Skeletal involvement can be difficult to diagnose because of lack of clinical symptoms and therefore needs to be confirmed by invasive testing, such as transiliac bone biopsy. If confirmed, bone oxalosis is the proof of disease severity and that combined liver-kidney transplantation should be performed.

A novel CYP24A1 genotype associated to a clinical picture of hypercalcemia, nephrolithiasis and low bone mass.

Mutations of the CYP24A1 gene, encoding for the enzyme 25(OH)D-24-hydroxylase, can cause hypercalcemia, hypercalciuria, nephrolithiasis and nephrocalcinosis. We report the case of a 22-year-old male patient with recurrent nephrolithiasis, nephrocalcinosis, hypercalcemia with low parathyroid hormone levels, hypercalciuria and low bone mass. Gene sequencing showed that the patient had compound heterozygous mutations including a novel genotype of the CYP24A1 gene. Genetic CYP24A1 testing and biochemical analyses were offered to other family members; the father was heterozygous for the same novel genotype and was also affected with recurrent nephrolithiasis.

The primary hyperoxalurias: A practical approach to diagnosis and treatment.

Although the primary hyperoxalurias (PH) are rare disorders, they are of considerable clinical importance in relation to calcium oxalate urolithiasis and as a cause of renal failure worldwide. Three distinct disorders have been described at the molecular level. The investigation of any child or adult presenting with urinary tract stones or nephrocalcinosis, must exclude PH as an underlying cause. This paper provides a practical approach to the investigation and diagnosis of PH, indicating the importance of distinguishing between the PH types for the purposes of targeting appropriate therapy. Conservative management is explored and the various transplant options are discussed.

Identification of SLC41A3 as a novel player in magnesium homeostasis.

Regulation of the body Mg(2+) balance takes place in the distal convoluted tubule (DCT), where transcellular reabsorption determines the final urinary Mg(2+) excretion. The basolateral Mg(2+) extrusion mechanism in the DCT is still unknown, but recent findings suggest that SLC41 proteins contribute to Mg(2+) extrusion. The aim of this study was, therefore, to characterize the functional role of SLC41A3 in Mg(2+) homeostasis using the Slc41a3 knockout (Slc41a3(-/-)) mouse. By quantitative PCR analysis it was shown that Slc41a3 is the only SLC41 isoform with enriched expression in the DCT. Interestingly, serum and urine electrolyte determinations demonstrated that Slc41a3(-/-) mice suffer from hypomagnesemia. The intestinal Mg(2+) absorption capacity was measured using the stable (25)Mg(2+) isotope in mice fed a low Mg(2+) diet. (25)Mg(2+) uptake was similar in wildtype (Slc41a3(+/+)) and Slc41a3(-/-) mice, although Slc41a3(-/-) animals exhibited increased intestinal mRNA expression of Mg(2+) transporters Trpm6 and Slc41a1. Remarkably, some of the Slc41a3(-/-) mice developed severe unilateral hydronephrosis. In conclusion, SLC41A3 was established as a new factor for Mg(2+) handling.

[Crystalluria]. / Cristallurie.

Crystalluria is the presence of crystals in urine. It results from excessive supersaturation and may induce kidney problems such as nephrolithiasis, nephrocalcinosis and sometimes acute or chronic kidney impairment able to result in end stage renal failure. Crystalluria is not, per se, a marker of a pathologic condition. For distinguishing between physiologic and pathologic crystalluria, various criteria may be considered (1) either related to the sample such as urine pH, crystal identity, crystal habit, abundance of the crystals, aggregation and also the occurrence of crystalluria judged on serial samples, (2) or related to the clinical context such as nephrolithiasis, nephrocalcinosis or renal failure. The choice of the sample and the pre-analytical conditions are critical to validate the results from a clinical point of view. In our experience, the first morning urine sample is often the best one to assess, through the crystalluria analysis, the main metabolic factors involved in the crystal and stone formation. The storage of urine sample should be less than two hours at room temperature following the voiding. Crystalluria examination allows identifying simply monogenic crystallogenetic pathologies, providing explanation for acute renal failure related to drug intake, helping the physician for identifying the main metabolic disorders involved in stone formation and assessing the efficacy of preventive measures proposed to avoid stone recurrence in stone former patients. Crystalluria study is by far the best marker for predicting stone recurrence during the follow-up of stone formers, offering the opportunity to adjust dietary advices or drug management and thus to prevent stone formation. In conclusion, crystalluria examination is a major tool for the diagnosis and the management of most lithogenetic diseases and pathologic conditions responsible for intratubular crystal formation and renal impairment.

Nephrocalcinosis is a risk factor for kidney failure in primary hyperoxaluria.

Stone formation and nephrocalcinosis are both very common features of primary hyperoxaluria, yet the extent of each disease varies markedly between patients. Here we studied whether kidney damage from nephrocalcinosis and/or stone related events contributed to end-stage kidney disease (ESKD). Clinical information was analyzed from 348 patients enrolled in the Rare Kidney Stone Consortium Primary Hyperoxaluria registry and included demographic, laboratory and imaging features. Among all patients there were 277 with type 1, 37 with type 2, and 34 with type 3 primary hyperoxaluria. Overall, 58% passed a stone (mean 0.3/year) and one or more urologic procedures were required by 70% of patients (mean 0.15/year). Nephrocalcinosis was found in 34% of patients, including 41% with type 1 primary hyperoxaluria. High urine oxalate was associated with increased risk for both nephrocalcinosis and stone number, while low urine citrate was a risk factor for stone events and stone number. After adjustment for the type of primary hyperoxaluria, diagnosis by family screening and age at first image, the overall adjusted hazard ratio for ESKD among those with a history of nephrocalcinosis was 1.7 [95% CI 1.0-3.0], while the risk was 4.0 [1.9-8.5] for new onset nephrocalcinosis during follow-up. In contrast, the number of stones and stone events were not significantly associated with ESKD risk. Thus, nephrolithiasis and nephrocalcinosis appear to be pathophysiologically distinct entities. The presence of nephrocalcinosis implies increased risk for ESKD.

A hypokalaemic woman with nephrocalcinosis: rebirth of old knowledge.

The coexistence of hypokalaemia and nephrocalcinosis poses a challenge in rapid diagnosis and appropriate management. We describe a 38-year-old woman who presented with thirst, intermittent carpopedal spasm, paresthaesia of both hands and progressive weakness of lower extremities for two years. She had a history of chronic hypokalaemia of unknown cause with intermittent potassium supplementation for 7-8 y and bilateral nephrocalcinosis notable for one year. She denied vomiting, diarrhoea or use of laxatives, alcohol or diuretics. Her blood pressure was normal. Laboratory investigations showed hypokalaemia (2.7 mmol/L) and metabolic alkalosis (HCO3(-) 32.6 mmol/L, pH 7.46). Two random urine samples both showed a consistently high urine K(+) excretion but with excretion rates of Na(+), Cl(-) and divalent cations which were high in one sample but not the other. Ingestion of furosemide 120 mg daily for body image for 7-8 y was uncovered. With furosemide cessation and potassium supplementation, her hypokalaemia with neuromuscular symptoms was corrected but nephrocalcinosis persisted. Surreptitious use of diuretics for various purposes should be kept in mind as an important cause of hypokalaemia and/or nephrocalcinosis. Measurement of electrolyte concentrations in at least two random urine samples is warranted to distinguish it from true renal tubular disorders and extrarenal causes.

Enamel renal syndrome: a rare case report.

Enamel renal syndrome is a very rare disorder associating amelogenesis imperfecta with nephrocalcinosis. It is known by various synonyms such as amelogenesis imperfecta nephrocalcinosis syndrome, MacGibbon syndrome, Lubinsky syndrome, and Lubinsky-MacGibbon syndrome. It is characterized by enamel agenesis and medullary nephrocalcinosis. This paper describes enamel renal syndrome in a female patient born in a consanguineous family.