Abstract
BackgroundMolecular 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.ResultsCharacterization 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.ConclusionsIn 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.
Highlights
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 [2,3,4]
In order to standardize a protocol for urinary exosome-like vesicles (uEVs) isolation in patients with polyuria we evaluated and refined different uEVs isolation methods based on differential centrifugation
Summary
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. Exosome research has increased notably to identify novel disease biomarkers but there is no consensus standardized protocols for uEVs isolation in patients with polyuria. The high number of rare kidney diseases, their low prevalence and, in many cases, their highly phenotypic variability leads to Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal-recessive disorder caused by mutations in either CLDN16 or CLDN19 genes [2,3,4]. Because kidney biopsy samples, which could allow the study of those intra-renal processes, are not clinically indicated in most patients with FHHNC, blood and urine are the only available samples for the discovery of surrogated markers that might indicate disease progression in those patients
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