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.

STAT3 phosphorylation at serine 727 activates specific genetic programs and promotes clear cell renal cell carcinoma (ccRCC) aggressiveness.

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor mainly activated by phosphorylation in either tyrosine 705 (Y705) or serine 727 (S727) residues that regulates essential processes such as cell differentiation, apoptosis inhibition, or cell survival. Aberrant activation of STAT3 has been related to development of nearly 50% of human cancers including clear cell renal cell carcinoma (ccRCC). In fact, phosho-S727 (pS727) levels correlate with overall survival of ccRCC patients. With the aim to elucidate the contribution of STAT3 phosphorylation in ccRCC development and progression, we have generated human-derived ccRCC cell lines carrying STAT3 Y705 and S727 phosphomutants. Our data show that the phosphomimetic substitution Ser727Asp facilitates a pro-tumoral phenotype in vitro, in a Y705-phosphorylation-independent manner. Moreover, we describe that STAT3 phosphorylation state determines the expression of different subsets of target genes associated with distinct biological processes, being pS727-dependent genes the most related to cellular hallmarks of cancer. In summary, the present study constitutes the first analysis on the role of overall STAT3 phosphorylation state in ccRCC and demonstrates that pS727 promotes the expression of a specific subset of target genes that might be clinically relevant as novel biomarkers and potential therapeutic targets for ccRCC.