The scratch assay is an in vitro technique used to assess the contribution of molecular and cellular mechanisms to cell migration. The assay can also be used to evaluate therapeutic compounds before clinical use. Current quantification methods of scratch assays deal poorly with irregular cell-free areas and crooked leading edges which are features typically present in the experimental data. We introduce a new migration quantification method, called ‘monolayer edge velocimetry’, that permits analysis of low-quality experimental data and better statistical classification of migration rates than standard quantification methods. The new method relies on quantifying the horizontal component of the cell monolayer velocity across the leading edge. By performing a classification test on in silico data, we show that the method exhibits significantly lower statistical errors than standard methods. When applied to in vitro data, our method outperforms standard methods by detecting differences in the migration rates between different cell groups that the other methods could not detect. Application of this new method will enable quantification of migration rates from in vitro scratch assay data that cannot be analysed using existing methods.

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.

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. 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. 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.

RESUMO Introdução: A doença renal crônica (DRC) em fase inicial é frequentemente assintomática e permanece subdiagnosticada em populações de alto risco. As vesículas extracelulares urinárias (uEVs) constituem uma fonte não invasiva de biomoléculas relacionadas ao rim e podem aprimorar as estratégias de detecção precoce. Avaliamos a interleucina-6 (IL-6), a catepsina D (CTD) e o fator de crescimento transformador-β (TGF-β) associados às uEVs como biomarcadores candidatos para a DRC em fase inicial, incluindo padrões estratificados por sexo. Métodos: Neste estudo transversal, 230 adultos de alto risco (diabetes mellitus tipo 2 e/ou hipertensão arterial sistêmica) foram classificados como tendo função renal normal (FRN) ou DRC nos estágios G1–G4. As uEVs foram isoladas por meio de um protocolo padronizado e caracterizadas por microscopia eletrônica de varredura, espectroscopia Raman, espalhamento dinâmico de luz e Western blot para Alix e flotilina-1. A proteína total associada às uEVs foi quantificada, e a CTD, a IL-6 e o TGF-β foram mensurados nas preparações de uEVs. Resultados: As uEVs apresentaram a morfologia e a expressão de marcadores esperadas. As distribuições de tamanho das partículas diferiram conforme o sexo e o estado da DRC. O conteúdo total de proteína das uEVs não diferiu significativamente entre os estágios da DRC. A IL-6 associada às uEVs foi significativamente mais elevada em G1 em comparação com a FRN (p ≤ 0,05). A CTD apresentou uma tendência decrescente de G1 a G4, com diferenças específicas de estágio e sexo, mais evidentes em mulheres no estágio G1. O TGF-β associado às uEVs não diferiu significativamente entre os estágios. Conclusões: A IL-6 e a CTD derivadas das uEVs mostram potencial como indicadores não invasivos de DRC em fase inicial, com padrões dependentes do sexo clinicamente relevantes, enquanto o TGF-β pode fornecer informação complementar. Fluxos de trabalho padronizados de isolamento e mensuração de uEVs são essenciais para a reprodutibilidade dos biomarcadores e para a futura translação clínica.