Eduard Sarró

Introduction. Androgenetic alopecia (AGA), the most common form of hair loss in women, is characterized by progressive hair thinning and loss of terminal hairs over frontal and parietal regions of the scalp. This study aimed to evaluate the efficacy of the nutritional supplement Pilopeptan® WOMAN 5αR (PPT5α) composed of specific plant extracts, vitamins, and minerals in women with initial to moderate AGA. Methods. This was a prospective, randomized, double‐blind, placebo‐controlled clinical trial study. Forty‐seven women (aged 25–59 years) with AGA grade I/II were randomized to daily receive PPT5α (n = 24) or placebo (n = 23), and the treatment safety, as well as the percentages of terminal hair in frontal and parietal areas from trichoscopic images, was assessed at 0, 3, and 6 months. Secondary outcomes included self‐assessed hair parameters and overall treatment satisfaction. Results. A total of 44 women completed the study. At 6 months, patients who received the nutritional supplement showed high percentages of terminal hair both in the frontal (77.6% vs 69.8%, p = 0.02) and parietal (77% vs 64.3%, p = 0.02) areas compared to the placebo group. Self‐assessed evaluation of hair parameters showed an improvement in the reduction of hair loss and hair thickness both at the 3‐month (p = 0.004 and p = 0.012) and 6‐month (p = 0.009 and p = 0.004) visits. At the 3 month visit, the intervention group also showed higher treatment satisfaction (p = 0.01). No significant adverse events were reported. Conclusion. These results evidence that the nutritional supplement PPT5α may be beneficial in preventing progression or even improving the condition of AGA in the early stages.

Dent disease 1 (DD1) is a rare X-linked renal proximal tubulopathy characterized by low molecular weight proteinuria and variable degree of hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressing to chronic kidney disease. Although mutations in the electrogenic Cl⁻/H⁺ antiporter ClC-5, which impair endocytic uptake in proximal tubule cells, cause the disease, there is poor genotype–phenotype correlation and their contribution to proximal tubule dysfunction remains unclear. To further discover the mechanisms linking ClC-5 loss-of-function to proximal tubule dysfunction, we have generated novel DD1 cellular models depleted of ClC-5 and carrying ClC-5 mutants p.(Val523del), p.(Glu527Asp) and p.(Ile524Lys) using the human proximal tubule-derived RPTEC/TERT1 cell line. Our DD1 cellular models exhibit impaired albumin endocytosis, increased substrate adhesion and decreased collective migration, correlating with a less differentiated epithelial phenotype. Despite sharing functional features, these DD1 cell models exhibit different gene expression profiles, being p.(Val523del) ClC-5 the mutation showing the largest differences. Gene set enrichment analysis pointed to kidney development, anion homeostasis, organic acid transport, extracellular matrix organization and cell-migration biological processes as the most likely involved in DD1 pathophysiology. In conclusion, our results revealed the pathways linking ClC-5 mutations with tubular dysfunction and, importantly, provide new cellular models to further study DD1 pathophysiology.

Dent disease 1 (DD1) is a rare X-linked renal proximal tubulopathy characterized by low molecular weight proteinuria (LMWP) and variable degree of hypercalciuria, nephrocalcinosis and/or nephrolithiasis with progression to chronic kidney disease (CKD). Although loss-of-function mutations in the gene CLCN5 encoding the electrogenic Cl ⁻ /H ⁺ antiporter ClC-5, which impair endocytic uptake in proximal tubule cells, cause the disease, there is poor genotype-phenotype correlation and their contribution to proximal tubule dysfunction remains unclear. Here, in order to discover the mechanisms leading to proximal tubule dysfunction due to ClC-5 loss-of-function, we have generated and characterized new human cellular models of DD1 by silencing CLCN5 and introducing the ClC-5 pathogenic mutants V523del, E527D and I524K into the human proximal tubule-derived cell line RPTEC/TERT1. Depletion of CLCN5 or expression of mutant ClC-5 impairs albumin endocytosis, increases substrate adhesion and decreases collective migration, which correlates with a less differentiated epithelial phenotype. Interestingly, although all conditions compromised the endocytic capacity in a similar way, their impact on gene expression profiles was different. Our DNA microarray studies show that ClC-5 silencing or mutant re-introduction alter pathways related to nephron development, anion homeostasis, organic acid transport, extracellular matrix organization and cell migration, compared to control cells. Cells carrying the V523del ClC-5 mutation show the largest differences in gene expression vs WT cells, which is in agreement with the more aggressive clinical phenotype observed in some DD1 patients. Overall, this work emphasizes the use of human proximal tubule derived cell models to identify the molecular processes underlying ClC-5 deficiency.

Restoration of kidney tubular epithelium following sublethal injury sequentially involves partial epithelial–mesenchymal transition (pEMT), proliferation, and further redifferentiation into specialized tubule epithelial cells (TECs). Because the immunosuppressant cyclosporine-A produces pEMT in TECs and inhibits the peptidyl-prolyl isomerase (PPIase) activity of cyclophilin (Cyp) proteins, we hypothesized that cyclophilins could regulate TEC phenotype. Here we demonstrate that in cultured TECs, CypA silencing triggers loss of epithelial features and enhances transforming growth factor β (TGFβ)-induced EMT in association with upregulation of epithelial repressors Slug and Snail. This pro-epithelial action of CypA relies on its PPIase activity. By contrast, CypB emerges as an epithelial repressor, because CypB silencing promotes epithelial differentiation, prevents TGFβ-induced EMT, and induces tubular structures in 3D cultures. In addition, in the kidneys of CypB knockout mice subjected to unilateral ureteral obstruction, inflammatory and pro-fibrotic events were attenuated. CypB silencing/knockout leads to Slug, but not Snail, downregulation. CypB support of Slug expression depends on its endoplasmic reticulum location, where it interacts with calreticulin, a calcium-buffering chaperone related to Slug expression. As CypB silencing reduces ionomycin-induced calcium release and Slug upregulation, we suggest that Slug expression may rely on CypB modulation of calreticulin-dependent calcium signaling. In conclusion, this work uncovers new roles for CypA and CypB in modulating TEC plasticity and identifies CypB as a druggable target potentially relevant in promoting kidney repair.

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.

Cyclophilins (Cyp) are peptidil-prolyl-isomerases and the intracellular receptors for the immunosuppressant Cyclosporine-A (CsA), which produces epithelial-mesenchymal-transition (EMT) and renal tubule-interstitial fibrosis. Since CsA inhibits Cyp enzymatic activity, we hypothesized that Cyp could be involved in EMT and fibrosis. Here, we demonstrate that CypB is a critical regulator of tubule epithelial cell plasticity on the basis that: i) CypB silencing caused epithelial differentiation in proximal tubule-derived HK-2 cells, ii) CypB silencing prevented TGFβ-induced EMT in HK-2, and iii) CypB knockdown mice exhibited reduced UUO-induced inflammation and kidney fibrosis. By contrast, silencing of CypA induces a more undifferentiated phenotype and favors TGFβ effects. EMT mediators Slug and Snail were up-regulated in CypA-silenced cells, while in CypB silencing, Slug, but not Snail, was down-regulated; thus, reinforcing the role of Slug in kidney fibrosis. CypA regulates Slug through its PPIase activity whereas CypB depends on its ER location, where interacts with calreticulin, a calcium modulator which is involved in TGFβ signaling. In conclusion, this work uncovers new roles for CypA and CypB in modulating proximal tubular cell plasticity.

Under-expression of CK2 subunit in ccRCC represents a complementary biomarker of p-STAT3 Ser727 that correlates with patient survival Under-expression of CK2β subunit in ccRCC represents a complementary biomarker of p-STAT3 Ser727 that correlates with patient survival

Immunosuppression based on calcineurin inhibitors (CNIs) has greatly improved organ transplantation, although subsequent nephrotoxicity significantly hinders treatment success. There are no currently available specific soluble biomarkers for CNI-induced nephrotoxicity and diagnosis relies on renal biopsy, which is costly, invasive and may cause complications. Accordingly, identification of non-invasive biomarkers distinguishing CNI-induced kidney tubular damage from that of other etiologies would greatly improve diagnosis and enable more precise dosage adjustment. For this purpose, HK-2 cells, widely used to model human proximal tubule, were treated with CNIs cyclosporine-A and FK506, or staurosporine as a calcineurin-independent toxic compound, and secretomes of each treatment were analyzed by proteomic means. Among the differentially secreted proteins identified, only fascin-1 was specifically released by both CNIs but not by staurosporine. To validate fascin-1 as a biomarker of CNI-induced tubular toxicity, fascin-1 levels were analyzed in serum and urine from kidney-transplanted patients under CNIs treatment presenting or not isometric vacuolization (IV), which nowadays represents the main histological hallmark of CNI-induced tubular damage. Patients with chronic kidney disease (CKD) and healthy volunteers were used as controls. Our results show that urinary fascin-1 was only significantly elevated in the subset of CNI-treated patients presenting IV. Moreover, fascin-1 anticipated the rise of sCr levels in serially collected urine samples from CNI-treated pulmonary-transplanted patients, where a decline in kidney function and serum creatinine (sCr) elevation was mainly attributed to CNIs treatment. In conclusion, our results point towards fascin-1 as a putative soluble biomarker of CNI-induced damage in the kidney tubular compartment.