A recent genome-wide association study identified a locus on chromosome 16 in the promoter region of the uromodulin ( UMOD ) gene that is associated with hypertension. Here, we examined the hypertension signal with functional studies in Umod knockout (KO) mice. Systolic blood pressure was significantly lower in KO versus wild-type (WT) mice under basal conditions (KO: 116.6±0.3 mm Hg versus WT: 136.2±0.4 mm Hg; P <0.0001). Administration of 2% NaCl did not alter systolic blood pressure in KO mice, whereas it increased in WT mice by ≈33%, P <0.001. The average 24-hour urinary sodium excretion in the KO was greater than that of WT mice ( P <0.001). Chronic renal function curves demonstrate a leftward shift in KO mice, suggesting that the relationship between UMOD and blood pressure is affected by sodium. Creatinine clearance was increased during salt loading with 2% NaCl in the KO mice, leading to augmented filtered Na ⁺ excretion and further Na ⁺ loss. The difference in sodium uptake that exists between WT and KO strains was explored at the molecular level. Urinary tumor necrosis factor-α levels were significantly higher in KO mice compared with WT mice ( P <0.0001). Stimulation of primary thick ascending limb of the loop of Henle cells with exogenous tumor necrosis factor-α caused a reduction in NKCC2A expression ( P <0.001) with a concurrent rise in the levels of UMOD mRNA ( P <0.001). Collectively, we demonstrate that UMOD regulates sodium uptake in the thick ascending limb of the loop of Henle by modulating the effect of tumor necrosis factor-α on NKCC2A expression, making UMOD an important determinant of blood pressure control.

The interest in implementing digital pathology (DP) workflows to obtain whole slide image (WSI) files for diagnostic purposes has increased in the last few years. The increasing performance of technical components and the Food and Drug Administration (FDA) approval of systems for primary diagnosis led to increased interest in applying DP workflows. However, despite this revolutionary transition, real world data suggest that a fully digital approach to the histological workflow has been implemented in only a minority of pathology laboratories. The objective of this study is to facilitate the implementation of DP workflows in pathology laboratories, helping those involved in this process of transformation to identify: (a) the scope and the boundaries of the DP transformation; (b) how to introduce automation to reduce errors; (c) how to introduce appropriate quality control to guarantee the safety of the process and (d) the hardware and software needed to implement DP systems inside the pathology laboratory. The European Society of Digital and Integrative Pathology (ESDIP) provided consensus-based recommendations developed through discussion among members of the Scientific Committee. The recommendations are thus based on the expertise of the panel members and on the agreement obtained after virtual meetings. Prior to publication, the recommendations were reviewed by members of the ESDIP Board. The recommendations comprehensively cover every step of the implementation of the digital workflow in the anatomic pathology department, emphasizing the importance of interoperability, automation and tracking of the entire process before the introduction of a scanning facility. Compared to the available national and international guidelines, the present document represents a practical, handy reference for the correct implementation of the digital workflow in Europe.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater^® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater^®. RNA sequencing libraries were generated applying the new Illumina TruSeq^® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater^® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R² = 0.97, and R² = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data obtained from FFPE kidney biopsies are comparable to data obtained from fresh stored material, thereby expanding the utility of archival tissue specimens.