Glomerular transcriptomics predicts long term outcome and identifies therapeutic strategies for patients with assumed benign IgA nephropathy.

Some patients diagnosed with benign IgA nephropathy (IgAN) develop a progressive clinical course, not predictable by known clinical or histopathological parameters. To assess if gene expression can differentiate between progressors and non-progressors with assumed benign IgAN, we tested microdissected glomeruli from archival kidney biopsy sections from adult patients with stable clinical remission (21 non-progressors) or from 15 patients that had undergone clinical progression within a 25-year time frame. Based on 1 240 differentially expressed genes from patients with suitable sequencing results, we identified eight IgAN progressor and nine non-progressor genes using a two-component classifier. These genes, including APOL5 and ZXDC, predicted disease progression with 88% accuracy, 75% sensitivity and 100% specificity on average 21.6 years before progressive disease was clinically documented. APOL lipoproteins are associated with inflammation, autophagy and kidney disease while ZXDC is a zinc-finger transcription factor modulating adaptive immunity. Ten genes from our transcriptomics data overlapped with an external genome wide association study dataset, although the gene set enrichment test was not statistically significant. We also identified 45 drug targets in the DrugBank database, including angiotensinogen, a target of sparsentan (dual antagonist of the endothelin type A receptor and the angiotensin II type 1 receptor) currently investigated for IgAN treatment. Two validation cohorts were used for substantiating key results, one by immunohistochemistry and the other by nCounter technology. Thus, glomerular mRNA sequencing from diagnostic kidney biopsies from patients with assumed benign IgAN can differentiate between future progressors and non-progressors at the time of diagnosis.

Systems analyses of the Fabry kidney transcriptome and its response to enzyme replacement therapy identified and cross-validated enzyme replacement therapy-resistant targets amenable to drug repurposing.

Fabry disease is a rare disorder caused by variations in the alpha-galactosidase gene. To a degree, Fabry disease is manageable via enzyme replacement therapy (ERT). By understanding the molecular basis of Fabry nephropathy (FN) and ERT's long-term impact, here we aimed to provide a framework for selection of potential disease biomarkers and drug targets. We obtained biopsies from eight control individuals and two independent FN cohorts comprising 16 individuals taken prior to and after up to ten years of ERT, and performed RNAseq analysis. Combining pathway-centered analyses with network-science allowed computation of transcriptional landscapes from four nephron compartments and their integration with existing proteome and drug-target interactome data. Comparing these transcriptional landscapes revealed high inter-cohort heterogeneity. Kidney compartment transcriptional landscapes comprehensively reflected differences in FN cohort characteristics. With exception of a few aspects, in particular arteries, early ERT in patients with classical Fabry could lastingly revert FN gene expression patterns to closely match that of control individuals. Pathways nonetheless consistently altered in both FN cohorts pre-ERT were mostly in glomeruli and arteries and related to the same biological themes. While keratinization-related processes in glomeruli were sensitive to ERT, a majority of alterations, such as transporter activity and responses to stimuli, remained dysregulated or reemerged despite ERT. Inferring an ERT-resistant genetic module of expressed genes identified 69 drugs for potential repurposing matching the proteins encoded by 12 genes. Thus, we identified and cross-validated ERT-resistant gene product modules that, when leveraged with external data, allowed estimating their suitability as biomarkers to potentially track disease course or treatment efficacy and potential targets for adjunct pharmaceutical treatment.

Network-Based Assessment of Minimal Change Disease Identifies Glomerular Response to IL-7 and IL-12 Pathways Activation as Innovative Treatment Target.

Background: Minimal change disease (MCD), a major cause of nephrotic syndrome, is usually treated by corticosteroid administration. MCD unresponsiveness to therapy and recurrences are nonetheless frequently observed, particularly in adults. To explore MCD-related pathogenetic mechanisms and to identify novel drug targets ultimately contributing to novel therapeutic avenues with a certain specificity for MCD, we compared glomerular transcriptomes from MCD with membranous nephropathy (MN) patients and healthy controls. Methods: Renal biopsies from adult patients with MCD (n = 14) or MN (n = 12), and non-diseased controls (n = 8) were selected from the Norwegian Kidney Biopsy Registry. RNA for 75 base-pair paired-end RNASeq were obtained from laser capture micro-dissected (LCM) glomeruli from FFPE sections. Transcriptional landscapes were computed by combining pathway-centered analyses and network science methodologies that integrate multiple bioinformatics resources. Results: Compared to normal glomeruli, cells from MCD displayed an inflammatory signature apparently governed by the IL1 and IL7 systems. While enrichment of IL1 production and secretion was a shared feature of MCD and MN compared to normal tissue, responses involving IL7 pathway activation were unique to MCD. Indeed, IL7R expressed by glomeruli was the most upregulated gene of the interleukin family in MCD versus normal controls. IL7 pathway activation was paralleled by significant enrichment in adaptive immune system processes and transcriptional regulation and depletion in pathways related to energy metabolism and transcription. Downregulation of these organ function-related themes again occurred predominately in MCD and was significantly less pronounced in MN. Immunofluorescence and immunohistochemistry, respectively, confirmed the expression of phosphorylated IL-7 receptor alpha (IL7RA, CD127) and IL12 receptor beta 1 (IL12RB1) proteins. Conclusions: Gene expression profiling of archival FFPE-biopsies identifies MCD-specific signatures with IL7RA and IL12RB1 as novel targets for MCD treatment.

Emplacement and associated sedimentary record of the Jurassic submarine salt allochthon of the Wurzeralm (Eastern Alps, Austria).

A fossil salt sheet emplaced in the Jurassic in submarine conditions is described in the Eastern Alps of Austria, providing unique insights into the emplacement of similar submarine structures and their potential control on depositional systems. The salt sheet is a plug-fed extrusion emplaced due to squeezing of a salt diapir under compression. The preserved mylonitic shear fabric in the evaporites indicates radial, south-directed emplacement of the salt sheet. Tectono-sedimentary relationships record the evolution of the salt structure, from initial diapiric growth, to salt sheet extrusion and posterior collapse. Syn-extrusion sediments record the variable bathymetry of the extruding salt sheet, with reefal carbonates building up on the crestal bulge while their deeper water equivalents accumulated on the extruding salt lobe. This is the first description of a salt allochthon still linked to its source diapir in the Eastern Alps.

A multiomics disease progression signature of low-risk ccRCC.

Clear cell renal cell carcinoma (ccRCC) is the most common renal cancer. Identification of ccRCC likely to progress, despite an apparent low risk at the time of surgery, represents a key clinical issue. From a cohort of adult ccRCC patients (n = 443), we selected low-risk tumors progressing within a 5-years average follow-up (progressors: P, n = 8) and non-progressing (NP) tumors (n = 16). Transcriptome sequencing, miRNA sequencing and proteomics were performed on tissues obtained at surgery. We identified 151 proteins, 1167 mRNAs and 63 miRNAs differentially expressed in P compared to NP low-risk tumors. Pathway analysis demonstrated overrepresentation of proteins related to "LXR/RXR and FXR/RXR Activation", "Acute Phase Response Signaling" in NP compared to P samples. Integrating mRNA, miRNA and proteomic data, we developed a 10-component classifier including two proteins, three genes and five miRNAs, effectively differentiating P and NP ccRCC and capturing underlying biological differences, potentially useful to identify "low-risk" patients requiring closer surveillance and treatment adjustments. Key results were validated by immunohistochemistry, qPCR and data from publicly available databases. Our work suggests that LXR, FXR and macrophage activation pathways could be critically involved in the inhibition of the progression of low-risk ccRCC. Furthermore, a 10-component classifier could support an early identification of apparently low-risk ccRCC patients.

AGAP2-AS1 as a prognostic biomarker in low-risk clear cell renal cell carcinoma patients with progressing disease.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cancer and one of the most common cancers. While survival for localized ccRCC is good, the survival of metastatic disease is not, and thirty percent of patients with ccRCC develop metastases during follow-up. Although current scoring methods accurately identify patients at low progression risk, a small subgroup of those patients still experience metastasis. We therefore aimed to identify ccRCC progression biomarkers in "low-risk" patients who were potentially eligible for adjuvant treatments or more intensive follow-up. METHODS: We assembled a cohort of ccRCC patients (n = 443) and identified all "low-risk" patients who later developed progressing tumors (n = 8). Subsequently, we performed genome-wide expression profiling from formalin-fixed samples obtained at initial surgery from these "low-risk" patients and 16 matched patients not progressing to recurrence with metastasis. The patients were matched for Leibovich sore, creatinine, age, sex, tumor size and tumor stage. Key results were confirmed with qPCR and external data from The Cancer Genome Atlas. RESULTS: Principal component analysis indicated that systematic transcriptomic differences were already detectable at the time of initial surgery. One thousand one hundred sixty-seven genes, mainly associated with cancer and immune-related pathways, were differentially expressed between progressors and nonprogressors. A search for a classifier revealed that overexpression of AGAP2-AS1, an antisense long noncoding RNA, correctly classified 23 of 24 samples, years (4.5 years average) in advance of the discovery of metastasis and without requiring larger gene panels. Subsequently, we confirmed AGAP2-AS1 gene overexpression by qPCR in the same samples (p < 0.05). Additionally, in external data from The Cancer Genome Atlas, overexpression of AGAP2-AS1 is correlated with overall unfavorable survival outcome in ccRCC, irrespective of other prognostic predictors (p = 2.44E-7). CONCLUSION: AGAP2-AS1 may represent a novel biomarker identifying high-risk ccRCC patients currently classified as "low risk" at the time of surgery.

Variable expression of eighteen common housekeeping genes in human non-cancerous kidney biopsies.

Housekeeping, or reference genes (RGs) are, by definition, loci with stable expression profiles that are widely used as internal controls to normalize mRNA levels. However, due to specific events, such as pathological changes, or technical procedures, their expression might be altered, failing to fulfil critical normalization pre-requisites. To identify RG genes suitable as internal controls in human non-cancerous kidney tissue, we selected 18 RG candidates based on previous data and screen them in 30 expression datasets (>800 patients), including our own, publicly available or provided by independent groups. Datasets included specimens from patients with hypertensive and diabetic nephropathy, Fabry disease, focal segmental glomerulosclerosis, IgA nephropathy, membranous nephropathy, and minimal change disease. We examined both microdissected and whole section-based datasets. Expression variability of 4 candidate genes (YWHAZ, SLC4A1AP, RPS13 and ACTB) was further examined by qPCR in biopsies from patients with hypertensive nephropathy (n = 11) and healthy controls (n = 5). Only YWHAZ gene expression remained stable in all datasets whereas SLC4A1AP was stable in all but one Fabry dataset. All other RGs were differentially expressed in at least 2 datasets, and in 4.5 datasets on average. No differences in YWHAZ, SLC4A1AP, RPS13 and ACTB gene expression between hypertensive and control biopsies were detected by qPCR. Although RGs suitable to all techniques and tissues are unlikely to exist, our data suggest that in non-cancerous kidney biopsies expression of YWHAZ and SLC4AIAP genes is stable and suitable for normalization purposes.

Fine needle aspirates of kidneys: a promising tool for RNA sequencing in native and transplanted kidneys.

BACKGROUND: Transcriptome analysis is emerging as emerging as a promising tool to enhance precision of diagnosis and monitoring in solid organ transplantation. Clinical progress has however been hampered by the current reliance on samples from core needle biopsies. This proof-of-principle study examined whether fine needle aspirates, being less invasive, permit the ascertainment of the identical molecular information as core biopsies. METHODS: We collected fine needles aspirates from various needle sizes (G19, 21, 23, 25) and the corresponding core biopsies (G16 needle) of non-tumor tissue of full nephrectomy specimens from patients suffering from clear cell renal cell carcinoma (n = 11). RNA expression patterns of two gene sets (156 genes) were executed using targeted RNA sequencing in samples from fine needle vs. core needle samples. A subgroup of kidneys (n = 6) also underwent whole transcriptome RNA sequencing from core biopsies of tumor and peri-tumoral normal tissue (Tru Seq RNA Access, Illumina). RESULTS: Samples from all needle sizes except two G25 aspirates yielded RNA potentially suitable for sequencing of both gene sets. The mRNA expression patterns of the two gene sets were highly correlated between fine needle aspirates (G23) and corresponding (G16) core biopsies (r = 0.985 and 0.982, respectively). This close correlation was further documented by heat map, Principal Component Analyses (PCA) and whole transcription RNA sequencing. The similarity between fine neddle aspirates and core needle biopsies was additionally confirmed in the subgroup with complete RNA sequencing. CONCLUSIONS: Fine needle biopsies yield similar genomic information to core needle biopsies. The less invasive nature of fine needle biopsies may therefore permit more frequent molecular monitoring and a more targeted use of core needle biopsies in native and especially in transplanted kidneys.

Expanding the Utilization of Formalin-Fixed, Paraffin-Embedded Archives: Feasibility of miR-Seq for Disease Exploration and Biomarker Development from Biopsies with Clear Cell Renal Cell Carcinoma.

Novel predictive tools for clear cell renal cell carcinoma (ccRCC) are urgently needed. MicroRNAs (miRNAs) have been increasingly investigated for their predictive value, and formalin-fixed paraffin-embedded biopsy archives may potentially be a valuable source of miRNA sequencing material, as they remain an underused resource. Core biopsies of both cancerous and adjacent normal tissues were obtained from patients (n = 12) undergoing nephrectomy. After small RNA-seq, several analyses were performed, including classifier evaluation, obesity-related inquiries, survival analysis using publicly available datasets, comparisons to the current literature and ingenuity pathway analyses. In a comparison of tumour vs. normal, 182 miRNAs were found with significant differential expression; miR-155 was of particular interest as it classified all ccRCC samples correctly and correlated well with tumour size (R² = 0.83); miR-155 also predicted poor survival with hazard ratios of 2.58 and 1.81 in two different TCGA (The Cancer Genome Atlas) datasets in a univariate model. However, in a multivariate Cox regression analysis including age, sex, cancer stage and histological grade, miR-155 was not a statistically significant survival predictor. In conclusion, formalin-fixed paraffin-embedded biopsy tissues are a viable source of miRNA-sequencing material. Our results further support a role for miR-155 as a promising cancer classifier and potentially as a therapeutic target in ccRCC that merits further investigation.

Clear Cell Renal Cell Carcinoma is linked to Epithelial-to-Mesenchymal Transition and to Fibrosis.

Clear cell renal cell carcinoma (ccRCC) represents the most common type of kidney cancer with high mortality in its advanced stages. Our study aim was to explore the correlation between tumor epithelial-to-mesenchymal transition (EMT) and patient survival. Renal biopsies of tumorous and adjacent nontumorous tissue were taken with a 16 g needle from our patients (n = 26) undergoing partial or radical nephrectomy due to ccRCC RNA sequencing libraries were generated using Illumina TruSeq® Access library preparation protocol and TruSeq Small RNA library preparation kit. Next generation sequencing (NGS) was performed on Illumina HiSeq2500. Comparative analysis of matched sample pairs was done using the Bioconductor Limma/voom R-package. Liquid chromatography-tandem mass spectrometry and immunohistochemistry were applied to measure and visualize protein abundance. We detected an increased generic EMT transcript score in ccRCC Gene expression analysis showed augmented abundance of AXL and MMP14, as well as down-regulated expression of KL (klotho). Moreover, microRNA analyses demonstrated a positive expression correlation of miR-34a and its targets MMP14 and AXL Survival analysis based on a subset of genes from our list EMT-related genes in a publicly available dataset showed that the EMT genes correlated with ccRCC patient survival. Several of these genes also play a known role in fibrosis. Accordingly, recently published classifiers of solid organ fibrosis correctly identified EMT-affected tumor samples and were correlated with patient survival. EMT in ccRCC linked to fibrosis is associated with worse survival and may represent a target for novel therapeutic interventions.

Development and confirmation of potential gene classifiers of human clear cell renal cell carcinoma using next-generation RNA sequencing.

OBJECTIVE: A previous study by this group demonstrated the feasibility of RNA sequencing (RNAseq) technology for capturing disease biology of clear cell renal cell carcinoma (ccRCC), and presented initial results for carbonic anhydrase-9 (CA9) and tumor necrosis factor-α-induced protein-6 (TNFAIP6) as possible biomarkers of ccRCC (discovery set) [Eikrem et al. PLoS One 2016;11:e0149743]. To confirm these results, the previous study is expanded, and RNAseq data from additional matched ccRCC and normal renal biopsies are analyzed (confirmation set). MATERIALS AND METHODS: Two core biopsies from patients (n = 12) undergoing partial or full nephrectomy were obtained with a 16 g needle. RNA sequencing libraries were generated with the Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using linear modeling (voom/Limma; R Bioconductor). RESULTS: The formalin-fixed and paraffin-embedded discovery and confirmation data yielded 8957 and 11,047 detected transcripts, respectively. The two data sets shared 1193 of differentially expressed genes with each other. The average expression and the log2-fold changes of differentially expressed transcripts in both data sets correlated, with R² = .95 and R² = .94, respectively. Among transcripts with the highest fold changes were CA9, neuronal pentraxin-2 and uromodulin. Epithelial-mesenchymal transition was highlighted by differential expression of, for example, transforming growth factor-ß1 and delta-like ligand-4. The diagnostic accuracy of CA9 was 100% and 93.9% when using the discovery set as the training set and the confirmation data as the test set, and vice versa, respectively. These data further support TNFAIP6 as a novel biomarker of ccRCC. TNFAIP6 had combined accuracy of 98.5% in the two data sets. CONCLUSIONS: This study provides confirmatory data on the potential use of CA9 and TNFAIP6 as biomarkers of ccRCC. Thus, next-generation sequencing expands the clinical application of tissue analyses.

Electroconvulsive therapy and corpus callosum aplasia: a 3-year followup.

Electroconvulsive Therapy (ECT) is a powerful treatment option in severe or chronic catatonic states and has been reported to be useful in oligophrenic patients. We report the followup medical history of a patient with corpus callosum aplasia (or agenesis) who was continuously treated with ECT over three years. First, he improved considerably after a series of ECT, but relapses of catatonia made a continuous, weekly ECT necessary. Due to the severity of the brain malformation, an add-on medication with benzodiazepines and second generation antipsychotics was necessary to treat catatonic symptoms. This case emphasises the benefits of long-term ECT in oligophrenic patients.

Influence of heating car seats on scrotal temperature.

OBJECTIVE: To evaluate the influence of a heated versus an unheated car seat on scrotal temperature under standardized experimental conditions. DESIGN: Controlled clinical study. SETTING: Healthy volunteers in an academic research environment. PATIENT(S): Thirty volunteers without a history of infertility and with a normal andrological examination. INTERVENTION(S): Scrotal temperatures were measured every minute with a portable data recorder connected to two thermistor temperature sensors, which were attached on either side of the scrotum. All volunteers started the experiment at the same time of day wearing standardized cotton wool trousers and shirts fitting to body size. Each volunteer performed two periods of 90 minutes in a randomized manner on either the heated or unheated car seat. RESULT(S): At the end of the sitting periods scrotal temperatures were significantly higher using the heated car seat versus the unheated seat (left scrotal side: 0.5 degrees C; right scrotal side: 0.6 degrees C). Maximum values recorded during sitting alone were exceeded on the heated seat already after one-third of the exposure time. CONCLUSION(S): The present study suggests that the frequent use of a heated car seat represents an additional scrotal, and consequently, testicular heat stress factor to that which is present by merely sitting for long periods.