AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-tomesenchymal transition (EMT) and inflammation – both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (aSMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgfb), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.publishedVersio

A Stochastic Hybrid Systems Framework for Analysis of Markov Reward Models

In this paper, we propose a framework to analyze Markov reward models, which are commonly used in system performability analysis. The framework builds on a set of analytical tools developed for a class of stochastic processes referred to as “Stochastic Hybrid Systems (SHS).” The state space of an SHS is composed of: i) a discrete state that describes the possible configurations/modes that a system can adopt, which includes the nominal (non-faulty) operational mode, but also those operational modes that arise due to component faults, and ii) a continuous state that describes the reward. Discrete state transitions are stochastic, and governed by transition rates that are (in general) a function of time and the value of the continuous state. The evolution of the continuous state is described by a stochastic differential equation, and reward measures are defined as functions of the continuous state. Additionally, each transition is associated with a reset map that defines the mapping between the pre- and post-transition values of the discrete and continuous states; these mappings enable the definition of impulses and losses in the reward. The proposed SHS-based framework unifies the analysis of a variety of previously studied reward models. We illustrate the application of the framework to performability analysis via analytical and numerical examples.National Science Foundation / CMG-0934491Ope

Hvordan forebygge og håndtere episoder med skadelige alger og maneter i oppdrettsnæringen

Prosjektleder: Trine DaleDette er hovedrapport for prosjektet «Hvordan forebygge og håndtere episoder med skadelige alger og maneter i oppdrettsnæringen» finansiert av Fiskeri og havbruksnæringens forskningsfinansiering. Hovedmålet i prosjektet har vært å sammenstille eksisterende kunnskap, erfaringer og teknologiske løsninger som er i bruk for å forebygge og håndtere episoder med skadelige alger og maneter og basert på dette utforme og formidle anbefalinger og om mulig beste praksis i ulike situasjoner. Det er utviklet en prototype av et verktøy som på en enkel og lettfattelig måte fremstiller anbefalinger og beste praksis samt kunnskapen som danner bakgrunn for disse.Fiskeri og havbruksnæringens forskningsfinansiering.publishedVersio

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9–mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.publishedVersio

New methods clear the dust off old biopsies. RNA sequencing of FFPE kidney biopsies

Background and aims: Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. We wanted to exploit renal biopsies also on the mRNA level to elucidate pathophysiological mechanisms and to ultimately define novel therapies of kidney diseases. The work in this thesis aimed to assess the technical feasibility of RNA sequencing per se and the quality of the respective mRNA data derived from extracted RNA of whole FFPE tissue sections. In the first paper the main aim was to test whether lower quality, partially degraded RNA obtained from archival formalin-fixed and paraffin-embedded (FFPE) renal tissues could serve as appropriate source of material for RNA sequencing. This was approached by testing transcriptome sequencing of RNA from concurrently harvested FFPE and fresh stored kidney biopsies. In the second paper we aimed to validate and expand the first analysis by investigating a second cohort of FFPE kidney biopsies from local ccRCC patients. The secondary aim of this thesis was to assess the technical feasibility and the quality of mRNA data obtained from LCM renal tissues. Further, the aim of the third paper was to evaluate the most appropriate method to extract RNA from FFPE renal tissues and to compare yield and quality of extracted RNA between the different methods with the target of conducting RNA sequencing, especially from LCM glomerular cross-sections. Methods: Kidney biopsies from resected tissues belonging to patients undergoing nephrectomy were obtained with a 16g core biopsy needle. In paper I, tumor samples and adjacent normal tissue specimens were FFPE or RNAlater® stored. In paper II, only FFPE kidney biopsies were used. In the third paper, FFPE biopsies from rat and human tissues were utilized. In all papers RNA sequencing libraries were built with the newly released Illumina’s TruSeq® Access library preparation kit (recently renamed RNA exome kit). Comparative analyses were done using voom/Limma package in R. Main results: In the first paper we demonstrated that the FFPE and RNAlater® datasets gave comparable numbers of detected genes, differentially expressed transcripts and affected pathways. The average expression and the differentially expressed genes had very high correlation between the FFPE and RNAlater® stored samples. In paper I and II the detected genes relevant for ccRCC were in accordance with the current literature. The number of detected transcripts in the “discovery/paper I” and “confirmation/paper II” data set gave 8957 and 11,047 detected transcripts, respectively. These data sets shared 1193 of differentially expressed genes. The average expression and the differentially expressed transcripts in both data sets correlated, with R2 of 0,95 and R2 of 0,94, respectively. In the third paper, several kits were eligible for RNA extraction from FFPE tissues from both whole kidney biopsy sections and from LCM samples. Conclusions: Gene expression data obtained from FFPE kidney biopsies are comparable to data obtained from freshly stored material, thus expanding the utility of archival tissue specimens. Next-generation sequencing expands the clinical application of tissue analyses from FFPE biopsies and gives results well in line with the current literature. RNA can be extracted from archival renal biopsies in sufficient quality and quantity from a single human kidney biopsy section and from around 100 LCM glomerular cross-sections to enable successful RNA library preparation and sequencing using commercially available RNA extraction kits

Abnormal Uroguanylin Immunoreactive Cells Density in the Duodenum of Patients with Diarrhea-Predominant Irritable Bowel Syndrome Changes following Fecal Microbiota Transplantation

Altered densities of enteroendocrine cells play an important role in patients with irritable bowel syndrome (IBS). Uroguanylin activates guanylate cyclase-C to regulate intestinal electrolyte and water transport. Aim. To quantify uroguanylin immunoreactive cells density in the duodenum of diarrhea-predominant IBS (IBS-D) patients compared to controls and to investigate the effect of fecal microbiota transplantation (FMT) on these cell densities. Method. Twelve patients with IBS-D according to Rome III criteria were included. The cause was identified as post infectious (PI, n=6) or idiopathic (n=6). They completed the IBS-symptom questionnaire before and 3 weeks after FMT. Thirty grams of fresh feces donated from healthy relatives were diluted with 60 ml normal saline and instilled via endoscope into the duodenum. Biopsies were taken from the patients’ duodenum before and 3 weeks after FMT. Duodenal biopsies taken from eight healthy controls were also included. The biopsies were immunostained for uroguanylin and quantified using computerized image analysis. Results. Uroguanylin immunoreactive cells were found both in duodenal villi and crypts in both controls and IBS-D patients. The densities of uroguanylin immunoreactive cells were significantly lower in the villi (P<0.0001) and higher in the crypts (P<0.0001) for the patients than the controls. Following FMT, the densities of uroguanylin immunoreactive cells for the total group and idiopathic subgroup decreased significantly in the duodenal crypts (P=0.049 and 0.04, respectively) but not in the villi. No significant changes were shown in the PI-IBS subgroups. The cells density in only the crypts correlated with diarrhea (r=0.97, P=0.001) and bloating (r=–0.91, P=0.01) in the PI-IBS subgroup before FMT and with abdominal pain (r=0.63, P=0.03) in the total group of IBS-D patients after FMT. Conclusion. Altered uroguanylin immunoreactive cells density was found in IBS-D patients compared to controls. Changes in these cells density following FMT correlated with IBS symptoms (diarrhea, bloating, and abdominal pain)

Abnormal Uroguanylin Immunoreactive Cells Density in the Duodenum of Patients with Diarrhea-Predominant Irritable Bowel Syndrome Changes following Fecal Microbiota Transplantation

Altered densities of enteroendocrine cells play an important role in patients with irritable bowel syndrome (IBS). Uroguanylin activates guanylate cyclase-C to regulate intestinal electrolyte and water transport. Aim. To quantify uroguanylin immunoreactive cells density in the duodenum of diarrhea-predominant IBS (IBS-D) patients compared to controls and to investigate the effect of fecal microbiota transplantation (FMT) on these cell densities. Method. Twelve patients with IBS-D according to Rome III criteria were included. The cause was identified as post infectious (PI, n = 6) or idiopathic (n = 6). They completed the IBS-symptom questionnaire before and 3 weeks after FMT. Thirty grams of fresh feces donated from healthy relatives were diluted with 60 ml normal saline and instilled via endoscope into the duodenum. Biopsies were taken from the patients’ duodenum before and 3 weeks after FMT. Duodenal biopsies taken from eight healthy controls were also included. The biopsies were immunostained for uroguanylin and quantified using computerized image analysis. Results. Uroguanylin immunoreactive cells were found both in duodenal villi and crypts in both controls and IBS-D patients. The densities of uroguanylin immunoreactive cells were significantly lower in the villi (P < 0:0001) and higher in the crypts (P < 0:0001) for the patients than the controls. Following FMT, the densities of uroguanylin immunoreactive cells for the total group and idiopathic subgroup decreased significantly in the duodenal crypts (P = 0:049 and 0.04, respectively) but not in the villi. No significant changes were shown in the PI-IBS subgroups. The cells density in only the crypts correlated with diarrhea (r = 0:97, P = 0:001) and bloating (r = –0:91, P = 0:01) in the PI-IBS subgroup before FMT and with abdominal pain (r = 0:63, P = 0:03) in the total group of IBS-D patients after FMT. Conclusion. Altered uroguanylin immunoreactive cells density was found in IBS-D patients compared to controls. Changes in these cells density following FMT correlated with IBS symptoms (diarrhea, bloating, and abdominal pain)

Transcriptomic analysis reveals partial epithelial–mesenchymal transition and inflammation as common pathogenic mechanisms in hypertensive nephrosclerosis and Type 2 diabetic nephropathy

Hypertensive nephrosclerosis (HN) and Type 2 diabetic nephropathy (T2DN) are the leading causes of chronic kidney disease (CKD). To explore shared pathogenetic mechanisms, we analyzed transcriptomes of kidney biopsies from patients with HN or T2DN. Total RNA was extracted from 10 μm whole kidney sections from patients with HN, T2DN, and normal controls (Ctrl) (n = 6 for each group) and processed for RNA sequencing. Differentially expressed (log2 fold change >1, adjusted p < 0.05) genes (DEG) and molecular pathways were analyzed, and selected results were validated by immunohistochemistry (IHC). ELISA on serum samples was performed on a related cohort consisting of patients with biopsy-proven HN (n = 13) and DN (n = 9), and a normal control group (n = 14). Cluster analysis on RNA sequencing data separated diseased and normal tissues. RNA sequencing revealed that 88% (341 out of 384) of DEG in HN were also altered in T2DN, while gene set enrichment analysis (GSEA) showed that over 90% of affected molecular pathways, including those related to inflammation, immune response, and cell-cycle regulation, were similarly impacted in both HN and T2DN samples. The increased expression of genes tied to interleukin signaling and lymphocyte activation was more pronounced in HN, while genes associated with extracellular matrix organization were more evident in T2DN. Both HN and T2DN tissues exhibited significant upregulation of genes connected with inflammatory responses, T-cell activity, and partial epithelial to mesenchymal transition (p-EMT). Immunohistochemistry (IHC) further confirmed T-cell (CD4+ and CD8+) infiltration in the diseased tissues. Additionally, IHC revealed heightened AXL protein expression, a key regulator of inflammation and p-EMT, in both HN and T2DN, while serum analysis indicated elevated soluble AXL levels in patients with both conditions. These findings underline the shared molecular mechanisms between HN and T2DN, hinting at the potential for common therapeutic strategies targeting both diseases.publishedVersio

Proteomic analysis unveils Gb3-independent alterations and mitochondrial dysfunction in a gla −/− zebrafish model of Fabry disease

Abstract Background Fabry disease (FD) is a rare lysosomal storage disorder caused by mutations in the GLA gene, resulting in reduced or lack of α-galactosidase A activity. This results in the accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids in lysosomes causing cellular impairment and organ failures. While current therapies focus on reversing Gb3 accumulation, they do not address the altered cellular signaling in FD. Therefore, this study aims to explore Gb3-independent mechanisms of kidney damage in Fabry disease and identify potential biomarkers. Methods To investigate these mechanisms, we utilized a zebrafish (ZF) gla −/− mutant (MU) model. ZF naturally lack A4GALT gene and, therefore, cannot synthesize Gb3. We obtained kidney samples from both wild-type (WT) (n = 8) and MU (n = 8) ZF and conducted proteome profiling using untargeted mass spectrometry. Additionally, we examined mitochondria morphology and cristae morphology using electron microscopy. To assess oxidative stress, we measured total antioxidant activity. Finally, immunohistochemistry was conducted on kidney samples to validate specific proteins. Results Our proteomics analysis of renal tissues from zebrafish revealed downregulation of lysosome and mitochondrial-related proteins in gla −/− MU renal tissues, while energy-related pathways including carbon, glycolysis, and galactose metabolisms were disturbed. Moreover, we observed abnormal mitochondrial shape, disrupted cristae morphology, altered mitochondrial volume and lower antioxidant activity in gla −/− MU ZF. Conclusions These results suggest that the alterations observed at the proteome and mitochondrial level closely resemble well-known GLA mutation-related alterations in humans. Importantly, they also unveil novel Gb3-independent pathogenic mechanisms in Fabry disease. Understanding these mechanisms could potentially lead to the development of innovative drug screening approaches. Furthermore, the findings pave the way for identifying new clinical targets, offering new avenues for therapeutic interventions in Fabry disease. The zebrafish gla −/− mutant model proves valuable in elucidating these mechanisms and may contribute significantly to advancing our knowledge of this disorder. Graphical Abstrac

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