Novel noninvasive marker of regression of clear cell renal cell carcinoma (ccRCC).

OBJECTIVE: Analyzing protein kinase C (PKC) alpha, iota, and zeta as well as levels of Mxi-2 and Vim3 in regressive clear cell renal carcinomas (ccRCCs) and urine samples. MATERIAL AND METHODS: Fresh samples of ccRCCs (predominantly pT1a/b) with different degrees of regression (<10%, 30%, 50%, and 70%) vs normal renal tissue and oncocytomas were studied by Western blot, using antibodies of different PKC isoforms. Urine samples from these tumors were analyzed by ELISA (PKC isoforms, Mxi-2, and Vim3). RESULTS: With increasing degree of regression beyond 10%, nuclear Mxi-2 and Vim3 were highly overexpressed in fresh tumor samples. In urine samples, Vim3 was significantly overexpressed in oncocytoma and downregulated in RCCs with 70% regression. Western blot analysis shows that PKC alpha and iota levels were significantly increased in fresh tumor tissue samples (tumors with 30% regression). PKC zeta was expressed in normal kidney and significantly increased in oncocytoma but not found in ccRCCs. In patients' urines, Mxi-2 was significantly reduced (regression > 50%), while PKC isoform alpha was significantly increased by advanced regression rate. PKC iota in patients' urine was overexpressed in oncocytoma and reduced in all ccRCC urines. CONCLUSION: Tumor regression in ccRCC tissue shows strong nuclear overexpression of Mxi-2, Vim3, and PKC alpha and iota. In respective urines, PKC alpha was overexpressed; PKC iota was decreased. Mxi-2 and Vim3 decreased with increasing regression rates. These reagents could serve as noninvasive ccRCC markers for regression.

High efficiency capture of biomarker miRNA15a for noninvasive diagnosis of malignant kidney tumors.

To date, there are no preoperative and quantitative dynamics in clinical practice that can reliably differentiate between a benign and malignant renal cell carcinoma (RCC). For monitoring different analytes in body fluids, more than 40 different molecular biomarkers have been identified, however, they are associated with limited clinical sensitivity and/or non-optimal specificity due to their leaky nature. Previous work on RCC demonstrated the miRNA15a to be reliable and novel biomarker with 98.1% specificity and 100% sensitivity. Despite the high potential of miRNA15a biomarker, its clinical application is considerably hampered by the insensitive nature of the detection methods and low concentration of biomarker in samples that is aggravated by the high level of contamination due to other solutes present in body fluids. In this work, a non-invasive quantitative approach is demonstrated to overcome such diagnostics issues through biotin-streptavidin binding and fluorescence active magnetic nanocarriers that ensured prompt isolation, enrichment and purification of the biomarker miRNA15a from urine. The study demonstrates that detectable low levels of these miRNAs through miRNA capturing nanocarriers can potentially function as advanced diagnostic markers for the non-invasive investigation and early detection of renal cancer.

Non-invasive urine markers for the differentiation between RCCs and oncocytoma.

BACKGROUND: Recently, our group showed that Vim3 is overexpressed in tissue samples of renal oncocytomas and Mxi-2 in clear cell renal carcinoma (ccRCC). The mechanism leading to the truncation of both proteins is known and involves with two miRs, both detectable in urine. Since the analysis of miRs is time-consuming, our aim was to identify the truncated proteins in urine instead. Furthermore, urine samples from small renal masses (SRMs) (n = 45, <4 cm) were analyzed to get a pre-surgical differentiation of the cancer subtypes. METHODS: Urines were accessed from the urological biobank (n = 350). Proteins were isolated from urine samples, and Western blots were performed. Each sample was analyzed with ELISA for the expression of Vim3 and Mxi-2. A lateral flow assay was established. For the detection of SRMs, the miRs were isolated and qRT-PCR was performed. RESULTS: A significant increase of Vim3 in urines from patients with oncocytoma (n = 20) was detectable with ELISA compared to all other subtypes of RCCs (chromophobe (n = 50), papillary (n = 40), ccRCC (n = 200), and controls (n = 40) (***p < 0.0001)). Mxi-2 was predominantly overexpressed in ccRCCs (***p < 0.0001). Lateral flow assay of Vim3 and Mxi-2 shows two bands in the case of oncocytoma and ccRCC indicating the specificity of this test. For SRMs, an overexpression of miR-15a/Mxi2 was detectable in urine samples from ccRCC and chromoRCC patients. In contrast to that, miR-498/Vim3 were predominantly overexpressed in oncocytoma patients. CONCLUSION: Both proteins (Vim3 and Mxi-2) were detectable in patients' urines and can be used for the non-invasive differentiation of kidney cancers.

Magnetic nanoparticle-based amplification of microRNA detection in body fluids for early disease diagnosis.

Circulating biomarkers such as microRNAs (miRNAs), short noncoding RNA strands, represent prognostic and diagnostic indicators for a variety of physiological disorders making their detection and quantification an attractive approach for minimally invasive early disease diagnosis. However, highly sensitive and selective detection methods are required given the generally low abundance of miRNAs in body fluids together with the presence of large amounts of other potentially interfering biomolecules. Although a variety of miRNA isolation and detection methods have been established in clinics, they usually require trained personnel and often constitute labor-, time- and cost-intensive approaches. During the last years, nanoparticle-based biosensors have received increasing attention due to their superior detection efficiency even in very low concentration regimes. This is based on their unique physicochemical properties in combination with their high surface area that allows for the immobilization of multiple recognition sites resulting in fast and effective recognition of analytes. Among various materials, magnetic nanoparticles have been identified as useful tools for the separation, concentration, and detection of miRNAs. Here, we review state-of-the-art technology with regard to magnetic particle-based miRNA detection from body fluids, critically discussing challenges and future perspective of such biosensors while comparing their handling, sensitivity as well as selectivity against the established miRNA isolation and detection methods.

Mxi-2 Dependent Regulation of p53 in Prostate Cancer.

BACKGROUND/AIM: Endothelin-1 (ET-1) is overexpressed in many types of cancer, inhibiting the release of the microRNA 15a (miR-15a) and inducing the production of Mxi-2. Our aim was to identify a molecular complex regulating p53 activity in prostate cancer (PCa). MATERIALS AND METHODS: DU145 cells were treated with ET-1, MAPK p38 inhibitor, Endothelin A receptor inhibitor (ETAR inhibitor) and Endothelin B receptor inhibitor (ETBR inhibitor). Extracts were analysed using Western Blot, immunoprecipitation and qRT-PCR. Furthermore, prostate cancer patient samples were analysed using qRT-PCR and ELISA. RESULTS: The hypothesised molecular complex was identified, with miR-15a, microRNA 1285 (miR-1285) and Mxi-2 levels up-regulated in patients in relation to increasing aggressiveness of PCa. CONCLUSION: A complex composed of Argonaut 2 (Ago2)/Mxi-2/miR-1285 is involved in PCa. The expression of Mxi-2 correlates with increasing PCa aggressiveness and might be used as a non-invasive marker for the diagnosis and progression of PCa.

Sudden death of a young adult with coronary artery vasculitis, coronary aneurysms, parvovirus B19 infection and Kawasaki disease.

We present a case of a 20-year-old man who suffered from Kawasaki disease (KD) associated with a florid parvovirus infection, and who died suddenly from thrombotic occlusion of the coronary arteries. The autopsy revealed several aneurysms of the coronary arteries, a chronic vasculitis and a myofibroblast proliferation leading to focal luminal narrowing. The inflammatory response as well as the detection of the viral particles by PCR in blood and in the lesional tissue demonstrated a possible cause by Parvovirus infection. The expression of endoglin on endothelial cells of neoangiogenesis indicates the involvement of the TGF-beta pathway, necessary for maintaining chronic inflammation. In addition, a possible connection between the intake of methylphenidate, arteritis and a possible pre-existing heart disease must be discussed. Furthermore, KD must also be considered as a cause of sudden death in the adult population.

Vimentin 3 Allows Differentiation between Normozoospermia and Oligoasthenoteratozoospermia.

Vimentin is a structural protein predominantly located in the head of sperms. The function and localization of the previously identified truncated version, Vimentin 3 (Vim3), are still unknown. To investigate whether the expression of Vim3 can be used as a reliable marker for the differentiation of sperm quality, we analyzed ejaculates from patients with oligoasthenoteratozoospermia (OAT) syndrome and normozoospermia. We identified sperms with head, neck, and tail changes, which were less positive for Vim3 in OAT syndrome compared to normozoospermia. The expression of Vim3 was significantly downregulated in patients with OAT syndrome compared to sperms from patients with normozoospermia (∗∗ p < 0.01). The ELISA analysis showed similar results as ejaculates from normozoospermic patients showed a significantly higher Vim3 concentration than patients with OAT syndrome (∗∗∗ p < 0.001). This study demonstrates that Vim3 is more highly expressed in ejaculates from patients with normozoospermia compared to ejaculates from patients with OAT syndrome. Therefore, we postulate that Vim3 can be used to determine ejaculate quality. Furthermore, we identified the marker, Vim3, to differentiate between mature sperms with no morphological changes and sperms with head, neck, and tail changes. A lateral flow assay that allows quick analysis is currently under development.

Leptin signaling impairs macrophage defenses against Salmonella Typhimurium.

The dynamic interplay between metabolism and immune responses in health and disease, by which different immune cells impact on metabolic processes, are being increasingly appreciated. However, the potential of master regulators of metabolism to control innate immunity are less understood. Here, we studied the cross-talk between leptin signaling and macrophage function in the context of bacterial infections. We found that upon infection with Gram-negative pathogens, such as Salmonella Typhimurium, leptin receptor (Lepr) expression increased in both mouse and human macrophages. Unexpectedly, both genetic Lepr ablation in macrophages and global pharmacologic leptin antagonization augmented lysosomal functions, reduced S Typhimurium burden, and diminished inflammation in vitro and in vivo. Mechanistically, we show that leptin induction activates the mTORC2/Akt pathway and subsequently down-regulates Phlpp1 phosphatase, allowing for phosphorylated Akt to impair lysosomal-mediated pathogen clearance. These data highlight a link between leptin signaling, the mTORC2/Phlpp1/Akt axis, and lysosomal activity in macrophages and have important therapeutic implications for modulating innate immunity to combat Gram-negative bacterial infections.

MicroRNAs as markers to monitor endothelin-1 signalling and potential treatment in renal disease: Carcinoma - proteinuric damage - toxicity.

This review highlights new developments in miRNA as diagnostic and surveillance tools in diseases damaging the renal proximal tubule mediated by endothelin in the field of renal carcinoma, proteinuric kidney disease and tubulotoxicity. A new mechanism in the miRNA regulation of proteins leads to the binding of the miRNA directly to the DNA with premature transcriptional termination and hence the formation of truncated protein isoforms (Mxi2, Vim3). These isoforms are mediated through miRNA15a or miRNA 498, respectively. ET-1 can activate a cytoplasmic complex consisting of NF-κB p65, MAPK p38α, and PKCα. Consequently, PKCα does not transmigrate into the nucleus, which leads to the loss of suppression of a primiRNA15a, maturation of this miRNA in the cytoplasm, tubular secretion and detectability in the urine. This mechanism has been shown in renal cell carcinoma and in proteinuric disease as a biomarker for the activation of the signalling pathway. Similarly, ET-1 induced miRNA 498 transmigrates into the nucleus to form the truncated protein Vim3, which is a biomarker for the benign renal cell tumour, oncocytoma. In tubulotoxicity, ET-1 induced miRNa133a down-regulating multiple-drug-resistant related protein-2, relevant for proteinuric and cisplatin/cyclosporine A toxicity. Current advantages and limitations of miRNAs as urinary biomarkers are discussed.

Beyond the 3'UTR binding-microRNA-induced protein truncation via DNA binding.

Here, we present a miR mechanism which is active in the nucleus and is essential for the production of intron included, C-terminal truncated and biologically active proteins, like e.g. Vim3. We exemplified this mechanism by miRs, miR-15a and miR-498, which are overexpressed in clear cell renal carcinoma or oncocytoma. Both miRs directly interact with DNA in an intronic region, leading to transcriptional stop, and therefore repress the full length version of the pre-mRNA, resulting in intron included truncated proteins (Mxi-2 and Vim3). A computational survey shows that this miR:DNA interactions mechanism may be generally involved in regulating the human transcriptome, with putative interaction sites in intronic regions for over 1000 genes. In this work, an entirely new mechanism is revealed how miRs can repress full length protein translation, resulting in C-terminal truncated proteins.

MicroRNAs as Urinary Biomarker for Oncocytoma.

The identification of benign renal oncocytoma, its differentiation from malignant renal tumors, and their eosinophilic variants are a continuous challenge, influencing preoperative planning and being an unnecessary stress factor for patients. Regressive changes enhance the diagnostic dilemma, making evaluations by frozen sections or by immunohistology (on biopsies) unreliable. MicroRNAs (miRs) have been proposed as novel biomarkers to differentiate renal tumor subtypes. However, their value as a diagnostic biomarker of oncocytoma in urines based on mechanisms known in oncocytomas has not been exploited. We used urines from patients with renal tumors (oncocytoma, renal cell carcinoma: clear cell, papillary, chromophobe) and with other urogenital lesions. miRs were extracted and detected via qRT-PCR, the respective tumors analyzed by immunohistology. We found isocitrate dehydrogenase 2 upregulated in oncocytoma and oncocytic chromophobe carcinoma, indicating an increased Krebs cycle metabolism. Since we had shown that all renal tumors are stimulated by endothelin-1, we analyzed miRs preidentified by microarray after endothelin-1 stimulation of renal epithelial cells. Four miRs are proposed as presurgical urinary biomarkers due to their known regulatory mechanism in oncocytoma: miR-498 (formation of the oncocytoma-specific slice-form of vimentin, Vim3), miR-183 (associated with increased CO2 levels), miR-205, and miR-31 (signaling through downregulation of PKC epsilon, shown previously).

Exclusive Phlebosclerosis of Submucosal Veins Leading to Ischemic Necrosis and Perforation of the Large Bowel: First European Case.

Phlebosclerotic colitis (PC) is a rare, potentially life-threatening disease of unclear pathogenesis almost exclusively reported in Asian patients of both genders. A fibrous degeneration of venous walls leads to threadlike calcifications along mesenteric vessels and colonic wall thickening, detectable by CT. This causes disturbed blood drainage and hemorrhagic infarction of the right-sided colonic wall. This is a report of PC in a Caucasian woman in Europe without Asian background and no history of herbal medications, a suspected cause in Asian patients. CT revealed no calcification of the mesenteric vein or its tributaries. Instead, submucosal veins of the left-sided colonic wall were calcified, leading to subsequent transmural necrosis. Clinically, the patient developed a paralytic ileus and sigmoidal perforation during a 2-week hospitalization due to a bleeding cerebral vascular aneurysm. This case of a European woman with PC is unique in its course as well as its radiologic, clinical, and pathologic presentation.

New Lineage of Lassa Virus, Togo, 2016.

We describe a strain of Lassa virus representing a putative new lineage that was isolated from a cluster of human infections with an epidemiologic link to Togo. This finding extends the known range of Lassa virus to Togo.

Exosomes as miRNA Carriers: Formation-Function-Future.

Exosomes, which are one of the smallest extracellular vesicles released from cells, have been shown to carry different nucleic acids, including microRNAs (miRNAs). miRNAs significantly regulate cell growth and metabolism by posttranscriptional inhibition of gene expression. The rapidly changing understanding of exosomes' formation and function in delivering miRNAs from cell to cell has prompted us to review current knowledge in exosomal miRNA secretion mechanisms as well as possible therapeutic applications for personalized medicine.

Vimentin 3, the new hope, differentiating RCC versus oncocytoma.

Vimentin is currently used to differentiate between malignant renal carcinomas and benign oncocytomas. Recent reports showing Vimentin positive oncocytomas seriously question the validity of this present diagnostic approach. Vimentin 3 is a spliced variant and ends with a unique C-terminal ending after exon 7 which differentiates it from the full length version that has 9 exons. Therefore, the protein size is different; the full length Vimentin version has a protein size of ~57 kDa and the truncated version of ~47 kDa. We designed an antibody, called Vim3, against the unique C-terminal ending of the Vimentin 3 variant. Using immune histology, immune fluorescence, Western blot, and qRT-PCR analysis, a Vim3 overexpression was detectable exclusively in oncocytoma, making the detection of Vim3 a potential specific marker for benign kidney tumors. This antibody is the first to clearly differentiate benign oncocytoma and the mimicking eosinophilic variants of the RCCs. This differentiation between malignant and benign RCCs is essential for operative planning, follow-up therapy, and patients' survival. In the future the usage of Vimentin antibodies in routine pathology has to be applied with care. Consideration must be given to Vimentin specific binding epitopes otherwise a misdiagnosis of the patients' tumor samples may result.

ET-1 Induced Downregulation of MRP2 via miRNA 133a - A Marker for Tubular Nephrotoxicity?

BACKGROUND: Multiple drug resistance (MDR), known from treating malignant tumors with chemotherapy, increases the efflux of reabsorbed reagents in tumor cells. This mechanism has been reported in the renal proximal tubule and may prevent therapeutic tubular protection in proteinuria. Since endothelin-1 (ET-1), a major component in the urine of proteinuric patients, stimulates proximal tubules, its influence on MDR was analyzed with emphasis on the multidrug resistance-associated protein 2 (MRP2), a prominent transporter in the human proximal tubule and microRNA (miRNA) 133a. METHODS: ET-1 stimulated, cultured human renal proximal tubule cells (RPTECs), were analyzed via Western blot for the expression of MRP2 and via qRT-PCR for miRNA 133a. For direct interaction between the miRNA 133a and the 3'UTR of MRP2, an immunoprecipitation was performed using FITC-labelled miRNA 133a as capture, followed by MRP2 PCR analysis and Sanger sequencing. Murine Adriamycin nephropathic model and human proteinuric samples showed high levels of miRNA 133a but low levels of MRP2. The increasing miRNA 133a levels were detectable in urine samples of humans and animals. RESULTS: ET-1 activates the miRNA 133a, which can bind to the 3'UTR of MRP2 and is therefore responsible for the detectable decrease of MRP2. CONCLUSION: This is the first report to analyze the correlation between ET-1-induced miRNA 133a overexpression in proteinuria resulting in MRP2 downregulation, which is a contributing factor for renal cytotoxicity. The detection of the miRNA 133a in urine samples can be possibly used as a monitor for cytotoxicity.

MicroRNA function in the profibrogenic interplay upon chronic liver disease.

In chronic liver disease leading to fibrosis, hepatic stellate cells (HSC) differentiate into myofibroblasts. Myofibroblastic HSC have taken center stage during liver fibrogenesis, due to their remarkable synthesis of extracellular matrix proteins, their secretion of profibrogenic mediators and their contribution to hypertension, due to elevated contractility. MicroRNAs (miRNAs) are small, noncoding RNA molecules of 19-24 nucleotides in length. By either RNA interference or inhibition of translational initiation and elongation, each miRNA is able to inhibit the gene expression of a wide panel of targeted transcripts. Recently, it was shown that altered miRNA patterns after chronic liver disease highly affect the progression of fibrosis by their potential to target the expression of extracellular matrix proteins and the synthesis of mediators of profibrogenic pathways. Here, we underline the role of miRNAs in the interplay of the profibrogenic cell communication pathways upon myofibroblastic differentiation of hepatic stellate cells in the chronically injured liver.

Potential complications of transcatheter aortic valve implantation (TAVI)-an autopsy perspective.

OBJECTIVE: Degenerative or post-endocarditic destruction of aortic valves with secondary left ventricular hypertension and cardiac insufficiency is seen more frequently in patients of increasing age. When conventional aortic valve replacement is no longer an option, because of age and co-morbidity, patients are increasingly treated with interventional aortic valve replacement using transcatheter aortic valve implantation (TAVI). METHODS AND RESULTS: TAVI has been performed in Cologne since 2008. We screened our autopsy registry for cases of TAVI, identifying and characterizing complications in connection with the TAVI procedure. We found 13 patients who underwent TAVI procedure. Five of these patients died of non-TAVI specific postoperative complications, whereas in 8 patients there was a direct relationship between TAVI complications and the cause of death. The Patients died within hours and few days after TAVI procedure respectively. Problems observed included predominantly complications due to calcifications of the aortic valve cusps as well as acute endocarditis in 20% of cases. In one case there was an irreversible compression of the implanted valve due to cardiac resuscitation and a malposition of the bioprosthesis. CONCLUSIONS: Future improvements of preoperative evaluation, especially concerning the degree of calcifications of the aortic valve, appear necessary to increase the chance of preventing such complications. Until then, autopsy analysis of complications may help to improve the TAVI procedure.

MicroRNAs: Small but amazing, and their association with endothelin.

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the expressional regulation of genes by inhibiting gene translation. MicroRNAs are recruited and incorporated into the miRISC, ribonucleoprotein complex, targeting specific mRNAs through mechanisms specific for a miRNA sequence. Here we review the biogenesis, regulation, and monitoring of miRNAs, as well as the current evidence for potential roles of miRNAs in human diseases associated with activation of the endothelin system. These diseases include cancer, kidney disease, cardiovascular diseases, inflammatory diseases, infectious diseases, and blood diseases, that may all be aggravated by aberrant miRNA expression. In this review we will also discuss regulatory mechanisms determining production of miRNA as well as measuring or targeting miRNAs as potential novel approaches for diagnosis and treatment. Targeting miRNAs possibly will allow one to detect diseases or to interfere with the progression of diseases associated with activation of the endothelin system.