Uloga interakcije receptora za fibroblastni faktor rasta sa neuralnim ćelijskim adhezionim molekulom u fibrozi bubrega

Progression of chronic kidney disease (CKD) remains an unsolved problem in clinical nephrology since approaches to reverse or repair chronic renal injury are not yet available. Independent of the underlying disease, loss of functional kidney parenchyma and tubulo-interstitial fibrogenesis are commonly observed when kidney injury progresses towards CKD. In this regard, epithelial-to-mesenchymal transition (EMT) in tubular epithelial cells (TECs) and consecutive G2/M arrest have been shown to determine maladaptive kidney repair in response to injury, ultimately associated with renal fibrogenesis and progression into CKD. Transforming growth factor β1 (TGF-β1) is considered as a key mediator of intrarenal EMT program and renal fibrosis. Neural cell adhesion molecule (NCAM) and fibroblast growth factor receptor (FGFR) signaling during EMT program have already been described and it has been noticed that both molecules are fundamental for EMT program in vitro. However, their cross-talk has been widely studied mainly in neural tissues and cancer cells, but there is a lacking of evidence for the contribution of their interplay to fibrogenesis, although several studies confirmed that both molecules can be separately involved in such process. Moreover, FGFR has been widely studied in many fields of research, including fibrosis, whereas NCAM contribution to renal fibrogenesis has been only suggested by two research groups (including our team). Thus, it encouraged us to investigate aforementioned molecules in human kidneys and to evaluate their significance in fibrotic response within the renal interstitial compartment. For the first time, here we present a functional significance of NCAM and FGFR co-operation in the induction of renal fibrosis, mediated by TGFβ-1. Material and methods In order to achieve goals of the study, we examined human kidney biopsy samples and performed cell culture experiments to clarify functional significance of NCAM/FGFR1 signaling in model of renal fibrosis. Immunostaining (immunohistochemistry and immunofluorescence) was performed to detect NCAM expressing cells in human kidneys in order to further characterize these cells and to label them for laser capture microdissection (LCM) which allows pure NCAM+ cells collecting for the subsequent qRT-PCR and gene expression analysis. Moreover, clinical relevance of both NCAM expressing renal interstitial cells and TGFβ-1 downstream effectors detection in human kidney biopsies were also examined. By using an established model of EMT program in human proximal tubular epithelial cells (HK- ii 2 cells) in response to TGF-β1 (10ng/mL) exposure, NCAM/FGFR1 signaling responses were analyzed by light microscopy, immunolabeling, qRT-PCR and scratch assays...Progresija hronične bubrežne insuficijencije predstavlja gotovo nerešiv problem kliničke nefrologije, imajući u vidu da su terapijski postupci koji bi doveli do zaustavljanja ili reverzibilnosti hroničnih oštećenja parenhima bubrega još uvek nedostupni. Sva hronična oboljenja bubrega dovode do progresivnog gubitka funkcionalnog parenhima i tubulointersticijske fibroze, indukujući pojavu hronične bubrežne insuficijencije. U tom pogledu, smatra se da je supstrat nemogućnosti regeneracije oštećenog parenhima bubrega zapravo fenomen epitelno-mezenhimne transformacije (EMT) sa sledstvenim G2/M zastojem u fazi ćelijskog ciklusa koji dovodi do nastanka fibroze i progresije hronične bubrežne insuficijencije. Smatra se da je glavni medijator EMT programa i fibroze u bubregu transformišući faktor rasta β1 (TGF-β1). Opisano je da se tokom EMT programa indukuje ekspresija neuralnog ćelijskog adhezionog molekula (NCAM) i receptora za fibroblastni faktor rasta (FGFR), koji imaju važnu ulogu u in vitro EMT. Međutim, značaj međusobnih interakcija NCAM i FGFR molekula do sada je prevashodno ispitivan u neuralnom tkivu i različitim malignim oboljenjima, dok nema podataka o značaju ovih interakcija tokom procesa fibroze, iako je nekoliko studija potvrdilo da individualno NCAM i FGFR molekul mogu biti uključeni u ovaj proces. Štaviše, FGFR je izučavan u mnogim patološkim procesima, uključujući i fibrozu, dok je povezanost NCAM molekula sa procesom fibroze u bubregu sugerisana od strane samo dva istraživačka tima (uključujući i naš). Stoga smo poželeli da ispitamo prethodno pomenute molekule u bubrezima čoveka i da procenimo njihov značaj u procesu fibrogeneze. Po prvi put, u ovoj studiji prikazujemo funkcionalni značaj NCAM/FGFR interakcija u indukciji fibroze bubrega, posredovanoj TGF-β1. Materijal i metode U svrhu ispitivanja zadatih ciljeva studije, korišćeni su uzorci humanih biopsija bubrega kao i eksperimenti na ćelijskim kulturama kako bi se razjasnio funkcionalni značaj NCAM/FGFR1 signalnog puta u modelu fibroze bubrega. Primenjene su imunohistohemisjke i imunofluorescentne tehnike detektovanja NCAM eksprimirajućih ćelija sa ciljem njihove bolje krakterizacije i vizuelizacije, kao i radi sprovođenja laserske mikrodiskcije (LCM) koja omogućava sakupljenje izolovanih NCAM+ ćelija za dalja ispitivanja genske ekspresije metodom real-time RT-PCR (qRT-PCR). Takođe, ispitan je klinički značaj prisustva NCAM+ ćelija u intersticijumu bubrega kao i značaj detekcije nishodnih efektora TGF-β1 v signalnog puta u biopsijskim uzorcima..

Copy number variation analysis identifies MIR9-3 and MIR1299 as novel miRNA candidate genes for CAKUT

Background Congenital anomalies of the kidney and urinary tract (CAKUT) represent a frequent cause of pediatric kidney failure. CNVs, as a major class of genomic variations, can also affect miRNA regions. Common CNV corresponding miRNAs (cCNV-miRNAs) are functional variants regulating crucial processes which could affect urinary system development. Thus, we hypothesize that cCNV-miRNAs are associated with CAKUT occurrence and its expressivity. Methods The extraction and filtering of common CNVs, identified in control samples deposited in publicly available databases gnomAD v2.1 and dbVar, were coupled with mapping of miRNA sequences using UCSC Genome Browser. After verification of the mapped miRNAs using referent miRBase V22.1, prioritization of cCNV-miRNA candidates has been performed using bioinformatic annotation and literature research. Genotyping of miRNA gene copy numbers for MIR9-3, MIR511, and MIR1299, was conducted on 221 CAKUT patients and 192 controls using TaqMan™ technology. Results We observed significantly different MIR9-3 and MIR1299 gene copy number distribution between CAKUT patients and controls (Chi-square, P = 0.006 and P = 0.0002, respectively), while difference of MIR511 copy number distribution showed nominal significance (Chi-square, P = 0.027). The counts of less and more than two of MIR1299 copy numbers were more frequent within CAKUT patients compared to controls (P = 0.01 and P = 0.008, respectively) and also in cohort of patients with anomalies of the urinary tract compared to controls (P = 0.016 and P = 0.003, respectively). Conclusions Copy number variations of miRNA genes represent a novel avenue in clarification of the inheritance complexity in CAKUT and provide potential evidence about the association of common genetic variation with CAKUT phenotypes

A preliminary study of the miRNA restitution effect on CNV-induced miRNA downregulation in CAKUT

Background The majority of CAKUT-associated CNVs overlap at least one miRNA gene, thus affecting the cellular levels of the corresponding miRNA. We aimed to investigate the potency of restitution of CNV-affected miRNA levels to remediate the dysregulated expression of target genes involved in kidney physiology and development in vitro. Methods Heterozygous MIR484 knockout HEK293 and homozygous MIR185 knockout HEK293 cell lines were used as models depicting the deletion of the frequently affected miRNA genes by CAKUT-associated CNVs. After treatment with the corresponding miRNA mimics, the levels of the target genes have been compared to the non-targeting control treatment. For both investigated miRNAs, MDM2 and PKD1 were evaluated as common targets, while additional 3 genes were investigated as targets of each individual miRNA (NOTCH3, FIS1 and APAF1 as hsa-miR-484 targets and RHOA, ATF6 and CDC42 as hsa-miR-185-5p targets). Results Restitution of the corresponding miRNA levels in both knockout cell lines has induced a change in the mRNA levels of certain candidate target genes, thus confirming the potential to alleviate the CNV effect on miRNA expression. Intriguingly, HEK293 WT treatment with investigated miRNA mimics has triggered a more pronounced effect, thus suggesting the importance of miRNA interplay in different genomic contexts. Conclusions Dysregulation of multiple mRNA targets mediated by CNV-affected miRNAs could represent the underlying mechanism behind the unresolved CAKUT occurrence and phenotypic variability observed in CAKUT patients. Characterizing miRNAs located in CNVs and their potential to become molecular targets could eventually help in understanding and improving the management of CAKUT

Correlations of CDKN1A and ADAM17 expression with a change of left ventricular remodeling echocardiographic parameters in PBMC of patients six months after the first myocardial infarction

Introduction: Myocardial infarction (MI) and consequential ischemia with cardiomyocyte loss are followed by left ventricular (LV) remodeling. LV remodeling is crucial process for cardiac function preservation, although when prolonged it can become maladaptive and lead to impaired systolic function and further cardiovascular complications. Echocardiographic parameters are used as a measure of LV structure and function. ADAM17 (a disintegrin and metalloprotease domain) and CDKN1A (cyclindependent kinase inhibitor 1A) have shown regulating role in DNA repair, inflammation, remodeling and fibrosis. The aim of this preliminary study was to investigate the potential effect of CDKN1 and ADAM17 mRNA in post MI heart remodeling. Methods: Sixty four patients with the first MI were prospectively followed-up 6 months after MI. Change (Δ) of echocardiographic parameters within 6 months was calculated as a difference between the value at 6-month follow-up and value at admission. Relative gene expression was detected using the TaqMan® technology. Statistical analyses were done by Statistica 8 software. Results: We have observed correlation between CDKN1A mRNA expression and change of LV enddiastolic diameter (ΔLVEDD, R=0.3, p=0.01) and LV end-systolic diameter (ΔLVESD, R=0.3, p=0.02), but not with LV ejection fraction and stroke volume. ADAM17 expression was not in correlation with analyzed parameters of LV remodeling. However, CDKN1A and ADAM17 mRNA expression in PBMC six months after MI were positively correlated (R=0.6, p<0.001). Conclusion: Preliminary resultssuggest that CDKN1 has a role in post MI LV remodeling, correlating with changes in echocardiographic parameters of LV structure. The validation on a larger sample size is required.The Second Congress of Molecular Biologists of Serbia; October 6-8, Belgrade, 2023

miRNA-free rare pathogenic CNVs could drive toward variable CAKUT phenotypes

Introduction: Genetic studies of congenital anomalies of the kidney and urinary tract (CAKUT) have demonstrated variable penetrability and expressivity of the associated genetic defects. Previously, it was shown that deletions of 17q12 and 22q11.2 regions were specific for kidney anomalies (KA) while 16p11.2 and 1q21.1 loci showed extensive pleiotropy in CAKUT phenotypes. CNVs affecting miRNA gene dosage have been described to have functional influence on gene expression. We aimed to conduct comprehensive in silico analysis using publicly available databases to analyze miRNA content of CAKUT-associated CNVs in quoted chromosomal loci with regard to pleiotropy. Methods: Extensive literature review was conducted to collect data about pathogenic rCNVs associated with CAKUT. UCSC genome browser tool was employed for mapping miRNAs onto collected rCNV regions. Results: Analysis of CNVs in CAKUT included four studies counting more than 2500 patients. In further analysis we included 191 patients harboring pathogenic CNVs. Surprisingly, CAKUT pleiotropic regions (16p11.2, 1q21.2) did not contain any miRNA. 22q11.2 showed the densest miRNAs content (n = 21). Conclusions: Absence of miRNAs may potentially pronounce the pleiotropy of the CAKUT genetic defects, thus leading to the variety of phenotypes. Contrary, abundancy of miRNAs in 22q11.2 might be associated with reproducible phenotype, such as KA, producing the functional effect when deleted. This assumption agrees with recent results of miRNA expression variability in 22q11.2 deletion syndrome.54th European Society of Human Genetics (ESHG) Conference; August 28-31, 2021; Virtual ConferenceAbstracts from the 54th European Society of Human Genetics (ESHG) Conference: e-Poster

Gene expression of chemokines CX3CL1 and CXCL16 and their receptors, CX3CR1 and CXCR6, in peripheral blood mononuclear cells of patients with relapsing-remitting multiple sclerosis - a pilot study

Background/Aim. In vitro and in vivo studies show that CX3CL1 and CXCL16 chemokines and their specific receptors, CX3CR1 and CXCR6, respectively, mediate mechanism of neuroinflammation during the pathogenesis of multiple sclerosis (MS). The aim of this study was to investigate relative messenger ribonucleic acid (mRNA) levels of CX3CL1, CXCL16, CX3CR1 and CXCR6 in peripheral blood mononuclear cells, as potential molecular markers of relapsing-remitting (RR) MS. Methods. The study included 43 unrelated RR MS patients, 20 of them with clinically active disease (relapse) and 23 with clinically stable disease (remission), and 28 unrelated healthy subjects as controls. Real-time polymerase chain reactions (PCR) were performed using TaqMan? gene expression assays. Relative expression (mRNA) level of each target gene in each sample of peripheral blood mononuclear cells was calculated as the mean normalized expression. Results. The levels of CX3CR1 mRNA were significantly higher in clinically active RR MS patients compared to controls [fold change = 1.38, p (Mann-Whitney U test) = 0.009], and significantly lower in clinically stable vs active RR MS patients [fold change = - 1.43, p (t-test) = 0.03]. Stable RR MS patients had significantly higher CXCL16 mRNA levels than controls [fold change = 1.33, p (Mann-Whitney U test) = 0.006]. A trend of increased CXCR6 gene expression was found in active RR MS patients compared to controls [fold change = 1.23, p (Mann-Whitney U test) = 0.08]. In either active or stable RR MS patients there were no significant correlations of the clinical parameters with expression levels of the target genes. Conclusion. The current results show that increased CX3CR1 mRNA levels in peripheral blood mononuclear cells could represent a proinflammatory molecular marker of clinically active RR MS

Effects of Single and Combined Losartan and Tempol Treatments on Oxidative Stress, Kidney Structure and Function in Spontaneously Hypertensive Rats with Early Course of Proteinuric Nephropathy

Oxidative stress has been widely implicated in both hypertension and chronic kidney disease (CKD). Hypertension is a major risk factor for CKD progression. In the present study we have investigated the effects of chronic single tempol (membrane-permeable radical scavenger) or losartan (angiotensin II type 1 receptor blocker) treatment, and their combination on systemic oxidative status (plasma thiobarbituric acid-reactive substances (pTBARS) production, plasma antioxidant capacity (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, pABTS), erythrocyte antioxidant enzymes activities) and kidney oxidative stress (kTBARS, kABTS, kidney antioxidant enzymes activities), kidney function and structure in spontaneously hypertensive rats (SHR) with the early course of adriamycin-induced nephropathy. Adult SHR were divided into five groups. The control group received vehicle, while the other groups received adriamycin (2 mg/kg, i.v.) twice in a 21-day interval, followed by vehicle, losartan (L, 10 mg/kg/day), tempol (T, 100 mg/kg/day) or combined T+L treatment (by gavage) during a six-week period. Adriamycin significantly increased proteinuria, plasma lipid peroxidation, kidney protein oxidation, nitrite excretion, matrix metalloproteinase-1 (MMP-1) protein expression and nestin immunostaining in the kidney. Also, it decreased kidney antioxidant defense, kidney NADPH oxidase 4 (kNox4) protein expression and abolished anti-inflammatory response due to significant reduction of kidney NADPH oxidase 2 (kNox2) protein expression in SHR. All treatments reduced protein-to-creatinine ratio (marker of proteinuria), pTBARS production, kidney protein carbonylation, nitrite excretion, increased antioxidant capacity and restored kidney nestin expression similar to control. Both single treatments significantly improved systemic and kidney antioxidant defense, bioavailability of renal nitric oxide, reduced kMMP-1 protein expression and renal injury, thus retarded CKD progression. Losartan improved blood pressure, as well astubular injury and restored anti-inflammatory defense by reverting kNox2 expression to the control level. Interestingly, tempol was more successful in reducing systemic oxidative stress, proteinuria, kMMP-1 and glomerulosclerosis. However, combined treatment failed to overcome the beneficial effects of single treatments in slowing down the progression of ADR-induced nephropathy in SHR

Identification of micro RNA from common copy number variants as risk factors for CAKUT

Introduction: Congenital anomalies of the kidney and urinary tracts(CAKUT) are a diverse spectrum of defects with complex etiology and not fully explained genetic background. miRNA-containing copy number variants (CNVs) are described as genetic risk factor for the disease development. We aimed to identify miRNAs with the maximum regulatory coverage of previously reported differentially expressed genes in CAKUT tissue compared to controls and bioinformatically characterize a set of these miRNAs which are located in common CNVs. Methods: Differentially expressed genes were identified from ureter tissue transcriptome open data GSE83946 from 15 CAKUT patients and 7 healthy controls, generated in house previously. miRPathDB v2.0 was used for identification of miRNAs with maximum coverage of DEGs(miRNAs which complimentarily regulate all DEGs). Mapping of maximum coverage miRNAs onto common CNVs (frequency >0.2) was performed using UCSC genome browser and gnomAD database. miRNA mapping common CNVs were further bioinformatically analyzed using miRPathDB v2.0. Results: In a maximum coverage set of 50 miRNAs interacting with DEGs in CAKUT, we have identified 3 miRNA geneslocated in the common CNVs(hsa-miR-663b, hsa-miR-3180-3p and hsa-miR-1302). Using Reactome database we identified all three miRNAsto be significantly enriched in the pathway Neuronal System: -log(p-value)>2.326 for hsa-miR-1302; -log(p-value)>1.556 for hsa-miR-3180-3p; and -log(pvalue)>1.703 for hsa-miR-663b. Conclusion: CAKUT is characterized with variable penetrability and expressivity and often followed with other comorbiditiessuch as neurodevelopmental disorders. miRNAsinvolved in DEG networks and prone to CNV effects could present modulating factors of the disease phenotype. Further studies should provide additional evidence about hsa-miR-1302, hsa-miR-3180-3p and hsa-miR-663b involvements in CAKUT etiologyThe Second Congress of Molecular Biologists of Serbia; October 6-8, Belgrade, 2023

Polymorphism rs10757278 in 9p21 region is associated with severe carotid atherosclerosis in sex specific manner: Preliminary results

Genome wide association studies have recognized the 9p21 rs10757278 polymorphism as a significant independent genetic prognostic marker for coronary artery disease. The aim of this study was to explore possible association of the rs10757278 polymorphism with advanced carotid atherosclerosis (CA) in the population of Serbia. The study group included 147 controls and 428 patients consecutively admitted for carotid endarterectomy. 9p21 rs10757278 polymorphism was genotyped using TaqMan technology on 7500 ABI Real Time PCR. There was no significant association of this polymorphism and CA, either in study group overall or in males. The GG genotype, according to recessive model of inheritance (AA+AG vs. GG), was significantly associated with advanced CA in females only (OR=2.15, 95% CI 1.07- 4.29, p=0.03). Preliminary results in this study suggest that rs10757278 GG genotype might be a significant predictive sex-specific marker for advanced CA in the population of Serbia.5th Congress of the Serbian Genetic Society : Book of abstracts; September 28 - October 2, Kladovo, 2014