Ekspresija neuralnih adhezionih molekula (NCAM) u zdravom i obolelom bubrežnom tkivu

Introduction: Kidney performs essential physiological roles that include metabolic waste excretion and maintenance of fluid and electrolyte balance. Different factors may lead to renal dysfunction. Kidney diseases are currently a global public health problem. Fortunately, dialysis and transplantation provide life-saving treatments, but these therapies are rife with limitations. Thus, new ways of therapies, such as stem cell treatment are more than required as an alternative and new glimmer of hope to all nephrology patients. Neural cell adhesion molecule (NCAM) is widely expressed during kidney development and it presents potential marker for renal stem/progenitor cells. Aim: Aim of this dissertation was evaluation of NCAM presence in different types of renal tissue (normal fetal or adult, and pathologically changed), and comparison and correlation of NCAM expression with already defined stem cell markers in order to more closely characterize renal fetal progenitor cells. Method: Specifically designed primers were used for reverse transcriptase PCR, after mRNA isolation from fetal, neonatal, adult normal, adult tissues with interstitial fibrosis and tumor tissues, to clarify presence of different NCAM on nucleotide level. Also, Western Blot tests were performed in order to identify expression of different NCAM protein isoforms in healthy and kidney with disease. Further, FACS analysis was done on renal cell lines to correlate NCAM expression with adult renal stem cell markers CD24 and CD133. Co-expression and co-localization of NCAM with other cell surface markers was also examined using immunohistochemistry and double immunofluorescent staining. Results: Our results showed aberrant NCAM expression in renal tumor tissues as well as in tissue with interstitial fibrosis. Interestingly in both types of mentioned tissues NCAM had co-expression with FGFR1, tyrosine kinase receptor, responsible for cell proliferation and aggressive behavior in some epithelial tumors. Considering NCAM expression on fetal samples possible renal progenitor population cell pool of NCAM+CD24+ cells was found, which was not the case with NCAM+CD133+ cell pool. Also, we had detected that NCAM molecules were post-trasnaltionally modified...UVOD: Bubreg ima značajnu fiziološku ulogu u izlučivanju štetnih produkata metabolizma putem urina, kao i ulogu u održanju ravnoteže vode i elektorlita. Različiti faktori mogu dovesti do poremećaja funkcije bubrega. Bolesti bubrega su trenutno globalni problem javnog zdravlja. Dijaliza i transplantacija su trenutno dominantni vidovi terapije koje se primenjuju u lečenju bubrežne isuficijencije, ali uz puno ograničenja. Novi načini terapije, kao što je tretman matičnim ćelijama, su više nego potrebni kao alternativa standardnim metodama lečenja i predstavljaju novi tračak nade za sve pacijente koji imaju neki oblik bubrežne disfunkcije. Neuralni ćelijski adhezioni molekul (NCAM) je široko ispoljen tokom razvoja bubrega i predstavlja potencijalni marker renalnih stem/progenitorskih ćelija. CILJ: Cilj ove disertacije je evaluacija NCAM ekspresije u različitim tipovima bubrežnog tkiva (fetalno, adultno normalno i adultno tkivo sa intersticijskom fibrozom i tumorsko tkivo), kao i njena korelacija sa ekspresijom već definisanih markera renalnih stem ćelija kako bi se preciznije definisale fetalne renalne progenitorske ćelije. METOD: Detekcija različitih NCAM izoformi na nivou nukleotida je vršena primenom RT-PCR metode, nakon izolacije iRNK iz fetalnog i normalnog adultnog tkiva, kao i tumorskog i tkiva sa intersticijskom fibrozom. Prisustvo NCAM isoformi na proteinskom nivou, u pomenutim tkivima detektovano je upotrebom Western blot-a i Imunoprecipitacije. Ko-ekspresija NCAM-a sa CD24 i CD133, markerima adultnih renalnih progenitora ispitivana je upotrebom protočne citomertije (FACS) na renalnim ćelijskim linijama. Imunohistohemijskim i dvostrukim imunofluorescentnim bojenjem ispitivana je koekspresija i kolokalizaciji NCAM-a sa drugim ćelijskim markerima. REZULTATI: RT-PCR rezultati ove doktorske disertacije su pokazale da sve ćelijske linije i ispitivana tkiva, osim tumorskog tkiva eksprimiraju tri glavne NCAM isoforme NCAM-120, NCAM-140 i NCAM-180. Takođe, detektovana je aberentnaprekomerna NCAM ekspresija u tumorskom, kao i u tkivu sa intersticijskom fibrozom. Interesantan nalaz čini pokazana koekspresija NCAM-a sa FGFR1, tirozin kinaznim receptorom, koji ima ulogu u ćelijskoj proliferaciji i agresivnom ponašanju pojedinih 7 epitelnih tumora

Losartan Improved Antioxidant Defense, Renal Function and Structure of Postischemic Hypertensive Kidney

Ischemic acute renal failure (ARF) is a highly complex disorder involving renal vasoconstriction, filtration failure, tubular obstruction, tubular backleak and generation of reactive oxygen species. Due to this complexity, the aim of our study was to explore effects of Angiotensin II type 1 receptor (AT1R) blockade on kidney structure and function, as well as oxidative stress in spontaneously hypertensive rats (SHR) after renal ischemia reperfusion injury. Experiments were performed on anaesthetized adult male SHR in the model of ARF with 40 minutes clamping the left renal artery. The right kidney was removed and 40 minutes renal ischemia was performed. Experimental groups received AT1R antagonist (Losartan) or vehicle (saline) in the femoral vein 5 minutes before, during and 175 minutes after the period of ischemia. Biochemical parameters were measured and kidney specimens were collected 24h after reperfusion. ARF significantly decreased creatinine and urea clearance, increased LDL and lipid peroxidation in plasma. Treatment with losartan induced a significant increase of creatinine and urea clearance, as well as HDL. Lipid peroxidation in plasma was decreased and catalase enzyme activity in erythrocytes was increased after losartan treatment. Losartan reduced cortico-medullary necrosis and tubular dilatation in the kidney. High expression of pro-apoptotic Bax protein in the injured kidney was downregulated after losartan treatment. Our results reveal that angiotensin II (via AT1R) mediates the most postischemic injuries in hypertensive kidney through oxidative stress enhancement. Therefore, blockade of AT1R may have beneficial effects in hypertensive patients who have developed ARF

Ticks (Acari: Argasidae, Ixodidae) parasitizing bats in the central Balkans

Ticks parasitizing bats have been largely understudied, especially in the central part of the Balkan Peninsula, where the last data from the field research date from almost 25 years ago. Bats are hosts to a large number of ectoparasites, including ticks, which can act as vectors of zoonotic agents. For this reason, it is important to identify the distribution of ticks and their relationship to different hosts, including wild animals, bats in particular. The present research was conducted at 16 localities throughout Serbia, Montenegro, Bosnia and Herzegovina, and Former Yugoslav Republic of Macedonia (FYROM). We examined 475 individuals of bats belonging to 13 species. A total of three tick species were identified, I. simplex being the most numerous and widespread, followed by I. vespertilionis and A. vespertilionis. To the best of our knowledge, the presented data include the first records of I. simplex in Serbia and Montenegro, I. vespertilionis for Montenegro and A. vespertilionis in FYROM. Also, we identify a new possible host/parasite association between I. simplex and Rhinolophus euryale

Effects of Automation on Sustainability of Immunohistochemistry Laboratory

The COVID-19 pandemic that hit the world recently caused numerous changes affecting the health system in every department. Reduced staff numbers, mostly due to illness, led to an increase in automation at every stage of laboratory work. The immunohistochemistry (IHC) laboratory conducts a high volume of slide staining every day. Therefore, we analyzed time and total costs required to obtain IHC slides in both the manual and automated way, comparing their efficiency by processing the same sample volume (48 microscope slides—the maximum capacity that an automated immunostainer—DAKO, Autostainer Link 48, Part No AS48030—can process over a single cycle). The total IHC procedure time to run 48 slides manually by one technician was 460 min, while the automated process finished a cycle within 390 min (15.22% less time). The final cost of a single manual IHC slide was 12.26 EUR and 7.69 EUR for slides labeled in the automated immunostainer, which reduced final costs by 37.27%. Thus, automation of the IHC procedure reduces the time and costs of the IHC process, contributing significantly to the sustainability of the healthcare system during the COVID-19 pandemic, overcoming insufficient human resources

Hard Ticks Parasitizing European Ground Squirrel, Spermophilus citellus (L.,1766) (Rodentia: Sciuridae) in Serbia

Specimens of the endangered species Spermophilus citellus (European ground squirrel) from populations inhabiting 12 localities in Vojvodina (Serbia) were inspected for the presence of attached hard ticks. A total of 1009 ticks were found on 151 infested animals. With respect to the level of infestation, over 60% of the squirrels were found with five or less ticks attached, while the distribution of the number of animals with a higher level of infestation tended to follow the 80/20 rule. A notably higher level of infestation was detected in males as compared to females. Nymphs were the predominant developmental stage of ticks parasitizing the inspected European ground squirrel specimens. The majority of removed ticks belonged to the nidicolous species Ixodes laguri (79%), while the remaining ticks were identified as Haemaphysalis concinna. In seeking molecular confirmation of morphological identification of Ixodes specimens, COI gene sequences of I. laguri were obtained and deposited in GenBank

Variable Expression of Neural Cell Adhesion Molecule Isoforms in Renal Tissue: Possible Role in Incipient Renal Fibrosis.

Rare neural cell adhesion molecule (NCAM) positive cells have been previously described within the normal human adult kidney interstitium, speculating that they could increase in the interstitium with incipient interstitial renal fibrosis (IRF). In the present study, among 93 biopsy samples of various kidney diseases, NCAM+ interstitial cells were detected in 62.4% cases. An increased number of NCAM+ cells was significantly observed only in incipient IRF compared to normal renal tissues and advanced IRF stages (p<0.001), independently of underlying diseases (p = 0.657). All three major NCAM isoforms' RT-PCR bands were visible either in normal or in kidneys with incipient IRF, albeit their mRNA expression levels measured by qRT-PCR were different. Applying qRT-PCR on pure NCAM+ cells population, obtained by laser capture microdissection, significant mRNA over-expression of NCAM140kD isoform was found in NCAM+ cells within incipient IRF (p = 0.004), while NCAM120kD and NCAM180kD isoforms were not changed significantly (p = 0.750; p = 0.704; respectively). Simultaneously, qRT-PCR also showed significant αSMA (p = 0.014) and SLUG (p = 0.004) mRNAs up-regulation within the NCAM+ cells of incipient IRF, as well as highly decreased matrix metalloproteinases (MMP) -2 and -9 mRNAs (p = 0.028; p = 0.036; respectively). However, using double immunofluorescence MMP-9 could still be detectable on the protein level in rare NCAM+ cells within the incipient IRF. Further characterization of NCAM+ cells by double immunofluorescent labeling revealed their association with molecules involved in fibrosis. Fibroblast growth factor receptor 1 (FGFR1) and α5β1 integrin were extensively expressed on NCAM+ cells within the incipient IRF areas, whereas human epididymis protein-4 (HE4) was found to be present in few NCAM+ cells of both normal and interstitium with incipient fibrosis. Heterogeneity of NCAM+ interstitial cells in normal and incipient IRF, concerning molecules related to fibrosis and variable expression of NCAM isoforms, could suggest diverse role of NCAM+ cells in homeostasis and in regulation of renal fibrosis in diseased kidneys

Biochemical parameters in experimental groups 24/reperfusion injury.

#<p><i>p</i><0.05,^###<i>p</i><0.001 compared with SHAM level; **<i>p</i><0.01, ***<i>p</i><0.001 compared with ARF. Data are presented as mean ± SEM. n-number of animals.</p

Histology of the kidney 24 h after reperfusion.

<p>(<b>A</b>) normal shape of the glomerulus and tubulointerstitium in the sham-operated animals (<b>B</b>) proximal tubular dilatation and necrosis, PAS-positive casts in the ARF control group (<b>C)</b> moderately intensive tubular necrosis in Losartan treated rats, reduced tubular dilatation and less number of PAS-positive casts (<b>D</b>) Histopathological score in experimental groups 24 h after reperfusion. SHAM, n = 7; ARF, n = 7; ARF+LOS, n = 9 (n-number of animals). Data are presented as mean ± SEM (^###p<0.001 compared with SHAMl; ***p<0.001 compared with ARF).</p

Oxidative stress parameters in experimental groups 24 h after reperfusion.

##<p><i>p</i><0.01 compared with SHAM level; *<i>p</i><0.05, **<i>p</i><0.01 compared with ARF. Data are presented as mean ± SEM; n-number of animals; p-plasma; e- erythrocyte.</p

Mean arterial pressure in experimental groups before and 24 h after reperfusion.

<p>SHAM, n = 7; ARF, n = 7; ARF+LOS, n = 9 (n-number of animals). Data are presented as mean ± SEM (###p<0.001 compared with SHAMl; ***p<0.001 compared with ARF).</p