Ivana Vrhovac

University of Split

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Davorka Breljak

Institute for Medical Research and Occupational Health

Ivan Sabolić

University Hospital Centre Zagreb

Dean Karaica

Institute for Medical Research and Occupational Health

Vedran Micek

Institute for Medical Research and Occupational Health

Marija Ljubojević

Institute for Medical Research and Occupational Health

Hrvoje Brzica

University of Arizona

Hermann Koepsell

University of Würzburg

Carol M. Herak–Kramberger

Institute for Medical Research and Occupational Health

Birgitta C. Burckhardt

University of Göttingen

Gerhard Burckhardt

University of Göttingen

Cooperative Institutions

University of Zagreb

Institute for Medical Research and Occupational Health

Chinese Academy of Sciences

University of Tübingen

Rudjer Boskovic Institute

University of Würzburg

University Children's Hospital Tübingen

University of Chinese Academy of Sciences

Central South University

Chinese Academy of Medical Sciences & Peking Union Medical College

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Baseline DNA damage levels in blood, liver and kidneys of adult sham-operated and gonadectomized rats

Marko Gerić Goran Gajski Jasna Jurasović Dean Karaica Marija Ljubojević

Baseline (sea)

Liver damage

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Role of hepatic and renal sat-1 in ethylene glycol-induced urolithiasis in rat

Ivan Sabolić Hrvoje Brzica Davorka Breljak Mila Lovrić Ivana Vrhovac

INTRODUCTION. Sodium-independent sulfate anion transporter (sat-1 ; Slc26a1) plays a major role in transport of oxalate (OX) across the cell membrane by exchanging OX for sulfate or bicarbonate. Sat-1 mRNA has been detected strongly in liver and kidney and weakly in few other organs in rats, mice, and humans. By immunocytochemistry (IC), the protein has been localized in rats to the sinusoidal membrane of hepatocytes and basolateral membrane of proximal tubules (PT) in the kidney cortex, with the male (M)-dominant expression. In liver, which is a major OX producer, sat-1 mediates the extrusion of OX and uptake of sulfate, whereas in PT, it mediates the OX uptake, mainly in exchange for intracellular sulfate. In middle-aged humans, OX is a major cause of sex-related urolithiasis ; men excrete more OX and have higher incidence of OX stones then women. Accordingly, in studies of experimental urolithiasis in ethylene glycol (EG)-treated rats, testosterone stimulated, whereas female (F) sex hormones inhibited the urine excretion of OX and formation of OX stones. The role of OX transporters that in liver and kidneys exhibit the sex-related expression, such as sat-1, in the development of OX urolithiasis is not known. In the present study, we tested the expression of sat-1 at the protein and mRNA levels in the liver and kidneys of EG-treated rats. METHODS. Adult M and female (F) Wistar strain rats were treated with EG (0, 75% v/v in drinking water) for one month. Controls drunk water without EG. 24-h urine was collected in metabolic cages a day before sacrificing. OX in the blood plasma and urine were determined by ion-chromatography. Tissue morphology and OX crystals in the urine sediment were checked by light microscopy. Sat-1 protein expression was studied using a polyclonal antibody by IC in p-formaldehyde-fixed tissue cryosections, and by Western blotting of total cell membranes isolated from tissue homogenates. Tissue expression of sat-1 mRNA was studied by real time RT-PCR. RESULTS. In control M and F animals, the M-dominant sex differences in the urine excretion of OX, in the number and size of urine OX crystals, and in the expression of sat-1 protein (but not mRNA) in liver and kidneys, were confirmed. Compared to controls, the EG-treated animals exhibited: a) in plasma, 100% (F) and 4-fold (M) higher concentration of OX, b) in urine, 100% (F) and 17-fold (M) higher OX excretion, c) in urine sediment, similar and low abundance of small OX crystals in F, and increased abundance of large OX crystals in M, d) in tissues of both sexes, un-affected morphology in liver, and dilatated PT and distended peritubular spaces in kidneys, e) upregulated expression of sat-1 protein in the F liver and kidneys to the level in M organs, f) unchanged expression of sat-1 protein in the M liver and kidneys, and g) unchanged expression of sat-1 mRNA in both organs of F and M animals. CONCLUSION. Whereas the blood and urinary parameters in EG-treated rats indicate the low and high oxaluric state in F and M rats, respectively, the EG-induced elevated expression of sat-1 protein in the F liver and kidneys, and unchanged expression of this protein in M organs, indicate that sat-1 plays no significant role in generation of OX urolithiasis in EG-treated animals.

Epithelial polarity

Apical membrane

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Effects of Microwave Heating on Antigen Retrieval in Cryosections of Formalin-Fixed Rat Tissues

Hrvoje Brzica Davorka Breljak Ivana Vrhovac Ivan Sabolić

Immunocytochemistry is a method for localizing proteins in the mammalian tissues and cultured cells. Samples for immunocytochemical studies are often fixed with formalin (2%-10% p-formaldehyde), frozen or embedded in paraffin, and sectioned. Fixation with formalin denatures proteins while preserving antigenicity, but it can ‘’mask’’ the antibody binding sites (epitopes), thus diminishing the availability of epitopes and fidelity of immunocytochemical findings. Hidden epitopes can be recovered by various antigen retrieval protocols before applying antibodies. These protocols are normally used with paraffin sections, and include heating in defined buffers, treatment with various alcohols, detergents, metals or high pressure, or various combinations of these procedures. Here we report on using similar antigen retrieval protocols on cryosections of the formalin-fixed rat kidney and liver tissues in order to reveal optimal immunostaining conditions for representative proteins localized in the cell membrane (Na/K-ATPase, aquaporin 1, megalin, cell adhesion molecule CAM105), cytoplasm (metallothionein), intracellular organelles (vacuolar H+-ATPase), and cytoskeleton (actin, tubulin). Our immunocytochemical data illustrate that: a) harsh antigen retrieval protocols, previously used with sections of the formalin-fixed paraffin-embedded tissues, can be efficiently used with cryosections of the formalin-fixed tissues, b) for each set of antigen-antibody, it is always necessary to test various antigen revealing protocols in order to determine optimal conditions for immunocytochemical presentation, and c) in most cases the protocols that include microwave heating give the best unmasking effects and staining intensity. Being the strongest unmasking method for different epitopes, microwave heating also generates conditions for significant reduction of the amount of primary antibodies.

Antigen retrieval

Antigenicity

Immunostaining

Metallothionein

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CELLULAR LOCALIZATION AND SEX-RELATED PROTEIN EXPRESSION OF ORGANIC CATION TRANSPORTERS OCT1 AND OCT2 IN MOUSE KIDNEYS AND LIVER

Davorka Breljak Vedran Micek Ivana Vrhovac Dean Karaica Giuliano Ciarimboli

Organic cation transporters (OCT ; family SLC22) mediate (re)absorption of various endogenous and exogenous organic cations. In rodents, Oct1/Slc22a1 and Oct2/Slc22a2 are expressed in major excretory organs including kidneys and liver. Due to absence of specific antibodies and corresponding knockout (KO) mice, cellular localization of these transporters and their sex-related protein expression in this species have not been characterized. Here we used adult wild-type (WT) and double Oct1/Oct2 KO mice of both sexes to study a) localization of Oct1 and Oct2 by immunocytochemistry in cryosections of the liver and kidneys, b) expression of these transporters by Western blotting of isolated total cell membranes (TCM), and c) sex-dependency in localization and expression of both transporters. Using specific antibodies, in WT mice the renal Oct1 and Oct2 were localized in the basolateral membrane (BLM) of proximal tubules (PT) exhibiting sex differences (malesfemales) in the staining intensity. However, Oct1 was detected in the PT S1/S2 segments, whereas Oct2 was expressed in the PT S2/S3 segments. In the liver, Oct1 was stained in the sinusoidal membrane of hepatocytes, whereas Oct2 was undetected. In TCM of WT mice, the 75-kDa Oct1 protein band was labeled in both organs, whereas the 75-kDa Oct2 protein band was labeled exclusively in kidneys. Both transporters were undetected in Oct1/Oct2 KO mice. Collectively, in the mouse kidneys, the Oct1 and Oct2 proteins are located in the BLM of specific PT segments with male-dominant expression, whereas in the mouse liver, only the sex-independent Oct1 expresssion was found in the hepatocyte sinusoidal membrane.

Apical membrane

Epithelial polarity

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EXPRESSION OF SODIUM-D-GLUCOSE COTRANSPORTER 1 IN MURINE TISSUES ; SEX-DEPENDENT EXPRESSION IN KIDNEYS

Ivana Vrhovac Davorka Breljak Dean Karaica Hermann Koepsell Ivan Sabolić

Sodium-D-glucose cotransporter SGLT1 (SLC5A1) in the brush-border membrane (BBM) of small intestinal enterocytes and renal proximal tubule (PT) epithelium is responsible for glucose absorption and reabsorption, respectively. Detailed localization of SGLT1 in other mammalian organs is poorly known. Since mice are frequently used experimental animals in preclinical testing, it is important to determine the expression of mouse Sglt1 (mSglt1) in their organs/tissues. To investigate the mSglt1 protein expression in different organs/tissues by immunocytochemistry (IC) in tissue cryosections and by Western blotting (WB) in isolated cell membranes, we used the adult wild-type (WT) and Sglt1 knock-out (KO) mice of both sexes. Specificity of the polyclonal mSglt1-antibody was confirmed in Sglt1 KO mice ; in WT mice, by WB in the jejunal and renal BBM the antibody labeled a single protein band of 75-kDa, whereas in the Sglt1 KO mice membranes, these protein bands were absent. The IC studies in the kidneys of WT mice revealed localization of the protein in PT BBM, exhibiting segmental (S2>S3), zonal (cortex>outer stripe) and sex (males>females) differences in staining intensity, and in the apical membrane of thick ascending limb of Henle and macula densa. However, contrary to the male-dominant protein expression, the renal Sglt1 mRNA expression, analyzed by qPCR, was female-dominant. The mSglt1-antibody further stained the luminal domain of bile and pancreatic ducts, whereas the spleen, cerebrum, cerebellum, fat and skeletal muscle, remained unstained. The observed distribution of mSglt1 protein in various mouse organs will enable further studies of its role in patho/physiological, pharmacological and toxicological conditions.

Distal convoluted tubule

Renal cortex

Brush border

Macula densa

Immunogold labelling

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Effect of melatonin and resveratrol on the expression of aquaporin 1 and 2 in scenecent male and female rats

Dean Karaica Davorka Breljak Ivana Vrhovac Vedran Micek Marija Ljubojević

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Influence of the grade and invasiveness of bladder cancer on disease course severity in patients with bladder tamponade resulting from a bleeding bladder cancer

Urology Annals (2022)

Dora Jakus Petra Čepin Ivana Vrhovac Ivana Alujević Jakus

Urinary bladder tamponade is a common urological emergency, but it has so far been insufficiently researched. The aim of our study was to show the association between the characteristics of bladder cancer (grade and invasiveness) and disease course severity based on blood hemoglobin (Hgb) count at admission, the need for red blood cell transfusion (RBCT), and the length of hospitalization in patients suffering from bladder tamponade.A retrospective, cross-sectional study was conducted, namely, including 25 adult patients surgically treated for bladder tamponade resulting from a bleeding bladder cancer.Patients with low-grade cancer had statistically significantly higher mean Hgb values at admission (101.14 ± 8.26 vs. 87.22 g/L ± 10.64 g/L, P = 0.005), as well as a lower mean number of received units of RBCT (0.71 ± 0.76 vs. 2.39 ± 1.46, P < 0.001) and a shorter hospitalization (2.43 ± 0.55 vs. 4.36 ± 1.04 days, P = 0.009) than those with high-grade cancer. Patients suffering from nonmuscle-invasive bladder cancer (NMIBC) had statistically significantly higher mean Hgb values at admission (96.69 ± 9.86 g/L vs. 81.22 ± 7.23 g/L, P = 0.001), as well as a lower mean number of received units of RBCT (1.31 ± 1.2 vs. 3 ± 1.41, P = 0.004) and a shorter hospitalization (3.31 ± 1.14 vs. 4.78 ± 0.97 days, P = 0.004) than those with muscle-invasive bladder cancer.Low-grade bladder cancer and NMIBC are associated with a milder clinical course of bladder tamponade.

10.4103/ua.ua_181_21

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Sex-independent expression of chloride/formate exchanger Cfex (Slc26a6) in rat pancreas, small intestine, and liver, and male-dominant expression in kidneys

Archives of Industrial Hygiene and Toxicology (2018)

Dean Karaica Davorka Breljak Jovica Lončar Mila Lovrić Vedran Micek

Abstract Chloride/formate exchanger (CFEX; SLC26A6) mediates oxalate transport in various mammalian organs. Studies in Cfex knockout mice indicated its possible role in development of male-dominant hyperoxaluria and oxalate urolithiasis. Rats provide an important model for studying this pathophysiological condition, but data on Cfex (rCfex) localisation and regulation in their organs are limited. Here we applied the RT-PCR and immunochemical methods to investigate rCfex mRNA and protein expression and regulation by sex hormones in the pancreas, small intestine, liver, and kidneys from intact prepubertal and adult as well as gonadectomised adult rats treated with sex hormones. rCfex cDNA-transfected HEK293 cells were used to confirm the specificity of the commercial anti-CFEX antibody. Various biochemical parameters were measured in 24-h urine collected in metabolic cages. rCfex mRNA and related protein expression varied in all tested organs. Sex-independent expression of the rCfex protein was detected in pancreatic intercalated ducts (apical domain), small intestinal enterocytes (brush-border membrane; duodenum > jejunum > ileum), and hepatocytes (canalicular membrane). In kidneys, the rCfex protein was immunolocalised to the proximal tubule brush-border with segment-specific pattern (S1=S2<S3), and both rCfex mRNA and protein expression exhibited male-dominant sex differences driven by stimulatory effects of androgens after puberty. However, urinary oxalate excretion was unrelated to renal rCfex protein expression. While the effect of male-dominant expression of rCfex in renal proximal tubules on urine oxalate excretion remains unknown, its expression in the hepatocyte canalicular membrane may be a pathway of oxalate elimination via bile.

Brush border

10.2478/aiht-2018-69-3157

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Sex-dependent expression of metallothioneins MT1 and MT2 and concentrations of trace elements in rat liver and kidney tissues: Effect of gonadectomy

Journal of Trace Elements in Medicine and Biology (2019)

Marija Ljubojević Tatjana Orct Vedran Micek Dean Karaica Jasna Jurasović

Metallothionein

10.1016/j.jtemb.2019.02.010

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The impact of age, sex, comorbidities, and use of antithrombotics on the clinical course severity among patients surgically treated for urinary bladder tamponade

Urology Annals (2024)

Dora Jakus Marijan Šitum Petra Čepin Ivana Vrhovac Josip A. Borovac

To examine the relationship between clinical patient characteristics and the severity of the disease course in patients hospitalized due to urinary bladder tamponade. The severity was assessed based on hemoglobin (Hgb) levels upon admission, the requirement for red blood cell transfusion (RBCT), and length of hospital stay.

10.4103/ua.ua_70_23

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