Expression of Bone Morphogenetic Protein-7, It´s Receptors and Smad1/5/8 in Normal Human Kidney and Renal Cell Cancer

Bone morphogenetic proteins (BMPs) are cytokines which are important for kidney homeostasis but also have role in the some renal diseases and renal cell carcinoma (RCC)1–5. In the last three decades incidence of RCC was constantly increased and the role of different molecular biomarkers in RCC is explored1. We analyzed expression of BMP-7, their receptors (BMPR-IA, BMPR-IB, BMPR-II) and proteins of their signaling pathway (pSmad1/5/8) in sixteen renal cancer samples and paired normal tissue. Tissue samples were analyzed by immunohistochemistry and Western blot. BMP-7, BMP receptors and pSmad1/5/8 were expressed in all structures of normal kidney but dominantly in the proximal tubular cells. In the cancer samples their expression was also noticed. Comparison of BMPs between different tissue showed increased expression of BMPR-IB and pSmad 1/5/8 and decreased expression of BMP-7 and BMPR-II in RCC compared to normal kidney. BMPR-IA was detected with immunohistochemistry but with Western blot attenuated signal was presented. BMP-7, BMP receptors and pSmad1/5/8 were showed in normal kidney and RCC. Detected alterations of BMP-7, BMP receptors and pSmad expression in RCC suggested their possible role in tumorigenesis of kidney cancer

Increased Bone Turnover Markers after Renal Transplantation

Bone remodeling is a process that occurs continuously in a seemingly inactive tissue like bone. Because of decreased vitamin D synthesis, phosphorus retention and decreased calcium blood concentration, patients with chronic renal failure (CRF) develop secondary hyperparathyroidism1–5. Elevated PTH levels shifts balance between osteoblast and osteoclast activity in favor of osteoclast activity and, therefore, bone resorption. Bone metabolic disorder that affects patients with CRF is called renal osteodystrophy (ROD)1–5. We presume that renal transplantation reverses bone metabolism disorder and our goal was to establish whether osteoblast and osteoclast activity returns to the levels of healthy individuals

Osteoprotegerin kao rani predskazatelj mineralno-koštanog poremećaja u bolesnika s kroničnom bubrežnom bolesti

Chronic kidney disease (CKD) is among the most significant health problems, with the associated cardiovascular disease and bone metabolism disorders being the leading cause of morbidity and mortality in these patients. The aim of the study was to determine markers of bone turnover in patient sera (phosphates, calcium, alkaline phosphatase, parathyroid hormone and osteoprotegerin (OPG)) in all stages of kidney failure including kidney transplant recipients. We also wanted to determine whether dialysis vintage affects recovery of bone markers one year after transplantation. There were 164 study patients, whereas 30 healthy individuals served as a control group. Serum OPG progressively increased with decline of the glomerular filtration rate. The highest OPG concentration was recorded in dialysis group. We observed a statistically significant OPG increase in stage 2 CKD. In kidney transplant group, there was positive correlation between OPG and dialysis vintage. We also found that serum OPG was lower in patients treated with dialysis for less than 4 years prior to transplantation. We confirmed that CKD-mineral and bone disorder began in stage 3 CKD with parathyroid hormone and OPG elevation, and a statistically significant OPG increase in stage 2 CKD might be an early sign of CKD-mineral and bone disorder. Dialysis vintage longer than 4 years is associated with more significant disturbances in mineral and bone metabolism.Kronična bubrežna bolest (KBB) važan je javnozdravstveni problem pri čemu su kardiovaskularne komplikacije i poremećaj mineralno-koštanog metabolizma vodeći uzroci pobola i smrtnosti ovih bolesnika. U ovom istraživanju mjerene su koncentracije biljega koštane pregradnje (kalcij, fosfati, alkalna fosfataza, paratireoidni hormon i osteoprotegerin (OPG)) u različitim stadijima kronične bubrežne bolesti uključujući i bolesnike nakon transplantacije bubrega. Temeljem tih rezultata namjera je bila odrediti u kojem je stadiju KBB potrebno započeti liječenje poremećaja mineralno-koštanog metabolizma. Također smo željeli utvrditi utječe li vrijeme provedeno na dijalizi na oporavak mineralno-koštanog metabolizma nakon učinjene transplantacije bubrega. U istraživanje je bilo uključeno 164 bolesnika te 30 zdravih ispitanika u kontrolnoj skupini. Utvrdili smo kako je koncentracija OPG-a obrnuto proporcionalna glomerularnoj filtraciji. Najviše koncentracije OPG-a utvrđene su u dijaliznoj skupini bolesnika, a statistički značajan porast koncentracije OPG-a utvrđen je već u drugom stadiju KBB. Također je utvrđeno da koncentracija OPG-a pozitivno korelira s vremenom trajanja hemodijalize. Ovim istraživanjem potvrdili smo da poremećaj mineralno-koštanog metabolizma počinje u trećem stadiju KBB. Statistički značajan porast koncentracije OPG-a u drugom stadiju KBB mogao bi biti rani znak poremećaja mineralno-koštanog metabolizma. Trajanje dijalize duže od četiri godine povezano je sa značajnijim poremećajem mineralno-koštanog metabolizma

The basics of bone biology

Koštano tkivo je najzastupljenije potporno tkivo u organizmu čovjeka. Ono izgrađuje koštani sustav čije su funkcije višestruke (potporni uređaj organizma, određuje veličinu i oblik tijela, zaštita organa, pokretanje u prostoru). Koštano tkivo razvija se iz mezenhima složenim procesom osteogeneze. Potonja je rezultat istovremenih procesa izgradnje i razgradnje koštanog tkiva. Izgradnja i razgradnja koštanog tkiva su, promatrajući njihovu svrhu u cjelini, sinergistički i fiziološki uravnoteženi procesi jer oba djeluju u cilju stvaranja i održavanja homeostaze koštanog sustava. Otklon od fiziološke ravnoteže ovih dvaju procesa očituje se u patološkoj osteogenezi pri kojoj je homeostaza u koštanom tkivu narušena, bez obzira na uzrok (osteoartroza, osteoporoza). Mnoga suvremena istraživanja usmjerena su na proučavanje ovih procesa, a njihovi rezultati omogućili su razumijevanje patofizioloških procesa koji zahvaćaju koštano tkivo. Rezultati istraživanja procesa osteoindukcije (koštani morfogenetski proteinii njihovi inhibitori) i osteokondukcije imaju uspješnu primjenu u koštanoj kirurgiji.Bone tissue is the most abundant supporting tissue in the human body. It builds the skeletal system, which performs several functions: it provides the framework of the body, determines the size and shape of the body, protects the organs, and enables movements. Bone tissue develops from the mesenchyme by a complex process of osteogenesis. Osteogenesis results from continuous processes of bone formation and resorption. Bone formation and resorption are, considering their purpose as whole, synergistically and physiologically balanced processes since they both act to create and maintain the skeletal system homeostasis. Physiological imbalance of these processes is evident as pathological osteogenesis when bone tissue homeostasis is disturbed, regardless of the cause (osteoarthrosis, osteoporosis). Many recent investigations have focused on the study of these processes and their results provided understanding of pathophysiological processes which affect bone tissue. The results of investigating osteoinduction (bone morphogenetic proteins and their inhibitors) and osteoconduction have found successful application in bone surgery

Contribution to the knowledge of comparative anatomy of the circulatory system of vertebrates

Građa i oblik cirkulacijskog sustava među životinjskim vrstama bitno se razlikuju, napose u pojedinim poredbeno anatomskim aspektima, čak do neprepoznatljivost. Budući da cirkulacijski sustav osigurava funkcionalnu i morfološku povezanost svih dijelova organizma, njegova konstrukcija bitno ovisi o obliku organizma i njegovim biološkim osobitostima. U ovome radu, u obliku diskusije, izneseni su neki komparativno-anatomski aspekti temeljnih načela cirkulacijskog sustava u kralježnjaka.Structure and shape of the circulatory system differ substantially among animal species, and particularly in some aspects of comparative anatomy they are unrecognizable. Since the circulatory system ensures the functional and morphological integrity of all parts of the organism, its structure depends essentially on the shape of the organism and its biological properties. This paper, in the form of a discussion, presents some aspects of comparative anatomy related to the basic principles of the vertebrate circulatory system

Contribution to the knowledge of comparative anatomy of the circulatory system of vertebrates

Građa i oblik cirkulacijskog sustava među životinjskim vrstama bitno se razlikuju, napose u pojedinim poredbeno anatomskim aspektima, čak do neprepoznatljivost. Budući da cirkulacijski sustav osigurava funkcionalnu i morfološku povezanost svih dijelova organizma, njegova konstrukcija bitno ovisi o obliku organizma i njegovim biološkim osobitostima. U ovome radu, u obliku diskusije, izneseni su neki komparativno-anatomski aspekti temeljnih načela cirkulacijskog sustava u kralježnjaka.Structure and shape of the circulatory system differ substantially among animal species, and particularly in some aspects of comparative anatomy they are unrecognizable. Since the circulatory system ensures the functional and morphological integrity of all parts of the organism, its structure depends essentially on the shape of the organism and its biological properties. This paper, in the form of a discussion, presents some aspects of comparative anatomy related to the basic principles of the vertebrate circulatory system

Uloga koštanog morfogenetskog proteina-7 u prevenciji oštećenja tkiva bubrega izazvanog hladnom ishemijom : doktorski rad

CILJ ISTRAŽIVANJA. Ispitati da li je perfuzijom bubrega s rhBMP-7 otopinom bolje očuvana struktura tkiva tijekom hladne ishemije u trajanju do 24 sata u odnosu na komercijalno korištenu UW otopinu. Ispitati mehanizam djelovanja rhBMP-7 na epitelne stanice kanalica bubrega i njegovu učinkovitost ovisno o dužini trajanja hladne ishemije. MATERIJALI I METODE. Istraživanje je provedeno na eksperimentalnom modelu hladne ishemije bubrega u štakora. Bubrezi su perfundirani sa slijedecim otopinama: fiziološkom, UW, rhBMP-7 i rhBMP-7+UW. Bubrezi su izloženi hladnoj ishemiji kroz 6, 12 i 24 sata. Učinjena su spektrofotometrijska mjerenja razine oštećenja lipida i proteina te razine antioksidativnih enzima. Western blotom izmjerene su razine ekspresije Hsp70, HIF-1alfa i aktivirane kaspaze 3. Metodom PCR-a analizirana je ekspresija mRNA BMP-7, TGF-β1, Smad1, Smad2, Smad3, Smad5 i Smad8. Imunohistokemijskom metodom prikazana je ekspresija i lokalizacija BMP-7, TGF-β1, E-cadherin, α-SMA i PCNA. REZULTATI. U epitelnim stanicama kanalica bubrega perfundiranih s rhBMP-7 i rhBMP-7+UW otopinom izražena je ekspresija BMP-7 i E-cadherin i nakon 24 sata hladne ishemije. U bubrezima koji nisu perfundirani s rhBMP-7 nalazi se pojačana ekspresija TGF- β1 i α-SMA. Također u tkivu bubrega koji je ispran s rhBMP-7 otopinom povećan je nivo ekspresije mRNA BMP-7. U istom tkivu dokazan je viši nivo ekspresije mRNA Smad1, Smad5 i Smad8, molekula unutarstaničnog signalnog puta za BMP-7. Nivo ekspresije mRNA BMP-7, Smad1, Smad5 i Smad8 podjednako je prisutan kroz cijelo vrijeme trajanja hladne ishemije. Nadalje, u bubrezima perfundiranim s rhBMP-7 otopinom nakon 6 i 12 sati nema promjene razine produkata lipidne peroksidacije i sadržaja karboniliranih proteina u odnosu na bubreg s održanom funkcijom. Razina produkata lipidne peroksidacije i sadržaja karboniliranih proteina je povišena u bubrezima tretiranim s UW otopinom, dok se razina istih povećava u bubrezima tretiranim rhBMP-7 otopinom tek nakon 24 sata hladne ishemije. Ispiranjem tkiva bubrega s rhBMP-7 i rhBMP-7+UW otopinom razina ekspresije Hsp70 i HIF-1alfa viša je u odnosu na bubrege perfundirane samo s UW otopinom. Takoder, u skupini bubrega perfundiranih s rhBMP-7 razina ekspresije aktivirane kaspaze 3 je niža u odnosu na bubrege perfundirane UW otopinom. ZAKLJUČAK. BMP-7 održava strukturu tkiva bubrega bolje nego UW otopina tijekom 24 sata hladne ishemije. BMP-7 sprječava epitelno-mezenhimalnu transformaciu čime održava epitelni fenotip stanica kanalica, smanjuje apoptozu stanica, potiče ekspresiju protektivnih činitelja i povećava antioksidacijski kapacitet tkiva.AIM. To analyze whether the perfusion of kidneys with rhBMP-7 solution can better preserve tissue structure during cold ischemia up to 24 hours compared to commercially used UW solution. To analyze mechanisms of BMP-7 activity in tubular epithelial cells and its proficiency considering the time of cold ischemia. MATERIAL AND METHODS. The study was conducted on experimental model of cold ischemia in the rat kidneys. Kidneys were perfused with solutions as follows: saline, UW, rhBMP-7 and rhBMP-7 + UW. Kidneys were exposed to cold ischemia during 6, 12 and 24 hours. Spectrophotometric measurements of lipid peroxidation products and protein carbonyl content and levels of antioxidant enzymes activity were performed. Using Western blot were measured levels of expression of Hsp70, HIF-1alpha and activated caspase 3. With PCR method was analyzed the expression of mRNA BMP-7, TGF-β1, Smad1, Smad2, Smad3, Smad5 and Smad8. Immunohistochemical analysis was used to show expression and localization of BMP-7, TGF-β1, E-cadherin, α-SMA and PCNA. RESULTS. In tubular epithelial cells of the kidneys perfused with rhBMP-7 and rhBMP-7+UW solution the expression of BMP-7 and E-cadherin was observed still after 24 hours of cold ischemia. In the kidneys not perfused with rhBMP-7 high expression of TGF-β1 and α-SMA was found. Also, in the kidneys perfused with rhBMP-7 solution level of mRNA BMP-7 expression was increased. In the same tissue higher level of mRNA Smad1, Smad5 and Smad8 expression, molecules of intracellular BMP-7 signal pathway, was proved. The levels of mRNA BMP-7, Smad1, Smad5 and Smad8 expression were equally present during whole time of cold ischemia. Furthermore, in the kidneys perfused with rhBMP-7 solution there was no changes of levels of lipid peroxidation products and protein carbonyl content after 6 and 12 hours compared to kidney with sustainable function. The levels of lipid peroxidation products and preotein carbonyl content were higher in the kidneys treated with UW solution, while in the kidneys treated with rhBMP-7 solution their levels increased only after 24 hours of cold ischemia. After reperfusion with rhBMP-7 and rhBMP-7+UW solutions the levels of Hsp70 and HIF-1alfa expression were higher in relation to the kidneys perfused only with UW solution. Also, in the group of the kidneys perfused with the BMP-7 solution the level of activated caspase 3 expression was lower in relation with the kidneys perfused with UW solution. CONCLUSION. BMP-7 maintains the morphology of the kidney tissue better than UW solution during 24 hours of cold ischemia. BMP-7 prevents epithelial to mesenchymal transformation and consequently maintains epithelial phenotype of tubular cells, reduces cell apoptosis, stimulates the expression of protective factors and increases the antioxidant capacity of the tissue

Serum expression of OPG/RANKL system in chronic kidney disease and transplanted kidney recipients: relation to IPTH serum levels and vitamin D therapy

Cilj: Ciljevi ove studije su istražiti ima li omjer serumskih vrijednosti RANKL/OPG bolje prognostičke vrijednosti uspoređen s razinom iPTH-a u serumu te ispitati ovise li razine OPG-a i RANKL-a u serumu o terapiji vitaminom D. Ispitanici i metode: Istraživanje je obuhvatilo 120 pacijenata iz Kliničkog bolničkog centra Rijeka, Hrvatska. Pacijenti su podijeljeni u dvije skupine; skupina pacijenata na hemodijalizi (HD) i skupina primatelja bubrežnog transplantata. Primatelji transplantata bubrega uključeni su u trenutku transplantacije te praćeni 12 mjeseci nakon transplantacije. Rezultati: Prema rezultatima ovog istraživanja, serumske razine RANKL/OPG u skupini pacijenata na hemodijalizi koji su imali iPTH > 33 pmol/l i iPTH 33 pmol/l i iPTH 33 pmol/l and iPTH33 pmol/l and iPTH<11 pmol/l were signifacantly higher compared to those who had iPTH11-33 pmol/l. Patients on hemodialysis and transplanted kidney recipients on vitamin D therapy had significantly higher serum levels of OPG and significantly lower sRANKL levels. Conclusion: Results suggest the beneficial effect of vitamin D on bone mass loss prevention in patients on hemodialysis and in kidney transplant recipients. The RANKL/OPG serum level ratio correlates with iPTH values, but it remains unclear why the RANKL/OPG ratio is low in moderately elevated iPTH values and further research is needed in order to get the answer to this question

Effects of cold ischemia during kidney transplantation: pathophysiology of damage and cold storage preservation solutions

Ishemijsko-reperfuzijska ozljeda jedan je od najvažnijih uzroka oslabljene funkcije transplantiranog bubrega. Dosad su identificirani mnogi čimbenici koji mogu povećati vjerojatnost njezina nastanka, a jedan od najvažnijih je način pohrane bubrega, odnosno duljina trajanja hladne ishemije. Osnovni uzrok oštećenja tkiva u hladnoj ishemiji je nedostatak kisika, koji dovodi do smanjenja koncentracije adenozin trifosfata (engl. adenosine triphosphate, ATP-a) te slabljenja aktivnosti Na/K-ATP-aze (Na/K crpke). Puno kompleksniji i teži oblik oštećenja tkiva nastaje ponovnom uspostavom cirkulacije, odnosno reperfuzijom, prilikom koje dolazi do pojačanog ulaska kisika u stanice te stvaranja velike količine slobodnih kisikovih radikala. Njihovo nakupljanje unutar tkiva uzrokuje oštećenja DNA, proteina i lipida stanične membrane. Ljudski organizam djelomično je zaštićen od djelovanja slobodnih kisikovih radikala pomoću vlastitih antioksidativnih enzimatskih sustava, poput superoksid dismutaze (SOD), glutation peroksidaze (GSH-Px) te katalaze. Kako bi se spriječio nastanak navedenih oštećenja, u kliničkoj se praksi za vrijeme trajanja ishemije bubrega koriste različite otopine za njegovo čuvanje.Ischemic-reperfusion injury is important cause of transplanted kidney decreased function. So far many risk factors have been identified, which may increase the probability of its occurrence, the most important one is duration of the cold ischemia. The main cause of tissue damage in cold ischemia is lack of oxygen, which leads to a decreased concentration of adenosine triphosphate (ATP) and decrease in Na/K-ATPase activity. More complex and severe form of tissue damage is caused by re-establishment of circulation, known as reperfusion, which leads to increased oxygen level in the cells and creation of reactive oxygen species (ROS). ROS cause damage of the DNA, proteins, and lipids of cell membrane. Human body is partially protected from effects of ROS by its own antioxidant enzymatic systems, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase. In order to prevent occurrence of these impairments, different organ preservation solutions can be used during cold ischemia period