OSTEOPROTEGERIN AS AN EARLY SIGN OF CHRONIC KIDNEY DISEASE-MINERAL AND BONE DISORDER.

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.

Expression of the BMP-2, -4 and -7 and their antagonists gremlin, chordin, noggin and follistatin during ectopic osteogenesis.

Molecular network of the osteogenic BMPs and extracellular inhibitors maintains homeostasis of the skeletal tissues. It is important to determine relationship between BMP-2, -4 and -7 and their inhibitors: gremlin, follistatin, chordin and noggin, during normal osteogenesis. To determine their expression pattern we conducted investigation by inducing ectopic bone formation in rats. The results shown that levels of the BMP-2 and BMP-4 expression in chondrocytes are similar to noggin and follistatin. The latter BMPs and inhibitors have increased levels of the expression at day 14th of the osteogenesis, which suggests their important roles in early phases of the chondrogenesis. Gremlin and chordin have shown increased levels of expression in late phase of chondrogenesis, which suggests their important role in regulation of the osteogenesis initiation. In this study, BMPs and inhibitors have the highest levels of the expression at 21st day in the osteocytes, which suggests their strong interactions in osteogenesis.

BMP signaling in rats with TNBS-induced colitis following BMP7 therapy.

Beyond stimulating bone formation, bone morphogenetic proteins (BMPs) are important in development, inflammation, and malignancy of the gut. We have previously shown that BMP7 has a regenerative, anti-inflammatory, and antiproliferative effect on experimental inflammatory bowel disease (IBD) in rats. To further investigate the BMP signaling pathway we monitored the effect of BMP7 therapy on the BMP signaling components in the rat colon during different stages of experimentally induced colitis by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The results showed a significantly decreased BMP7 expression in the acute phase, followed by a significantly increased BMP2 and decreased BMP6 expression during the chronic phase of colitis. BMP7 therapy influenced the expression of several BMPs with the most prominent effect on downregulation of BMP2 and upregulation of BMP4 in the chronic phase of colitis. Importantly, connective tissue growth factor and noggin expression were elevated in the acute stage and significantly decreased upon BMP7 therapy. BMP receptor I expression was unchanged, whereas BMP receptor II was decreased at day 2 and increased at days 14 and 30 of TNBS inflammation. However, an opposite pattern of expression following BMP7 therapy has been observed. BMP7 increased the expression of BR-Smad including Smad3 and Smad4. Inhibitory Smads were increased in colitis and significantly decreased following BMP7 therapy at later stages of the disease. We suggest that BMP signaling was altered during TNBS-induced colitis and was recovered with BMP7 administration, suggesting that IBD is a reversible process.

The influence of clinical and anthropometric parameters on the serum levels of the endothelin-1 in pregnant women and their newborns.

Pregnancy induced hypertension (PIH) is major contributor to maternal death in developing countries. Endothelin-1 (ET-1) is the most potent vasoconstriction agent known and its serum levels are increased in PIH. Therefore it is important to elucidate maternal and neonatal factors which influence endothelin-1 serum levels. 100 pathological pregnancies and 88 controls were analyzed for blood endothelin-1 and their anthropometric and clinical data were collected. In maternal blood ET-1 levels were strongly predicted by diagnosis, therapy and BMI, while umbilical cord ET-1 levels were strongly predicted by gestational age, therapy and delivery termination. Positive correlation between BMI and ET-1 levels suggest that obese pregnant women have increased risk for cardiovascular diseases. Inverse relationship between Apgar and umbilical ET-1 indicates that ET-1 could be considered as a prognostic marker in cases of neonatal asphyxia.

Hepatoregenerative role of bone morphogenetic protein-9.

Bone morphogenetic protein-9 (BMP-9) is a member of the transforming growth factor beta (TGF-ß) superfamily of cytokines, which regulate cell growth and differentiation during embryogenesis. Apart of that, the hypoglycemic potential of BMP-9 is of great interest. It has been confirmed that BMP-9, like insulin, improves glycemia in diabetic mice and regulates directional glucose metabolism in hepatocytes; therefore it is proposed to be a candidate hepatic insulin-sensitizing substance (HISS). In liver fibrosis, due to the portocaval shunt, insulin bypasses the organ and the liver undergoes atrophy. Parenteral administration of insulin reverses atrophy by stimulating mitogenic activity of the hepatocytes. Because BMP-9 has a signaling pathway similar to other BMPs and insulin, it is to be expected that BMP-9 has a certain regenerative role in the liver, supporting the above-mentioned is evidence of BMP-9 expression in Dissè's spaces and BMP-7's mitogenic activity in mucosal cells. However, further studies are needed to confirm the possible regenerative role of BMP-9.

mRNA expression of bone morphogenetic proteins and their receptors in human renal cell carcinoma.

INTRODUCTION: Bone morphogenetic proteins (BMPs) have been studied in several cancers, but only limited information is available about renal cell carcinomas (RCCs). We determined the expression of mRNA of several BMP ligands and BMP receptors (BMPRs) in healthy kidney tissue and RCCs, and data were compared to clinicopathological parameters. MATERIAL AND METHODS: Sixty-four samples of RCCs and healthy renal tissues were prospectively examined. The expression of BMP2, BMP4, BMP6, BMP7, BMPRIA, BMPRIB and BMPRII mRNA was determined using semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: The expression levels of different BMP ligands and BMPRs were considerably higher in RCCs than in normal kidney tissue. BMP ligands showed elevated expression in clear-cell RCCs, whereas all three BMPRs showed higher expression levels in non-clear-cell RCCs. In clear-cell RCCs, the expression levels of BMP2 progressively increased and expression levels of BMP6, BMP7 and BMPRIB were lost with higher tumor stage. CONCLUSIONS: All BMPs and their receptors have stronger expression levels in RCC. The expression level of BMP2 is strongly elevated in kidney cancer.

Histomorphometric analysis of subchondral bone of the femoral head in osteoarthritis and osteoporosis.

There have been reports both supporting and refuting an inverse relationship between hip fracture and hip osteoarthritis (OA). We have investigated this relationship using histomorphometric study of femoral head subchondral bone. We studied 74 subjects with hip fracture (74% females) and 24 subjects with osteoarthritis (45% females). By histomorphometric analysis of parafined sections, we analysed followed subhondral trabecular bone parameters bone volume (BV), bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th.), trabecular number (Tb.N.) and trabecular separation (Tb.S.). The subjects with osteoarthritis and subjects with hip fracture had BV/TV 31.3% and 19.6% respectively. BV/TV of osteoarthritis group was rather uniform whereas BV/TV of hip fracture group was greatly ranged and we divided it into three subgroups, 13.2%, 19.8% and 25.9% respectively. The OA group and hip fracture groups had Tb.Th. as followed 0.205 mm, 0.148 mm, 0.170 mm and 0.183 mm respectively. The OA group and hip fracture three subgroups had Tb.N. as followed 1.454/mm, 0.897/mm, 1.170/mm and 1.425/mm respectively. The OA group and hip fracture three subgroups had Tb.S. as followed 0.518 mm, 0.681 mm, 0.620 mm and 0.550 mm respectively. The results of our study support an inverse relationship between hip fracture and hip osteoarthritis.

Treatment of osteoporosis with a modified zeolite shows beneficial effects in an osteoporotic rat model and a human clinical trial.

The severity of osteoporosis in humans manifests in its high incidence and by its complications that diminish quality of life. A societal consequence of osteoporosis is the substantial burden that it inflicts upon patients and their families. Several bone-modifying drugs have been prescribed to patients with osteoporosis. However, evidence for their anti-fracture efficacy remains inconclusive. To the contrary, long-term use of anti-osteoporotic drugs such as bisphosphonates and Denosumab, an RANKL inhibitor, have resulted in adverse events. We now present an alternative and adjuvant approach for treatment of osteoporosis. The data derive from in vivo studies in an ovariectomized rat model and from a randomized double blind, placebo-controlled human clinical study. Both studies involved treatment with Panaceo Micro Activation (PMA)-zeolite-clinoptilolite, a defined cation exchange clinoptilolite, which clearly improved all bone histomorphometric parameters examined from ovariectomized animals, indicative for increased bone formation. Moreover, intervention with PMA-zeolite-clinoptilolite for one year proved safe in humans. Furthermore, patients treated with PMA-zeolite-clinoptilolite showed an increase in bone mineral density, an elevated level of markers indicative of bone formation, a significant reduction in pain, and significantly improved quality of life compared with patients in the control (placebo) group. These encouraging positive effects of PMA-zeolite-clinoptilolite on bone integrity and on osteoporosis warrant further evaluation of treatment with PMA-zeolite-clinoptilolite as a new alternative adjuvant therapy for osteoporosis.

How activity of inflammatory bowel disease influences bone loss.

Bone loss is a common problem for individuals with inflammatory bowel disease (IBD). The aim of our study was to assess bone mineral density (BMD) in patients with IBD and to investigate the role of corticosteroid (CS) use and duration and activity of disease on BMD. Ninety-two patients (56 men and 36 women) with IBD, of whom 32 had ulcerative colitis (UC) and 60 had Crohn's disease (CD), underwent clinical assessment. Lumbar and femoral neck BMDs were measured by dual-energy X-ray absorptiometry. Osteopenia was observed in 14 patients (43%) with UC and in 24 patients (40%) with CD (p=0.187). Four patients (12%) with UC and 7 patients (11%) with CD had osteoporosis (p=0.308). Femoral BMD decreased in patients with long duration of CS use and correlated inversely with disease activity. Multiple regression analysis of BMD showed that statistically significant risk factors were duration of active disease and body mass index as well. Based on our results, it is necessary to take into account the risk of decreased BMD in patients with IBD. It is most important to achieve disease remission as soon as possible in addition to nutritional support.

Bone quality assessment in individuals of different age, gender and body constitution.

The concept of bone quality describes the sets of the characteristics of the osseous tissue that influence bone strength. The aim was to explore the influence of anthropometric parameters and age on the parameters of the bone architecture and bone mineral properties in the lumbar vertebral bone of men and women. Vertebral bone samples underwent bone histomorphometry, bone densitometry and atomic absorption spectrometry. Men have greater values of the bone volume and thicker bone trabeculae in relation to women, which indicates that vertebral bone architecture is better preserved in men than in women. Age is the best predictor of changes that affect bone architecture and bone mineral properties. Bone mineral density value and calcium concentration are both negatively predicted by age, but positively predicted by body mass index. Such result supports the opinion that low body mass index is associated with conditions of bone deficit such are osteopenia and osteoporosis.

Histomorphological analysis of the osteophytic appositions in patients with lumbar lateral recess syndrome.

Patients with lumbar lateral recess syndrome (LRS) can be successfully cured by removing osseous excrescences that grow on the peripheral edge of articular surface of the facet joint. They cause narrowing of the lateral recess and compress a root of the spinal nerve. Their appearance is related to the instability of respective dynamic vertebral segment. The aim of this study was to analyze the osteophytic composition morphohistochemically and elucidate cellular processes that lead to this new formation appearance. It is necessary to find a possible causative-consequential relation between the osteophyte and instability. The ideal object to explore was the osteophyte in the lateral recess because it had to be removed during operative treatment. The group of 30 patients with clinical feature of LRS was chosen. Each patient had clinically verified LRS with consequential radiculopathy. Bony outgrowths were removed surgically and analyzed by histological and immunohistochemical methods: toluidine blue, Goldner trichrome, TRAP, indirect peroxidase with antibodies against BMP 3 and BMP 7. The outgrowths that caused lateral recess stenosis were composed of fibrous and hyaline cartilage and cancellous bone. The changes in cartilage and bone, and occurrence of intramembranous bone formation in sense of enlargement of trabeculae, leads to the conclusion that marginal osteophytic formations could be an adaptation to changed conditions in the dynamic vertebral segment and an attempt to stabilize this segment by enlargement of articular surface.

Role of bone morphogenetic proteins in human prostate cancer pathogenesis and development of bone metastases: immunohistochemical study.

Bone morphogenetic proteins (BMP) have the ability to induce ectopic bone formation. The findings of their expression in prostate cancers have been linked with specifically tumor progression to bone and development of osteosclerotic metastases. We investigated the expression pattern of BMP-2/4, -6 and -7 and the receptors BMPR-IA,-IB and -II in normal human prostate, organ-localized and metastatic prostate cancers. The expression we also examined in skeletal metastases caused by prostate cancer. In localized prostate cancers we found increased expression of BMP-6 and decreased expression of BMP-2/4 and -7. In metastatic prostate cancers the expression of examined BMPs decreased. The expression of BMPRs showed the tendency to be lower with progression of prostate cancer but the expression of BMPR-II was completely absent in metastatic prostate cancers. In bone metastases caused by prostate cancer we found high expression of BMP-2/4, -6 and -7. Decreased expression of BMPs and lose of BMPR-II expression, could suggest that the influence of BMPs on prostate cancer cells is inhibited and plays an important role in prostate cancer pathogenesis. High expression of osteogenic BMPs in prostate cancer bone metastases could explain their osteosclerotic properties.

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 hyperparathyroidism. 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). 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.

Expression of bone morphogenetic protein-7, its 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). In the last three decades incidence of RCC was constantly increased and the role of different molecular biomarkers in RCC is explored'. 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.

Bone morphogenetic protein-7 expression in human pyelonephritis.

Bone morphogenetic protein 7 (BMP- 7) is a member of the transforming growth factor (TGF) beta superfamily and is involved in regeneration, repair, and development of specific tissues, for example kidney and skeleton. The experimental studies have shown its protective role against fibrotic processes. Tubulointerstitial changes are present in the pyelonephritic kidney which progresses to fibrosis. Renal fibrosis may lead to the loss of renal function. The aim of this study was to investigate BMP-7 expression in acute and chronic pyelonephritis in humans. Seven patients with acute pyelonephritis and 7 with chronic pyelonephritis were treated in Department of Nephrology Clinical Hospital, Rijeka. Tissue biopsy was taken and renal tissue was studied histopathologically by use of hematoxylin and eosin and scored for diagnosis of pyelonephritis. BMP-7 expression was studied by immunohistochemical staining. BMP-7 expression was observed in the tubular area of the pyelonephritic kidneys. The expression of BMP- 7 was stronger in the acute pyelonephritic group and less in the chronic pyelonephritic group of patients. The results imply that BMP-7 has a role in chronic pyelonephritis. Tubular BMP-7 expression had a negative correlation with fibrosis and tubular, atrophy. Our results are suggesting that BMP- 7 plays an important protective role in renal inflammatory diseases preventing greater damage and fibrosis.

Properties of the celiac trunk--anatomical study.

Although anatomical properties and vessel variations of the celiac trunk are well explored in the literature, there is not so much information on the arterial diameters, and this data is important for surgical procedures and angiographic examinations. The aim of this study was to investigate properties of the celiac trunk in humans by using anatomical dissection. Ninety cadavers were dissected for the celiac trunk identification and arterial diameter measurements. The results of anatomical examination showed that in 72% of all cases the celiac trunk divides into the splenic artery and the common hepatic artery, while the left gastric artery arises as a first branch and had origin between aorta, all over the celiac trunk up to a bifurcation. From the 90 cadavers, 4 presented anatomical variations. Where normal anatomy was present, the mean length of the celiac trunk was 1.9 +/- 0.08 cm and its mean arterial diameter was 0.78 +/- 0.08 cm. The splenic artery had the largest diameter (0.61 +/- 0.05 cm) and the left gastric artery had the smallest diameter (0.38 +/- 0.03 cm). Our data represent original results about anatomical variations and arterial diameter of the celiac trunk and its main branches provided by anatomical dissection.

Treatment of tibial bone defect with rotational vascular periosteal graft in rabbits.

It is well known that the periosteum is capable of bone formation. In the present study, the value of the vascularized periosteal graft in healing the long-bone defect filled with the allogenous bone graft was studied. The aim of the study was to verify the efficiency of the rotational vascularized periosteal graft as a optional surgical method as well as to prove its advantages in comparison with the nonvascularized periosteal graft. The study was undertaken on 40 rabbits. Four rabbits served as allogenous bone graft donors, while the remaining 36 were divided into two equal groups. In the control group, the experimentally created bone defect on the junction of the tibial proximal and median thirds was filled with allogenous bone graft and then covered with avascular periosteal graft. In the experimental group, the allogenous bone graft filled defect was covered with the rotational vascular periosteal graft. Groups of 6 rabbits from each group were sacrificed after 2, 5 and 12 weeks following surgery. The results were evaluated with radiographical, histological and morphometric methods. The results obtained 2 and 5 weeks after surgery demonstrated better tibial bone defect healing in the experimental group. The defect was bridged in both analysed groups after 5 weeks and was completed after 12 weeks with no difference between the control and the experimental groups. The obtained results have confirmed the efficiency of the method using the rotational vascularized periosteal graft in the treatment of tibial bone defect in rabbits. The advantage of the vascularized periosteal graft as compared to the avascular one has been proved by better quality of bone healing in the early stage.

Microfracture technique in combination with intraarticular hyaluronic acid injection in articular cartilage defect regeneration in rabbit model.

Although articular hyaline cartilage typically has low potential for regeneration, numerous methods and techniques have been proposed to induce the reparation process. In our work, we used microfracture techniques in combination with intraarticular application of hyaluronic acid in rabbit knee articular cartilage defect. In comparison with the control group, after 6 and 10 weeks we observed a higher potential of healing in the experimental group, with thicker and more organized repair tissue filling the defect. In conclusion, a combination of the microfracture technique and application of hyaluronic acid might be potentially beneficial in inducing articular cartilage defect reparation.

Expression of bone morphogenetic proteins, cartilage-derived morphogenetic proteins and related receptors in normal and osteoarthritic human articular cartilage.

Newborn and adult articular cartilage expresses bone (BMPs) and cartilage derived morphogenetic proteins (CDMPs). These morphogenetic proteins act over membrane receptors (BMPRs). We examined the expression pattern of BMP-7, BMP-3, CDMP-1, CDMP-2 and their receptors in adult normal and osteoarthritic, articular, knee cartilage. Immunostaining was carried out using polyclonal antibodies. The expression of BMP-7,-3, CDMP-1,-2 was detected in all layers of normal articular cartilage with the strongest expression in chondrocytes of the transitional layer. BMP-7 and CDMPs expression decreased in osteoarthritic articular cartilage whereas BMP-3 expression was absent. BMPR-IA and BMPR-II were strongly expressed in both normal and osteoarthritic articular cartilage. BMPR-IB was not expressed in osteoarthritic (OA) cartilage. BMPs and CDMPs with intact signalling play an important role in articular cartilage homeostasis, preventing cartilage degeneration.

Anthropometric parameters as predictors for iliopsoas muscle strength in healthy girls and in girls with adolescent idiopathic scoliosis.

In this study iliopsoas muscle strength was measured by portable dynamometer and it was explored to what extent independent predictors (age, body weight, body height and body mass index) affect iliopsoas strength in healthy subjects and in subjects with adolescent idiopathic scoliosis. The study population was consisted of 183 girls (90 healthy girls and 93 girls with adolescent idiopathic scoliosis). Student t test analysis showed no differences in maximal voluntary isometric contraction between healthy girls and girls with scoliosis. Independent variables predicted significantly iliopsoas strength in healthy group (r=0.96, p<0.01) and in scoliosis group (r=0.94, p<0.001). Separate analysis with respect to types of scoliosis demonstrated that independent variables significantly predict iliopsoas strength in right thoracic (r=0.97, p<0.01), left thoracic (r=0.98, p=0.004), right thoracic lumbar (r=0.97, p<0.01) and left lumbar (r=0.96, p<0.01) scoliosis subgroups. In healthy girls iliopsoas strength was mostly predicted by body weight, followed by body height and body mass index. In girls with scoliosis body weigth was the strongest predictor of iliopsoas strength and was followed by curvature angle degree.