Sabe-se que a ativação do Sistema Nervoso Simpático (SNS) induz osteopenia via adrenoceptores b2 (b2-AR). Para investigar se o hormônio tireoideano (HT) interage com o SNS para regular a massa óssea, estudamos o efeito do HT em associação com isoproterenol ou propranolol (agonista e antagonista b-adrenérgicos) e avaliamos o efeito do HT em camundongos com elevado tônus simpático, devido à dupla inativação gênica do a2A-AR e a2C-AR (a2A/a2C-AR-/-), autorreceptores que inibem a liberação de noradrenalina. Vimos que esses animais apresentam um fenótipo de alta massa óssea, apesar do elevado tônus simpático e de intacta sinalização b2-adrenérgica, sugerindo que o a2A-AR e/ou a2C-AR, além do b2-AR, possam mediar ações do SNS no osso. O propranolol limitou e o isoproterenol acentuou os efeitos deletérios do HT no esqueleto, já os animais a2A/a2C-AR-/- apresentaram resistência à osteopenia induzida pela tireotoxicose, o que sugere que há interação entre SNS e o HT para regular a massa óssea, e que esta depende tanto do b2-AR como do a2A- e/ou a2C-AR. / It is known that the sympathetic nervous system (SNS) activation induces ostepenia, via b2-adrenoceptors (b2AR). To investigate if thyroid hormone (TH) interacts with the SNS to regulate bone mass, we studied the effect of TH in association with isoproterenol or propranolol (b-adrenergic agonist and antagonist) and evaluated the effect of TH in mice with a chronic elevated sympathetic tone, due to double disruption of a2A-AR and a2C-AR (a2a/a2c-AR-/-), autoreceptors that inhibit noradrenalin release. We showed that KO mice present a high bone mass phenotype in spite of an elevated sympathetic tone and of intact b2-adrenergic signaling, which suggests that a2A- and/or a2C-AR, besides b2-AR, may also mediate the SNS actions in the bone. Propranolol limited and isoproterenol accentuated the deleterious effects of TH in the skeleton, while a2A/a2C-AR-/- mice presented resistance to the T3-induced osteopenia, which suggest that there is an interaction between the SNS and TH to regulate bone mass, and that it is dependent on b2-AR and a2A-AR and/or a2C-AR signaling.

<p>Intra-articular glucocorticoid injection therapy is frequently used to relieve symptoms of arthritis, but there is considerable variation in injection routines among physicians. One issue of debate concerns the importance of synovial fluid aspiration during the injection procedure. In the present randomised controlled study of patients with rheumatoid arthritis (RA), a significantly reduced risk for arthritis relapse was observed when arthrocentesis was included in the intra-articular injection procedure of the knee. </p><p>Furthermore, there is no consensus about the post-injection regimes. Previous studies have shown beneficial effects of post-injection rest of the knee, but also injection routines for other joints often include such recommendations. The present randomised controlled trial showed that 48-hour rest in elastic orthosis after intra-articular injection in the wrist did not improve the outcome. Thus, the effect of post-injection rest varies between different joints. </p><p>The improved treatment result of post-injection rest of the knee is supposed to be caused by retarded steroid resorption from the joint. In order examine the metabolic effects in cartilage, bone and the hypothalamic-piuitary-adrenal (HPA)-axis, resting and mobile RA patients were studied after intra-articular knee injections. Serum levels of the injected glucocorticoid, triamcinolone hexacetonide (THA), were analysed, as well as cartilage oligomeric matrix protein (COMP) as a marker of cartilage turnover, osteocalcin for bone formation and deoxypyridinoline for bone resorption. The HPA-axis was assessed using serum levels of cortisol and adrenocorticotropine hormone. The result showed a short term and reversible suppression of the HPA-axis and bone formation, whereas bone resorption was unaffected. No differences between mobile and resting patients were observed. In both groups reduction of COMP levels were seen, but these were significantly more pronounced in resting patients, suggesting a cartilage-protective effect. The THA levels increased similarly in both groups, indicating that rest did not affect glucocorticoid resorption. </p><p>Consequently, another explanation for the beneficial effects of postinjection rest of knee synovitis should be considered. In the present material the incidence of infectious complications of intra-articular treatment was less than 1/12,000 injections. </p><p>The findings in this thesis can be applied in the clinical practice and should be considered when new guidelines for intra-articular glucocorticoid therapy are created.</p>

Intra-articular glucocorticoid injection therapy is frequently used to relieve symptoms of arthritis, but there is considerable variation in injection routines among physicians. One issue of debate concerns the importance of synovial fluid aspiration during the injection procedure. In the present randomised controlled study of patients with rheumatoid arthritis (RA), a significantly reduced risk for arthritis relapse was observed when arthrocentesis was included in the intra-articular injection procedure of the knee. Furthermore, there is no consensus about the post-injection regimes. Previous studies have shown beneficial effects of post-injection rest of the knee, but also injection routines for other joints often include such recommendations. The present randomised controlled trial showed that 48-hour rest in elastic orthosis after intra-articular injection in the wrist did not improve the outcome. Thus, the effect of post-injection rest varies between different joints. The improved treatment result of post-injection rest of the knee is supposed to be caused by retarded steroid resorption from the joint. In order examine the metabolic effects in cartilage, bone and the hypothalamic-piuitary-adrenal (HPA)-axis, resting and mobile RA patients were studied after intra-articular knee injections. Serum levels of the injected glucocorticoid, triamcinolone hexacetonide (THA), were analysed, as well as cartilage oligomeric matrix protein (COMP) as a marker of cartilage turnover, osteocalcin for bone formation and deoxypyridinoline for bone resorption. The HPA-axis was assessed using serum levels of cortisol and adrenocorticotropine hormone. The result showed a short term and reversible suppression of the HPA-axis and bone formation, whereas bone resorption was unaffected. No differences between mobile and resting patients were observed. In both groups reduction of COMP levels were seen, but these were significantly more pronounced in resting patients, suggesting a cartilage-protective effect. The THA levels increased similarly in both groups, indicating that rest did not affect glucocorticoid resorption. Consequently, another explanation for the beneficial effects of postinjection rest of knee synovitis should be considered. In the present material the incidence of infectious complications of intra-articular treatment was less than 1/12,000 injections. The findings in this thesis can be applied in the clinical practice and should be considered when new guidelines for intra-articular glucocorticoid therapy are created.

Das RANKL/RANK/OPG-System spielt eine entscheidende Rolle in der Steuerung der Osteoklastendifferenzierung und -aktivierung durch Osteoblasten/ Knochenmarkbindegewebszellen im Rahmen des Knochenremodelings. Osteoblasten/Knochenmarkbindegewebszellen exprimieren RANKL. Dieses hat im Körper zwei Rezeptoren: RANK und OPG. RANKL kann durch Bindung an RANK auf Osteoklasten/Osteoklastenvorläuferzellen in Gegenwart von M-CSF seine osteoklastenstimulierende Wirkung entfalten. Der ebenfalls von Osteoblasten gebildete „decoy“-Rezeptor OPG blockiert als freies Protein durch Bindung an RANKL dessen Interaktion mit RANK und verhindert somit die Osteoklastogenese und Osteoklastenaktivierung. Das RANKL/RANK/OPG-System erfüllt im Körper noch weitere Funktionen im Immunsystem, in der Organentwicklung lymphatischer Gewebe und in der Entwicklung der laktierenden Brustdrüse. Viele Zytokine greifen hemmend oder aktivierend in die Osteoklastogenese ein. Sie können dies zum einen durch die Beeinflussung des RANKL/OPG-Verhältnisses, zum anderen durch direkte Interaktion mit Osteoklasten/Osteoklastenvorläuferzellen tun. Zytokine, die die Osteoklastogenese begünstigen, werden vor allem bei inflammatorischen Prozessen ausgeschüttet. Zusammen mit dem, bei diesen Zuständen von aktivierten T-Zellen produzierten RANKL kann dies längerfristig zu einem Knochenverlust führen, welcher sich im klinischen Bild der Osteoporose äußert. Aus den in der vorliegenden Dissertation durchgeführten Untersuchungen ergeben sich folgende Schlussfolgerungen: 1. Monozyten der Zelllinie RAW 264.7 lassen sich, wie bereits in der Literatur beschrieben, durch Zugabe von M-CSF und RANKL zu osteoklastenähnlichen Zellen differenzieren. 2. Die Osteoklastogenese lässt sich anhand der Veränderung verschiedener osteoklastenspezifischer Parameter charakterisieren. Es zeigt sich bei den mit M-CSF und RANKL stimulierten Monozyten eine erhöhte Transkription von CTR (Calcitoninrezeptor)- und TRAP (tartratresistente saure Phosphatase)-mRNA, eine erhöhte Expression des CTR-Proteins, eine erhöhte TRAP-Aktivität und eine Formierung TRAP-positiver mehrkerniger Riesenzellen, die in diesen Eigenschaften Osteoklasten entsprechen. Die zusätzliche Zugabe von TGF-b1 in Kombination mit M-CSF und RANKL resultiert in einer verstärkten Expression von CTR-mRNA und CTR-Protein. TRAP-mRNA-Expression und TRAP-Aktivität bleiben davon unbeeinflusst. 3. Als funktionelles Merkmal der in vitro differenzierten Osteoklasten können ihre Fähigkeit zur Ausbildung von Aktinringen und die Resorption von mineralisiertem Kollagen nachgewiesen werden. 4. Im Verlauf ihrer Differenzierung sekretieren Osteoblasten unterschiedliche Mengen an OPG. Das Maximum der Synthese liegt bei Tag 11. Freies RANKL lässt sich in Überständen von MC3T3-E1-Osteoblasten nicht nachweisen. 5. Das von Osteoblasten in das Medium abgegebene OPG ist in der Lage, die durch RANKL induzierte Osteoklastogenese von RAW-Monozyten zu hemmen. 6. In Kokulturen von MC3T3-E1-Osteoblasten und RAW-Monozyten kann keine Osteoklastogenese beobachtet werden, wahrscheinlich durch Fehlen der RANKLExprimierung oder zu starke OPG-Sekretion durch Osteoblasten. Besonders in der westlichen Welt mit ihrer hohen Lebenserwartung haben Krankheiten mit Knochenverlust sowie bösartige Neubildungen mit Knochenbefall eine große medizinische Bedeutung. Die Beeinflussung des RANKL/RANK/OPG-Systems bietet eine vielversprechende Möglichkeit zur Entwicklung hochwirksamer und nebenwirkungsarmer Medikamente zur Behandlung dieser Zustände.

Made available in DSpace on 2014-12-17T14:16:33Z (GMT). No. of bitstreams: 1 MelinaBL_DISSERT.pdf: 4468439 bytes, checksum: 05a71c9a22430196d211e6d7c8feea61 (MD5) Previous issue date: 2008-09-22 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Diabetes Mellitus (DM) and osteoposes are chronic diseases with great socioeconomic consequences, mainly due to the late complications and consequent disabilities. The potential effects of DM on bone metabolism remain a very conroversial issue, and disagreement exists with regard to the clinical implications of diabetic osteopenia and the mechanism of its ocurrence. The issue is further complicated by the contribuicion of the especific factors, such as duration of disease an dthe degree of metabolic control. The objective of this study is to identify the osteopathy in children and adolescents with DM 1 assisted in the hospital of pediatrics, UFRN, through biochemical markers of bone and mineral metabolism and the extent of bone mineral density. The study was composed by 74 diabetics type 1 patients (DM1) of both gender and aged 6 to 20 yars. Normoglic?mic group was composed by 97 healthy subjects of both genders, which showed the same age range of DM1, in addition to same socioeconomic class. These individuals qere students from the networks of public education in the city of Natal-RN, randomly invited to paticipate in our study. Both groups DM1 and NG were divided intofour subgroups, according to the classification of tanner , T1, T2, T3, T4 for achieving a benchmark. Diabetic individuals showed up with a poor glycemic control. the group DN1 T4 showed an incresead value for total protein, albumin, urea and microalbumiuria are predictors of grumelura injury in DM1 patients . The total alkaline phosphatase activitywas kept on high levels for both groups because they are in a stature development age. For osteocalcin there were decreased levels for groups Dm1 T1, T2, and T3 when compared to their NG (s), suggesting that this decrease could be associated with reduction in the number and/or differentiation os osteoblasts thereby contributing to reducing bone formation. There were no changes in the activity of TRAP. The serum concentrations of total and ionized calcium, phosphorus and magnesium were included within the RV. It was observed that the BMD (Z- SCORE ) has always been within the RV for both groups, despite to DM1 T4. Taking all together, our results support the hypothesis that children and adolescents with type 1 DM present the risk in the long run to suffer a reduction in the bone mass, associated to poor glicemic control and disease duration. It could limit the bone growth and increase the probality of development of osteopenia, as well as other complications surch as retinopathy and renal failure / Diabetes mellitus (DM) a osteoporose s?o doen?as cr?nicas com grandes consequ?ncias socioecon?micas, sobretudo devido ? complica??es tardias e consequente desabilidades. Os efeitos potenciais do DM no metabolismo ?sseo continua a ser uma quest?o controversa, e ainda n?o existe um consenso no que diz respeito ?s implica??es cl?nicas da osteopenia diab?tica e os mecanismos da sua ocorr?ncia. Entretanto, a contribui??o de fatores espec?ficos, tais como a dura??o da doen?a e o grau de controle metab?lico tem sido muito discutidos. O objetivo do presente estudo foi identificar precocemente a osteopatia diab?tica em crian?as e adolescentes com DM 1 atendidos no Hospital de Pediatria da UFRN atrav?s de marcadores bioqu?micos do metabolismo mineral e ?sseo, marcadores da fun??o renal e da medida da densidade mineral ?ssea (DXA; Z-score L1-L4) . O estudo foi constitu?do por uma amostra de 74 pacientes diab?ticos tipo 1 (DM1) de ambos os sexos, com faixa et?ria entre 6 a 20 anos. O grupo normoglic?mico (NG) foi constitu?do por 97 indiv?duos saud?veis, de ambos os sexos, os quais apresentaram a mesma faixa et?ria do DM1, al?m de compreenderem a mesma classe socioecon?mica. Estes indiv?duos eram alunos de escolas da rede p?blica de ensino da cidade de Natal-RN, convidados aleatoriamente a participar do nosso estudo. Tanto o grupo DM1 quanto o NG foram divididos em quatro subgrupos, de acordo com a Classifica??o de Tanner, T1, T2, T3, T4, para viabilizar uma avalia??o comparativa. Os indiv?duos diab?ticos apresentaram um controle glic?mico insatisfat?rio, com valores de glicemia e HbA1C significativamente superiores aos obtidos para os NG. O grupo DM1 T4 apresentou valores aumentados de prote?nas totais, albumina, ur?ia e microalbumin?ria, sugerindo assim um in?cio de comprometimento renal, visto que os valores elevados de microalbumin?ria s?o preditores de les?o glomerular em pacientes DM1. A atividade da fosfatase alcalina total mostrou-se acima dos VR nos grupos DM1 e NG por estes estarem numa faixa et?ria de desenvolvimento estatural. Observa-se uma diminui??o da concentra??o de osteocalcina para os grupos DM1 T1, T2 e T3 quando comparados aos respectivos NG (s), sugerindo que esta diminui??o estaria associada a diminui??o do n?mero e/ou da diferencia??o dos osteoblastos no seu est?gio final de matura??o, contribuindo consequentemente para a redu??o da forma??o ?ssea. N?o foram observadas altera??es na atividade da TRAP. As concentra??es s?ricas de c?lcio total e ionizado, f?sforo e magn?sio estavam compreendidos dentro dos VR, mas os grupos diab?ticos apresentaram hipozincemia e hiperzinc?ria. A DMO (Z-score L1-L4; DXA) esteve sempre dentro dos VR para os grupos estudados, entretanto os grupos DM1 apresentaram sempre valores abaixo do seu respectivo NG, alca?ando uma diferen?a significativa para DM1 T4. O conjunto de resultados obtidos indicam que o baixo controle glic?mico e o tempo de doen?a representaram fatores de risco importantes para o desenvolvimento precoce da osteopenia diab?tica, bem como para o comprometimento renal e sinais de retinopatia.

Das RANKL/RANK/OPG-System spielt eine entscheidende Rolle in der Steuerung der Osteoklastendifferenzierung und -aktivierung durch Osteoblasten/ Knochenmarkbindegewebszellen im Rahmen des Knochenremodelings. Osteoblasten/Knochenmarkbindegewebszellen exprimieren RANKL. Dieses hat im Körper zwei Rezeptoren: RANK und OPG. RANKL kann durch Bindung an RANK auf Osteoklasten/Osteoklastenvorläuferzellen in Gegenwart von M-CSF seine osteoklastenstimulierende Wirkung entfalten. Der ebenfalls von Osteoblasten gebildete „decoy“-Rezeptor OPG blockiert als freies Protein durch Bindung an RANKL dessen Interaktion mit RANK und verhindert somit die Osteoklastogenese und Osteoklastenaktivierung. Das RANKL/RANK/OPG-System erfüllt im Körper noch weitere Funktionen im Immunsystem, in der Organentwicklung lymphatischer Gewebe und in der Entwicklung der laktierenden Brustdrüse. Viele Zytokine greifen hemmend oder aktivierend in die Osteoklastogenese ein. Sie können dies zum einen durch die Beeinflussung des RANKL/OPG-Verhältnisses, zum anderen durch direkte Interaktion mit Osteoklasten/Osteoklastenvorläuferzellen tun. Zytokine, die die Osteoklastogenese begünstigen, werden vor allem bei inflammatorischen Prozessen ausgeschüttet. Zusammen mit dem, bei diesen Zuständen von aktivierten T-Zellen produzierten RANKL kann dies längerfristig zu einem Knochenverlust führen, welcher sich im klinischen Bild der Osteoporose äußert. Aus den in der vorliegenden Dissertation durchgeführten Untersuchungen ergeben sich folgende Schlussfolgerungen: 1. Monozyten der Zelllinie RAW 264.7 lassen sich, wie bereits in der Literatur beschrieben, durch Zugabe von M-CSF und RANKL zu osteoklastenähnlichen Zellen differenzieren. 2. Die Osteoklastogenese lässt sich anhand der Veränderung verschiedener osteoklastenspezifischer Parameter charakterisieren. Es zeigt sich bei den mit M-CSF und RANKL stimulierten Monozyten eine erhöhte Transkription von CTR (Calcitoninrezeptor)- und TRAP (tartratresistente saure Phosphatase)-mRNA, eine erhöhte Expression des CTR-Proteins, eine erhöhte TRAP-Aktivität und eine Formierung TRAP-positiver mehrkerniger Riesenzellen, die in diesen Eigenschaften Osteoklasten entsprechen. Die zusätzliche Zugabe von TGF-b1 in Kombination mit M-CSF und RANKL resultiert in einer verstärkten Expression von CTR-mRNA und CTR-Protein. TRAP-mRNA-Expression und TRAP-Aktivität bleiben davon unbeeinflusst. 3. Als funktionelles Merkmal der in vitro differenzierten Osteoklasten können ihre Fähigkeit zur Ausbildung von Aktinringen und die Resorption von mineralisiertem Kollagen nachgewiesen werden. 4. Im Verlauf ihrer Differenzierung sekretieren Osteoblasten unterschiedliche Mengen an OPG. Das Maximum der Synthese liegt bei Tag 11. Freies RANKL lässt sich in Überständen von MC3T3-E1-Osteoblasten nicht nachweisen. 5. Das von Osteoblasten in das Medium abgegebene OPG ist in der Lage, die durch RANKL induzierte Osteoklastogenese von RAW-Monozyten zu hemmen. 6. In Kokulturen von MC3T3-E1-Osteoblasten und RAW-Monozyten kann keine Osteoklastogenese beobachtet werden, wahrscheinlich durch Fehlen der RANKLExprimierung oder zu starke OPG-Sekretion durch Osteoblasten. Besonders in der westlichen Welt mit ihrer hohen Lebenserwartung haben Krankheiten mit Knochenverlust sowie bösartige Neubildungen mit Knochenbefall eine große medizinische Bedeutung. Die Beeinflussung des RANKL/RANK/OPG-Systems bietet eine vielversprechende Möglichkeit zur Entwicklung hochwirksamer und nebenwirkungsarmer Medikamente zur Behandlung dieser Zustände.

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Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducers and Activators of Transcription 3 (STAT3), a transcription factor expressed in many cell types, including osteoblasts and osteoclasts, is emerging as a key regulator of bone mass and strength. STAT3 mutations cause a rare human immunodeficiency disease characterized by extremely elevated levels of IgE in serum that have associated craniofacial and skeletal features, such as reduced bone mineral density and recurrent pathological fractures. Our microarray data and immunohistochemical staining using a normal rat model have shown that STAT3 mRNA and protein levels markedly increase in response to mechanical loading. In addition, as indicated by STAT3 phosphorylation in MC3T3-E1 osteoblastic cells, STAT3 activity significantly increases in response to 30 to 90 minutes fluid shear stress. In order to further study the role that STAT3 plays in bone responsiveness to loading, tissue-selective STAT3 knockout (KO) mice, in which inactivation of STAT3 occurs in osteoblasts, were generated by breeding the transgenic mice in which Cre recombinase cDNA was cloned downstream of a 3.6 or 2.3 kb fragment of the rat Col1a1 promoter (Col3.6-Cre and Col2.3-Cre, respectively) with a strain of floxed mice in which the two loxP sites flank exons 18-20 of the STAT3 gene were used. Mice engineered with bone selective inactivation of STAT3 in osteoblasts exhibited significantly lower bone mineral density (7-12%, p<0.05) and reduced ultimate force (21-34%, p<0.01) compared to their age-matched littermate controls. The right ulnae of 16-week-old bone specific STAT3 KO mice and the age-matched control mice were loaded with peak forces of 2.5 N and 2.75 N for female and male mice, respectively, at 2 Hz, 120 cycles/day for 3 consecutive days. Mice with inactivation of STAT3 specific in bone were significantly less responsive to mechanical loading than the control mice as indicated by decreased relative mineralizing surface (rMS/BS, 47-59%, p<0.05) and relative bone formation rate (rBFR/BS, 64-75%, p<0.001). Bone responsiveness was equally decreased in mice in which STAT3 is inactivated either in early osteoblasts (Col3.6-Cre) or in mature osteoblasts (Col2.3-Cre). Accumulating evidence indicates that bone metabolism is significantly affected by activities in mitochondria. For instance, although STAT3 is reported to be involved in bone formation and resorption through regulation of nuclear genes, inactivation of STAT3 is shown to disrupt mitochondrial activities and result in an increased level of reactive oxygen species (ROS). Inactivation of STAT3 suppressed load-driven mitochondrial activity, which led to an elevated level of ROS in cultured primary osteoblasts. Oxidative stress induced by administration of buthionine sulfoximine (BSO) significantly inhibits load-induced bone formation in wild type mice. Taken together, the results support the notion that the loss-of-function mutation of STAT3 in osteoblasts and osteocytes diminishes load-driven bone formation and impairs the regulation of oxidative stress in mitochondria.

Diabetic vasculopathy (DV) is the most important consequence of chronic hyperglycemia in patients with diabetes mellitus (DM). This thesis explores the interaction of blood, bone and kidney in the pathogenesis of DV by i) reviewing the current understanding of pathogenesis of macrovascular and microvascular diseases in DM to identify gaps in literature and generate hypotheses relating to various facets of DV ii) undertaking a series of prospective studies to examine these hypotheses iii) analysing the findings and integrating any new information obtained from the clinical studies into the current knowledge base and iv) generating hypotheses upon which future work might be based. The literature search was carried out with the aim of understanding current concepts of pathogenesis of DV and its potential modulators. The original reviews resulting from this process are presented in chapters 2 to 4. A series of pilot studies reported in chapters 7 to 11, were then carried out to interrogate hypotheses originating from this process. The first study was carried out in healthy individuals to define the biological variation of potential modulators of DV, namely erythropoietin (EPO), parathyroid hormone, 25 hydroxyvitamin D and 1, 25-dihydroxyvitamin D to facilitate the design and interpretation of subsequent studies. It revealed a wide biological variation of these modulators in the healthy population thus,emphasizing the need to have a control group in the subsequent study population. To examine whether tubulointerstitial dysfunction occurs before the onset of microalbuminuria, a measurement of the above mentioned parameters was carried out along with markers of tubulointerstitial injury in patients with type 1 and type 2 DM without microalbuminuria and in non-diabetic controls. It was found that tubulointerstitial dysfunction with low levels of EPO and 1, 25-dihydroxyvitamin D and higher excretion of tubular injury markers, occurs before the onset of microalbuminuria. Subsequently, diabetic and nondiabetic chronic kidney disease (CKD) patients with EPO deficiency anaemia were examined to study the effects of EPO therapy on the excretion of tubular injury markers. However, in these patient groups, we were unable to demonstrate an effect of EPO therapy on the markers of tubular injury in spite of a beneficial haematological response. To examine whether vascular calcification (VC) and bone mineral density (BMD) were linked in patients with diabetes mellitus and to explore their relationship to modulators of DV, an assessment of VC and BMD was undertaken in patients with type 2 DM with different degrees of proteinuria and normoalbuminuria. VC was assessed by CT scan and BMD by a DEXA scan. Modulators of DV were measured including serum Osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-b-ligand (RANKL). The findings were i) a high prevalence of VC and osteopenia in normoalbuminuric type 2 DM patients with normal serum creatinine ii) a weak inverse relationship between VC and osteopenia iii) proteinuric patients had worse VC but not osteopenia iv) weak relationships between OPG levels and both VC and osteopenia, masked by age in multivariate analysis. The final study examined the relationship between modulators of DV, including OPG and RANKL, and the degree of CKD. It was found that abnormalities of OPG and RANKL occur before the onset of microalbuminuria and progress with deterioration of renal function. Compared to nondiabetics, DM patients have higher OPG levels in the predialysis phase and lower levels in haemodialysis phase, a phenomenon that might indicate endothelial exhaustion in dialysis patients with DM. The derangements associated with DV seem to occur earlier than previously thought. Further work is required to untangle these complexities and to define the contribution of factors such as the adverse blood milieu, the vasculature, abnormal bone and mineral metabolism, and early tubulointerstitial damage. The findings from the studies reported here may help in the formulation of new hypotheses, which might contribute to future work in this area.