AGE-RAGE Interaction Does Not Explain the Clinical Improvements after Therapeutic Fasting in Osteoarthritis.

BACKGROUND: Therapeutic fasting improves joint pain in patients with osteoarthritis (OA), but the underlying mechanisms are unknown. Interactions of advanced glycation end products (AGEs) and their receptors (RAGE) play a role in OA pathogenesis. This study aimed to investigate whether the benefits of fasting in OA can be explained by changes in AGEs or RAGE. PATIENTS AND METHODS: 37 patients with OA underwent fasting for 8 days. Serum levels of an AGE (N-ε-(carboxymethyl)-lysine; CML) and the soluble RAGE (sRAGE) as well as clinical outcome parameters such pain characteristics (measured by visual analogue scale; VAS), joint function (determined by the Western Ontario and McMaster Universities Arthritis Index; WOMAC), and quality of life (via the 36-Item Short-Form Health Survey (SF-36) questionnaire) were assessed. The variables were measured at baseline, the end of fasting, and at follow-up at 4 weeks. RESULTS: The CML levels did not significantly change from baseline to the end of intervention (Δ = -25.6 ± 92.2 ng/ml; p = 0.10). In contrast, the sRAGE levels (Δ = -182.7 ± 171.4 ng/ml; p < 0.0001) and the sRAGE/CML ratio (Δ = -0.4 ± 0.6; p < 0.001) significantly decreased, but they returned to baseline levels 4 weeks after the end of fasting. The scores for pain, WOMAC, and the physical subscale of the SF-36 significantly improved during fasting. There was no correlation between the clinical outcomes and changes in serum levels of CML, sRAGE, or the sRAGE/CML ratio. CONCLUSIONS: Fasting resulted in a significant but non-sustained reduction of sRAGE levels and the sRAGE/CML ratio in OA, while the CML levels did not change. Improvement in clinical endpoints of OA does not correlate with changes in CML or sRAGE.

Growth arrest specific 2-like protein 1 expression is upregulated in podocytes through advanced glycation end-products.

Background: Growth arrest specific 2-like protein 1 (GAS2L1) protein is a member of the GAS2 family of proteins, known to regulate apoptosis and cellular cytoskeleton reorganization in different cells. Recently we identified that Gas2l1 gene expression in podocytes is influenced by advanced glycation end product-bovine serum albumin(AGE-BSA). Methods: The study was performed employing cultured podocytes and diabetic ( db/db ) mice, a model of type 2 diabetes. Akbuminuria as wellas urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion as measured with specific ELISAs. Gene expression was analysed via semiquantitative and real-time polymerase chain reaction. The protein levels were determined by western blotting and immunostaining. Results: We found that the Gas2l1 α isoform is expressed in podocytes. Treatment with AGE-BSA induced Gas2l1 α and Gas2 mRNA levels compared with controls incubated with non-glycated control BSA (Co-BSA). Moreover, application of the recombinant soluble receptor of AGEs (sRAGE), a competitor of cellular RAGE, reversed the AGE-BSA effect. Interestingly, AGE-BSA also increased the protein levels of GAS2L1α in a RAGE-dependent manner, but did not affect the GAS2 expression. Periodic acid-Schiff staining and albuminuria as well as urinary NGAL excretion revealed that db/db mice progressively developed diabetic nephropathy with renal accumulation of N ε -carboxy-methyl-lysine (immunohistochemistry, western blots). Analyses of GAS2L1α and GAS2 proteins in diabetic mice revealed that both were significantly elevated relative to their non-diabetic littermates. In addition, GAS2L1α and GAS2 proteins positively correlated with the accumulation of AGEs in the blood plasma of diabetic mice and the administration of sRAGE in diabetic mice reduced the glomerular expression of both proteins. Conclusions: We show for the first time that the protein expression of GAS2L1α in vitro and in vivo is regulated by the AGE-RAGE axis. The suppression of AGE ligation with their RAGE in diabetic mice with progressive nephropathy reversed the GAS2L1α expression, thus suggesting a role of GAS2L1α in the development of diabetic disease, which needs to be further elucidated.

Vitamin D3 Partly Antagonizes Advanced-Glycation Endproducts-Induced NFκB Activation in Mouse Podocytes.

BACKGROUND/AIMS: We have previously shown that advanced glycation-endproducts (AGEs) induced NFκB activation in differentiated mouse podocytes. This NFκB activation may contribute to the progression of renal disease and mediation of fibrosis by various mechanisms. This study was undertaken to test whether this detrimental response may be reversed by vitamin D3 or its analogue paricalcitol. METHODS: Differentiated mouse podocytes were challenged with glycated bovine serum albumin (AGE-BSA), or non-glycated control BSA (in the presence or absence of various concentrations of vitamin D3 (decostriol, 1α,25-dihydroxyvitamin D3)) or its active analog paricalcitol. Quantitative mRNA expressions were measured by real-time PCR, whereas protein expressions were determined by Western blotting followed by densitometry. Cytoplasmic and nuclear protein expression of the NFκB subunit p65 (Rel A) were determined by Western blotting. Furthermore, the ratio of phosphorylated to non-phosphorylated IκB-α was measured using specific antibodies. Electrophoretic mobility shift assays and a capture ELISA assay were used to assess NFκB transactivation in vitro. In addition, NFκB transactivation was also monitored in HEK-NFκBIA reporter cells using live cell luminometry. RESULTS: Podocytes expressed the receptor for vitamin D. The vitamins did not suppress receptor for AGEs (RAGE) expression; instead, they rather upregulated RAGE. Although vitamin D3 and paricalcitol partly and differentially modified some of the studied parameters, both hormones inhibited AGE-BSA-induced NFκB transactivation, presumably by various mechanisms including the upregulation of IκB-α protein, keeping NFκB sequestered in an inactive state in the cytoplasm. CONCLUSION: Vitamin D3 or its analog paricalcitol partly prevented AGE-mediated NFκB activation, an important feature of diabetic nephropathy (DN). Whether this in vitro finding is of clinical relevance to prevent/treat DN requires further studies.

Preconditioned suppression of prolyl-hydroxylases attenuates renal injury but increases mortality in septic murine models.

BACKGROUND: Septic conditions contribute to tissue hypoxia, potentially leading to multiple organ failure, including acute kidney injury. The regulation of cellular adaptation to low oxygen levels is regulated by hypoxia-inducible transcription factors (HIFs). While the role of HIFs in ischaemia/reperfusion is more studied, their function in sepsis-induced renal injury is not well characterized. In this study, we investigated whether pharmacological activation of HIFs by suppression of prolyl-hydroxylases (PHDs) protects against septic acute kidney injury. METHODS: Two models of sepsis-caecal ligation and punction and peritoneal contamination and infection-were induced on 12-week-old C57BL6/J mice. Pharmacological inhibition of PHDs, leading to HIF activation, was achieved by intraperitoneal application of 3,4-dihydroxybenzoate (3,4-DHB) before sepsis. A quantitative real-time reverse transcription polymerase chain reaction, immunohistology and enzyme-linked immunosorbent assays were utilized to detect gene expression, renal protein levels and renal functional parameters, respectively. Tissue morphology was analysed by periodic acid-Schiff reaction. Early kidney injury was estimated by kidney injury molecule-1 analyses. Apoptosis was detected in situ by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling stain. The systemic effect of 3,4-DHB pretreatment in sepsis was analysed by 72-h survival studies. RESULTS: Pharmacological activation of HIFs before sepsis induction attenuated sepsis-related vacuolization and dilation of the proximal tubules, reduced tubular apoptosis and correlated to lower T-cell infiltration in renal tissue compared with the non-treated septic animals. PHD suppression elevated the basal renal HIF-1α expression and basal plasma concentrations of HIF targets erythropoietin and vascular endothelial growth factor. Whereas it preserved renal structure in both models, it improved renal function in a model-dependent manner. Moreover, inhibition of PHDs led to increased mortality in both models. Analysis of liver function showed increased organ destruction with massive glycogen loss and hepatocyte's apoptosis due to 3,4-DHB administration before sepsis induction. CONCLUSIONS: In summary, the pharmacological activation of HIFs by 3,4-DHB administration, although it showed renoprotective effects in sepsis-related kidney injury, induced more severe problems in other organs such as the liver during sepsis, leading to increased mortality.

Activation of the receptor for advanced glycation end products induces nuclear inhibitor of protein phosphatase-1 suppression.

The activation of the receptor for advanced glycation end products (RAGE) is involved in the development of diabetic nephropathy. Analysis of protein phosphatase-1 indicated that advanced glycation end products did not affect its expression, but increased its phosphatase activity. Using differential display analysis we previously demonstrated that stimulation of RAGE in podocytes modulates the expression of numerous genes, among others nuclear inhibitor of protein phosphatase-1 (NIPP1). Here we found that silencing of NIPP1 induced podocyte hypertrophy, cell cycle arrest, and significantly increased protein phosphatase-1 activity. NIPP1 downregulation was associated with increased p27(Kip1) protein expression. Reporter assays revealed a transcriptional activation of nuclear factor-κB in podocytes after suppression of NIPP1. The protein level of NIPP1 was also significantly reduced in podocytes of diabetic mice. Blocking the RAGE in vivo by a soluble analog elevated the NIPP1 protein in podocytes of diabetic mice. Thus, activation of the RAGE by advanced glycation end products or other ligands suppresses NIPP1 expression in diabetic nephropathy, contributes to podocyte hypertrophy, and glomerular inflammation.

Clinical and endocrine correlates of circulating sclerostin levels in patients with type 1 diabetes mellitus.

AIM: Type 1 diabetes mellitus (T1DM) increases fragility fractures due to low bone mass, micro-architectural alterations and decreased bone formation. Sclerostin is expressed by osteocytes and inhibits osteoblastic bone formation. We evaluated serum sclerostin levels in T1DM and their association with bone mineral density (BMD), bone turnover, glycaemic control and physical activity. PATIENTS AND METHODS: In a cross-sectional study, 128 men and premenopausal women with long-standing T1DM (mean age 43·4 ± 8·8 years, diabetes duration 22·4 ± 9·5 years) and 77 age-, BMI (Body Mass Index) and gender-matched healthy individuals were evaluated. RESULTS: Serum sclerostin levels were higher in T1DM compared with controls, irrespective of gender (male 0·55 ± 0·17 vs 0·49 ± 0·12 ng/ml, P = 0·046; female 0·52 ± 0·19 ng/ml vs 0·43 ± 0·12 ng/ml, P = 0·012). Partial correlation analysis adjusted for age and gender revealed a positive correlation between serum sclerostin levels and BMD at lumbar spine and femoral neck in T1DM and between BMD at lumbar spine, femoral neck and total hip in controls. Bone turnover markers, parathyroid hormone, calcium and vitamin D did not correlate with serum sclerostin levels in T1DM or controls. Physical activity was not associated with serum sclerostin levels. A multivariate analysis revealed that only the interaction of T1DM and age affects serum sclerostin levels but not T1DM alone. The influence of age on serum sclerostin levels was more pronounced in T1DM compared with controls. CONCLUSIONS: Sclerostin serum levels were increased in patients with T1DM, and the positive correlation of age with serum sclerostin levels was stronger in T1DM. There was no effect of serum sclerostin levels on markers of bone metabolism and they do not explain the detrimental effects of T1DM on BMD.

Erythropoietin ameliorates podocyte injury in advanced diabetic nephropathy in the db/db mouse.

Podocyte damage and accumulation of advanced glycation end products (AGEs) are characteristics of diabetic nephropathy (DN). The pathophysiology of AGE-challenged podocytes, such as hypertrophy, apoptosis, and reduced cell migration, is closely related to the induction of the cell cycle inhibitor p27(Kip1) and to the inhibition of neuropilin 1 (NRP1). We have previously demonstrated that treatment with erythropoietin is associated with protective effects for podocytes in vitro. db/db mice with overt DN aged 15-16 wk were treated with either placebo, epoetin-ß, or continuous erythropoietin receptor activator (CERA) for 2 wk. db/db mice compared with nondiabetic db/m control mice revealed the expected increases in body weight, blood glucose, albumin-to-creatinine ratio, and AGE accumulation. Whereas there were no differences in body weight, hyperglycemia and AGEs were observed among diabetic mice that received epoetin-ß compared with CERA and placebo treatment, indicating that epoetin-ß/CERA treatment does not interfere with the development of diabetes in this model. However, the albumin-to-creatinine ratio was significantly lower in db/db mice treated with epoetin-ß or CERA. Furthermore, kidney weights in db/db mice were increased compared with db/m control mice, indicating renal hypertrophy, whereas the increase in renal weight in epoetin-ß- or CERA-treated db/db mice was significantly lower than in placebo-treated control mice. Induction of p27(Kip1) and suppression of NRP1 were significantly reduced in the epoetin-ß treatment group versus the CERA treatment group. Furthermore, erythropoietin treatment diminished the diabetes-induced podocyte loss. Together, independently from hematopoetic effects, epoetin-ß or CERA treatment was associated with protective changes in DN, especially that NRP1 and p27(Kip1) expressions as well as numbers of podocytes returned to normal levels. Our data show, for the first time, that medication of overt DN with erythropoietin for a short time can ameliorate albuminuria and podocyte loss.

Advanced glycation end products and ß(2)-microglobulin as predictors of carpal tunnel syndrome in hemodialysis patients.

BACKGROUND/AIMS: Carpal tunnel syndrome (CTS) is a common clinical presentation of dialysis-related amyloidosis. It was determined whether ß(2)-microglobulin (ß2M) and advanced glycation end products in serum are predictors of CTS in dialysis patients. METHODS: A total of 385 hemodialysis patients were screened for CTS. ß2M in serum was determined by a competitive enzyme-linked immunoassay, CML by a competitive enzyme-linked immunosorbent assay and total pentosidine by reverse-phase high-performance liquid chromatography. RESULTS: 127 patients (33%) were treated with biocompatible membranes, 174 (45%) with high-flux dialysis. 122 patients (31.7%) had clinical signs of CTS. Significant predictors of CTS were: age, female gender, serum ß2M, total protein, dialysis with non-biocompatible high-flux dialysis compared to non-biocompatible low-flux dialysis, Kt/V and serum concentration of CML (OR 2.47 for the 3rd vs. 1st quartile, 95% CI 1.229-4.961, p = 0.011). CONCLUSION: The prevalence of CTS as a possible manifestation of dialysis-related amyloidosis is still high. Serum concentration of CML may be a predictor of CTS besides ß2M and malnutrition.

Angiotensin II differentially regulates Morg1 expression in kidney cells.

BACKGROUND: The mitogen-activated protein kinase organizer 1 (Morg1) belongs to the WD-40 repeat protein family and is a scaffold molecule for the extracellular regulated kinase signaling pathway. Morg1 also binds to prolyl-hydroxylase 3 (PHD3) and regulates the hypoxia-inducible factor-1α (HIF-1α) expression via PHD3 stabilization. Morg1 has been detected in the kidney as well as in other cell tissues but its expression in renal cells has not been well investigated. It has been widely shown that angiotensin II (ANG II) mediates renal damage. We have previously shown that ANG II downregulates the expression of PHD3 in PC12 cells. The aim of this study was to analyze whether ANG II regulates Morg1 expression in mouse mesangial cells (MMC), mouse proximal tubular cells (MTC) and in differentiated podocytes. The correlation between the expression of Morg1 and PHD3 activity was also addressed. METHODS: Effect of ANG II on the Morg1 mRNA expression level was assessed by real-time PCR. Morg1 and HIF-1α cellular localization was analyzed by immunohistochemistry. HIF-1α promoter activity was investigated using a reporter gene system. PHD3 hydroxylase activity test was measured with a hydroxylation-coupled decarboxylation assay. RESULTS: ANG II differentially regulates Morg1 expression in MMC, MTC and differentiated podocytes. We detected a biphasic effect of ANG II on Morg1 mRNA expression which was time dependent. While 9-hour ANG II treatment downregulated Morg1 expression in MMC, it induced Morg1 expression in MTC. Conversely, 24-hour ANG II stimulation upregulated the expression of Morg1 mRNA in MMC, but showed an opposite effect in MTC and differentiated podocytes. In addition, we found that ANG II signals mostly through the AT(1) receptor subtype in MMC and via the AT(2) subtype in MTC. PHD3 activity correlated to Morg1 expression patterns. Our data also demonstrate that HIF-1α transcriptional activity in MTC contrasted to PHD3 activity at 9 and 24 h, whereas in the MMC and in podocytes we did not find any correlation between PHD3 HIF-1α hydroxylation ability and HIF-1α transcriptional activation, suggesting a different mechanism of regulation in these cell types. Interestingly, the reduced expression of Morg1 in mesangial cells isolated from Morg1 (+/-) heterozygous mice correlated with a reduced PHD3 enzymatic activity and an increased HIF-1α transcriptional activity compared with mesangial cells originated from wild-type (Morg1 +/+) mice. CONCLUSIONS: We show for the first time in various renal cells that ANG II modulates Morg1 expression and HIF-1α transcriptional activity via cell type-specific mechanisms, demonstrating a novel mechanism by which ANG II may contribute to renal disease.

The balance between soluble receptors regulating IL-6 trans-signaling is predictive for the RANKL/osteoprotegerin ratio in postmenopausal women with rheumatoid arthritis.

The objective of this study is to investigate the relationship between soluble components of the interleukin 6 (IL-6) system mediating and modifying IL-6 trans-signaling and the RANKL-RANK-osteoprotegerin system in postmenopausal women with rheumatoid arthritis (RA). The following parameters were investigated in 126 postmenopausal women with RA: IL-6, soluble IL-6-receptor (sIL-6R), soluble glycoprotein 130 (sgp130), sRANKL, osteoprotegerin (OPG), osteocalcin, erythrocyte sedimentation rate and C-reactive protein in sera, pyridinolin and desoxypyridinolin crosslinks in the morning urine. Bone mineral density (BMD) was measured by dual X-ray absorptiometry at the lumbar spine (BMD-LS) and at the femoral neck (BMD-FN). Predictors of RANKL/OPG ratio and BMD were evaluated by multiple linear regression analysis. The following determinants of the RANKL/OPG ratio were identified: sIL-6R/sgp130 ratio and daily glucocorticoid (GC) dose as positive determinants in the whole group (R (2) = 0.56; P = 0.001), sIL-6R/sgp130 ratio as the exclusive positive determinant in patients with GC therapy (R (2) = 0.48; P = 0.001) and sgp130 as negative determinant in patients without GC (R (2) = 0.42; P = 0.031). Sgp130 was highly significantly positively correlated with OPG in the whole group (P < 0.001) as well as in patients with (n = 70; P < 0.05) and without GC therapy (n = 56; P < 0.01). sIL-6R was the main negative predictor of BMD-LS (R (2) = 0.41; P = 0.019). High sIL-6R/sgp130 ratio and/or low sgp130 are associated with a high sRANKL/OPG ratio in sera of postmenopausal women with RA indicating the critical significance of IL-6 trans-signaling for an increase in the RANKL/OPG ratio and of bone resorption. Inhibition of IL-6 trans-signaling may be an effective bone-protecting principle in postmenopausal women with RA.

Podocytes of AT2 receptor knockout mice are protected from angiotensin II-mediated RAGE induction.

BACKGROUND/AIMS: The interaction of 'advanced glycation end products' (AGEs) and their receptor 'RAGE' plays an important role in diabetic nephropathy. We have previously found that in cultured differentiated podocytes, angiotensin II (ANG II) induces RAGE expression via an AT2 receptor-mediated pathway. METHODS: To further confirm our results in an in vivo study, AT2 receptor knockout mice (AT2(-/-)) and wild-type mice were infused with ANG II by osmotic minipumps for 14 days. RESULTS: As shown by immunohistochemistry, ANG II treatment of wild-type animals (C57BL6) allowed a significantly increased RAGE expression in renal podocytes in comparison to sham-operated C57BL6 mice. In contrast, RAGE expression in podocytes of ANG II-treated knockout mice (AT2(-/-)) was only moderately higher than in control animals, but significantly lower than in ANG II-treated wild-type mice. For the AGE species Nε-carboxymethyllysine, a similar immunohistochemical staining pattern was found. There was no significant change in glomerular AT1a receptor expression. However, no difference in RAGE mRNA expression could be found between ANG II-infused wild-type and AT2(-/-) animals by real-time PCR using whole kidney mRNA, presumably due to the low abundance of podocyte mRNA in these preparations. No effects were seen on glomerular apoptosis. CONCLUSION: These data support the fact that ANG II-mediated RAGE induction in podocytes occurs via AT2 receptors. The present findings may suggest that not all ANG II-mediated changes in diabetic nephropathy can be treated with AT1 receptor blockers.

Erythropoietin protects podocytes from damage by advanced glycation end-products.

BACKGROUND: Podocyte damage and accumulation of advanced glycation end-products (AGEs) are implicated in the development and progression of diabetic nephropathy. We have previously shown that changes in podocyte pathophysiology, such as hypertrophy and reduced migration, are closely linked with the induction of the cell cycle inhibitor p27(Kip1) and a decrease in neuropilin-1 (NRP1) expression. We investigated whether the erythropoietin receptor activators CERA and epoetin-ß may prevent AGE-mediated changes in podocytes. METHODS: Differentiated mouse podocytes in culture were challenged by AGE-modified bovine serum albumin (BSA) or control BSA in the presence or absence of CERA as well as epoetin-ß. Cell cycle analysis and determination of apoptosis markers were performed. p27(Kip1) and NRP1 expression was measured by RT-PCR and Western blots. RESULTS: Differentiated mouse podocytes in culture expressed erythropoietin receptors which were phosphorylated after incubation with CERA or epoetin-ß. CERA or epoetin-ß prevented the p27(Kip1)-dependent cell cycle arrest and cellular hypertrophy induced by AGE-BSA incubation. Furthermore, the p27(Kip1)-dependent AGE-BSA-induced decrease in cell viability and decrease in cell proliferation was ameliorated in the presence of CERA or epoetin-ß. Following erythropoietin treatment, AGE-BSA failed to further reduce NRP1 expression, resulting in improved podocyte migration. CONCLUSION: Treatment with the erythropoietin receptor activators epoetin-ß or CERA protected podocytes from AGE-BSA-mediated damage via an effect on p27(Kip1) and NRP1 expression. Consequently, early treatment with erythropoietin may help to prevent diabetic nephropathy.

Influence of Rituximab on markers of bone remodeling in patients with rheumatoid arthritis: a prospective open-label pilot study.

Immune system and bone are interacting in a complex way. Rheumatoid arthritis is characterized not only by joint destruction, but also by development of systemic osteopenia and osteoporosis. The CD20-depleting antibody Rituximab (Rtx) is a novel therapeutic option able significantly to slow the destructive joint process of rheumatoid arthritis. However, there are little data whether Rtx influences systemic bone remodeling. In the present prospective study, we evaluated the influence of Rtx on markers of bone metabolism with a follow-up of 3-15 months after Rtx therapy (2 dose of each 1,000 mg) in 13 patients with rheumatoid arthritis. There was no significant change of the bone formation markers bone alkaline phosphatase and c-terminal propeptide of collagen I. However, a non-significant tendency of decrease of RANKL (with no chance of osteoprotegerin) and a significant decrease of the bone degradation marker desoxypyridinolin crosslinked collagen I was observed 15 months after Rtx application. These initial results provide no evidence of a negative systemic influence of Rtx on bone remodeling. In contrast, it appears that Rtx lowered osteoclast activity often found increased in active rheumatoid arthritis contributing to osteoporosis in this disease.

Advanced glycation end-products and the kidney.

BACKGROUND: Advanced glycation end-products (AGEs) are increased in situations with hyperglycemia and oxidative stress such as diabetes mellitus. They are products of nonenzymatic glycation and oxidation of proteins and lipids. The kidney plays an important role in clearance and metabolism of AGEs. METHODS: Medline and other relevant databases were searched. In addition, key review articles were scanned for relevant original publication. Finally, original data from our research group were also included. RESULTS: Kidney podocytes and endothelial cells express specific receptors for AGEs. Their activation leads to multiple pathophysiological effects including hypertrophy with cell cycle arrest and apoptosis, altered migration, and generation of proinflammatory cytokines. AGEs have been primarily implicated in the pathophysiology of diabetic nephropathy and diabetic microvascular complications. AGEs are also involved in other primary renal diseases as well as in the development and progression of atherosclerosis. However, serum or plasma concentrations of AGEs do not correlate well with cardiovascular events in patients with chronic kidney disease (CKD). This is likely due to the fact that serum concentrations failed to correlate with AGEs deposited in target tissues. Several inhibitors of the AGE-RAGE axis are currently tested for various indications. CONCLUSION: AGEs and their receptors are involved in the pathogenesis of vascular and kidney disease. The role of circulating AGEs as biomarkers for cardiovascular risk estimation is questionable. Whether putative inhibitors of AGEs will get the maturity for its therapeutic use in the future remains open.

Vitamin B6 metabolism in chronic kidney disease--relation to transsulfuration, advanced glycation and cardiovascular disease.

BACKGROUND: Vitamin deficiency is common in chronic kidney disease (CKD). Data on B(6) supply and possible relationships to cardiovascular events (CVE) in CKD are rare. Pyridoxamine exerts inhibitory effects on the formation of advanced glycation endproducts (AGE) implicated in the pathogenesis of CKD and atherosclerosis. METHODS: In 48 CKD patients at stage 2-4, 72 hemodialysis patients (HD), 38 renal transplant recipients (RTR) and 141 healthy controls (mean age 58 +/- 13, 61 +/- 12, 50 +/- 12 and 54 +/- 16 years, respectively), plasma and red blood cell (RBC) concentrations of pyridoxal-5'-phosphate (PLP), pyridoxal (PL), 4-pyridoxic acid (PA), pyridoxamine-5'-phosphate (PMP) and of the AGE pentosidine were measured by high-performance liquid chromatography, N(epsilon)-(carboxymethyl)lysine and imidazolone by an ELISA, and total homocysteine and cystathionine by gas chromatography-mass spectrometry. RESULTS: Despite routine low-dose vitamin supplementation in HD, plasma PLP was decreased in HD (79 +/- 69 nmol/l) compared with CKD stage 2-4 patients (497 +/- 944 nmol/l), RTR (416 +/- 604 nmol/l) and controls (159 +/- 230 nmol/l; p < 0.001). Plasma PA was significantly increased in HD (11,667 +/- 17,871 nmol/l) in comparison with CKD stage 2-4 (435 +/- 441 nmol/l), RTR (583 +/- 668 nmol/l) and controls (46 +/- 49 nmol/l; p < 0.001). B(6) forms were significantly affected by renal function (R = 0.792, p < 0.001 for CKD stage 2-4). There was no relation of vitamers with a history of CVE. Relationships between B(6) forms and AGE (RBC-PMP with pentosidine in CKD stage 2-4: R = -0.351, p < 0.05) were found. CONCLUSION: HD patients showed a deficiency in PLP in plasma but not in RBC. Prospective trials are needed to elucidate the potential role of elevated PA on cardiovascular and renal outcome in CKD. Vitamin B(6) supplementation might be successful in preventing AGE-related pathologies.

Advanced glycation end products induce cell cycle arrest and proinflammatory changes in osteoarthritic fibroblast-like synovial cells.

INTRODUCTION: Advanced glycation end products (AGEs) have been introduced to be involved in the pathogenesis of osteoarthritis (OA). The influence of AGEs on osteoarthritic fibroblast-like synovial cells (FLS) has been incompletely understood as yet. The present study investigates a potential influence of AGE-modified bovine serum albumin (AGE-BSA) on cell growth, and on the expression of proinflammatory and osteoclastogenic markers in cultured FLS. METHODS: FLS were established from OA joints and stimulated with AGE-BSA. The mRNA expression of p27Kip1, RAGE (receptor for AGEs), nuclear factor kappa B subunit p65 (NFkappaB p65), tumor necrosis factor alpha (TNF-alpha, interleukin-6 (IL-6), receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin was measured by real-time PCR. The respective protein expression was evaluated by western blot analysis or ELISA. NFkappaB activation was investigated by luciferase assay and electrophoretic mobility shift assay (EMSA). Cell cycle analysis, cell proliferation and markers of necrosis and early apoptosis were assessed. The specificity of the response was tested in the presence of an anti-RAGE antibody. RESULTS: AGE-BSA was actively taken up into the cells as determined by immunohistochemistry and western blots. AGE-induced p27Kip1 mRNA and protein expression was associated with cell cycle arrest and an increase in necrotic, but not apoptotic cells. NFkappaB activation was confirmed by EMSAs including supershift experiments. Anti-RAGE antibodies attenuated all AGE-BSA induced responses. The increased expression of RAGE, IL-6 and TNF-alpha together with NFkappaB activation indicates AGE-mediated inflammation. The decreased expression of RANKL and osteoprotegerin may reflect a diminished osteoclastogenic potential. CONCLUSIONS: The present study demonstrates that AGEs modulate growth and expression of genes involved in the pathophysiological process of OA. This may lead to functional and structural impairment of the joints.

Angiotensin II upregulates RAGE expression on podocytes: role of AT2 receptors.

BACKGROUND: Advanced glycation end products (AGEs) play an important role in diabetic nephropathy. The receptor for AGEs, called RAGE, is present on podocytes. We investigated whether angiotensin II (ANG II) modulates RAGE expression on cultured differentiated podocytes. RESULTS: Cultured podocytes expressed AT1 and AT2 receptors. Surprisingly, ANG II induced RAGE mRNA and protein expression through AT2 receptors. ANG II had no influence on proliferation or protein content of podocytes. The increase in RAGE expression depended on stimulated transcriptional activity. Using various mutant reporter constructs of the RAGE promoter region, it was shown that a NF-kappaB binding site at -1519 was essential for ANG II-induced transcriptional activity. Preincubation with ANG II increased the expression of tumor necrosis factor-alpha mRNA and protein expression induced by AGE, indicating that the ANG II-mediated upregulation of RAGE has functional consequences. AGE-BSA was incorporated into cells as measured by Western blots for N epsilon-carboxymethyllysine, but ANG II did not influence this process. ANG II in the absence or presence of AGE-BSA did not induce apoptosis of podocytes. CONCLUSION: Our study revealed aninteraction between the renin-angiotensin system and the AGE/RAGE axis in podocytes. Since intraglomerular ANG II levels are increased in diabetic nephropathy, this interaction may have pathophysiological consequences for podocyte injury and inflammation associated with the development of diabetic nephropathy.

Advanced glycation end-products suppress neuropilin-1 expression in podocytes.

Advanced glycation end products (AGEs) have been linked to the pathogenesis of diabetic nephropathy. Here we tested the effect of AGE-modified bovine serum albumin (AGE-BSA) on differentiated mouse podocytes in culture. Differential display and real-time PCR analyses showed that in addition to neuropilin-1, the entire signaling receptor complex of neuropilin-2, semaphorin-3A, and plexin-A1, was significantly reduced by AGE-BSA as was neuropilin-1 protein. The effect was specific for podocytes compared to isolated mesangial and tubular epithelial cells. Further, AGE-BSA was not toxic to podocytes. Neuropilin-1 expression was decreased in glomeruli of diabetic db/db mice compared to their non-diabetic littermates. Transcripts of both neuropilins were found to be decreased in renal biopsies from patients with diabetic nephropathy compared to transplant donors. Podocyte migration was inhibited by AGE-BSA with similar results found in the absence of AGE-BSA when neuropilin-1 expression was down-regulated by siRNA. In contrast, podocyte migration was stimulated by overexpression of neuropilin-1 even in the presence of AGE-BSA. Our study shows that AGE-BSA inhibited podocyte migration by down-regulating neuropilin-1. The decreased migration could lead to adherence of uncovered areas of the glomerular basement membrane to Bowman's capsule contributing to focal glomerulosclerosis.

Significance of risk factors for osteoporosis is dependent on gender and menopause in rheumatoid arthritis.

The aim of our study was to compare the significance of risk factors for osteoporosis according to gender and menopausal state in patients with rheumatoid arthritis (RA). Bone mineral density (dual X-ray absorptiometry), cumulative glucocorticoid dose, age, disease duration, body mass index (BMI) and parameters of disease activity and bone turnover were registered in 343 postmenopausal women, 100 premenopausal women and 108 men with RA. Osteoporosis was found in a significantly higher percentage in postmenopausal women (55.7%) and in men (50.5%) in comparison with premenopausal women (18%; P < 0.001). The following risk factors for osteoporosis were found: older age, low BMI and high cumulative glucocorticoid dose in postmenopausal women, low BMI and high cumulative glucocorticoid dose in men and low BMI in premenopausal women. There is a very high prevalence of osteoporosis not only in postmenopausal women but also in men with RA. Osteoporosis risk factors are strongly dependent from gender and menopausal state.

Serum levels of the advanced glycation end products Nepsilon-carboxymethyllysine and pentosidine are not influenced by treatment with the angiotensin receptor II type 1 blocker irbesartan in patients with type 2 diabetic nephropathy and hypertension.

BACKGROUND/AIMS: The aim of this post-hoc analysis of a prospective study in patients with type 2 diabetic nephropathy was to investigate whether treatment with the angiotensin II type 1 receptor blocker irbesartan leads to a reduction in the serum levels of the advanced glycation end products (AGEs) pentosidine and N(epsilon)-carboxymethyllysine (CML). METHODS: One hundred and ninety-six patients of the Irbesartan in Diabetic Nephropathy Trial cohort (mean age 61 +/- 6.5 years, 62 female, 134 male) with a mean estimated glomerular filtration rate of 47.7 ml/min were treated with irbesartan (n = 65), the calcium channel blocker amlodipine (n = 61) or by placebo (n = 70). Serum levels of pentosidine and CML were measured at baseline and after follow-up (23.4 months). RESULTS: Estimated glomerular filtration rate decreased in all groups by a mean of 8.6 ml/min. Serum levels of AGEs increased significantly (p < 0.001) during follow-up. After controlling for renal function and total protein concentration, changes were 53, 55, 50% for pentosidine and 29, 24, 23% for CML (irbesartan, amlodipine and placebo group, respectively). The increase was not significantly different between the treatment groups. CONCLUSION: Irbesartan did not alter the increase in pentosidine and CML in serum of type 2 diabetic patients with progressive nephropathy. This finding suggests that angiotensin receptor blockade alone is insufficient to reduce serum levels of AGEs in diabetic nephropathy.