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.

Rosiglitazone increases PPARgamma in renal tubular epithelial cells and protects against damage by hydrogen peroxide.

BACKGROUND/AIMS: Thiazolidinediones (TZD) are ligands known to bind to and activate the nuclear peroxisome proliferator-activated receptor gamma (PPARgamma), and are currently used as insulin sensitizers in type 2 diabetes. Recently, several studies have shown that TZD may have a role in renal protection in various experimental models. However, the precise mechanisms by which TZD may possibly affect tubular cell survival after injury remain unclear. We studied the influence of the TZD rosiglitazone on PPARgamma expression and cell function with cellular damage induced by increasing hydrogen peroxide (H2O2) concentrations in bovine renal tubular epithelial cells (bEPC) to determine whether rosiglitazone is cytoprotective under these conditions. METHODS: bEPC were cultured in the presence of H2O2 after pretreatment with or without 25 microM rosiglitazone. The expression of PPARgamma mRNA and protein were determined using RT-PCR or Western blots, respectively, after 6 and 24 h. Some cells also received actinomycin D or cycloheximide and PPARgamma protein expression was tested. Proliferation rates of cultures were compared after 15 h and after a recovery phase of 6 days. Apoptosis was assessed by DNA fragmentation. Nuclear PPARgamma activity was evaluated by electrophoretic mobility shift assay (EMSA), and the cellular location was detected using immunofluorescence. RESULTS: Incubation of bEPC with H2O2 concentrations up to 0.75 mM did not induce apoptosis as tested by DNA fragmentation assay, but significantly and dose-dependently reduced proliferation 15 h after injury as measured by [3H]thymidine incorporation. 25 microM rosiglitazone alone also reduced proliferation and failed to attenuate the H2O2-mediated inhibition of proliferation. However, rosiglitazone facilitates recovery of tubular cells 6 days after H2O2-induced injury. Rosiglitazone (25 microM) increased PPARgamma mRNA and protein expression in bEPC in the absence of H2O2. Rosiglitazone failed to increase PPARgamma mRNA in cells with oxidative stress, but Western blots revealed an increase in cellular PPARgamma protein content in the presence of rosiglitazone and increasing concentrations of H2O2. This increase in PPARgamma protein content was almost totally abolished in the presence of 1 microg/ml cycloheximide, but was only marginally reduced by 0.1 microg/ml actinomycin D. EMSA showed a robust increase in nuclear PPARgamma protein binding in vitro to its consensus site after rosiglitazone whereas H2O2 treatment reduced PPARgamma activation. Rosiglitazone treatment of cells with oxidative stress preserved nuclear transactivation of PPARgamma. CONCLUSIONS: Rosiglitazone increases the PPARgamma content in bEPC after H2O2-induced injury by a posttranscriptional mechanism. Activation of PPARgamma facilitates the long-term recovery of tubular cells 6 days after oxidative injury, but had no effect on the attenuated proliferation shortly after injury. TZD cannot prevent oxidative injury to tubular cells, but may be important mediators to enhance cellular recovery after oxidative stress.

Renal Angiotensin receptor type 1 and 2 upregulation in intrauterine growth restriction of newborn piglets.

Epidemiological and experimental studies suggest that intrauterine growth restriction (IUGR) is associated with abnormalities in kidney development which is thought to be linked with alterations causing adult cardiovascular diseases. The renin-angiotensin system (RAS) plays an important role in the development of renal vascular and tubular structures, and is known to be altered by experimentally induced IUGR. These experimental models of IGUR have been criticized because they may have a more severe impact on intrauterine development than that which is normally encountered in humans. Therefore, we asked whether naturally occurring small-for-gestational-age newborn piglets exhibit features of altered RAS activity. We investigated the regional renal expression of angiotensin II type 1 (AT1) and AT2 receptors in normal-weight and IUGR piglets. The AT1 receptor mRNA expression was markedly enhanced in IUGR piglets, in the renal cortex by 64% and in the renal medulla by 52% (p < 0.05, compared with normal littermates). In contrast, mRNA expression for the AT2 receptor was similar in both the normal-weight and IUGR piglets. A significantly higher AT1 receptor protein expression was found in the IUGR piglets (p < 0.05) in the glomeruli, in the proximal and distal tubules, as well as in the collecting ducts by immunohistochemistry. Furthermore, AT2 receptor protein expression was significantly higher in the IUGR piglets (p < 0.05) in the subcapsular nephrogenic zone and in the distal tubules and collecting ducts. Thus, IUGR is accompanied by an upregulation of angiotensin II receptor expression in the kidneys of newborn piglets. This may indicate an alteration of the RAS in newborns suffering from naturally occurring IUGR.

Advanced glycation end products: a possible link to angiotensin in an animal model.

Advanced glycation end products (AGEs) have been associated with progressive vascular and renal damage in a variety of pathological conditions such as renal failure and diabetes mellitus. The formation of AGEs is generally attributed to increased oxidative and carbonyl stress or hyperglycemia. Activation of the cellular receptor of AGE (RAGE) leads to subsequent cellular activation and proinflammatory responses. Angiotensin (Ang) produces cellular oxidative stress and similarly promotes end organ damage via its type 1 receptor. We investigated the interrelation between these two systems in a new transgenic rat (TGR) model with Ang II-dependent hypertension and renal damage and in nontransgenic controls. TGR showed increased systolic blood pressure (approximately 210 mmHg), proteinuria, and increased renal collagen I mRNA expression compared with normotensive nontransgenic controls. Immunohistochemical staining of kidney sections showed colocalization for Nepsilon-carboxy(methyl)lysine, RAGE, and NF-kappaB in TGR glomeruli. These features were absent in nontransgenic controls. Our observations suggest a possible link between Ang II-dependent end-organ damage and the AGE/RAGE axis in vivo. TGRs provide an excellent model to study the interrelation between the renin-angiotensin system and the AGE/RAGE axis in promoting cardiovascular end-organ damage, which would otherwise not be possible in humans.

Fibronectin splice variants--prognostic markers for the stage of renal interstitial fibrosis in the rat.

BACKGROUND/AIMS: Renal interstitial fibrosis (RIF) is the main cause for progressive renal failure, but its pathogenic factors are not well known. In animal models of renal fibrogenesis done thus far an increase of total fibronectin (FN) mRNA has been proved. Recent studies have pointed to a key role of the splice variant EIIIA(+)-FN and EIIIB(+)-FN for the development of organ fibrosis. However, a broader knowledge of the distribution of these different FN mRNA isoforms is still lacking. Our aim was to study the particular expression of the EIIIA(+)-FN and EIIIB(+)-FN during the process of fibrogenesis in two rat models and to evaluate the FN isoforms as diagnostic/prognostic marker for the stage of interstitial damage in rat kidneys. METHODS: Kidneys of unilateral ureteral obstruction (UUO) and control rats were removed in intervals of 5, 14 or 21 days after surgery. For the investigation of kidney damage due to uranyl nitrate (UN), rats obtained a single i.p. dose of 5 mg/kg body weight UN and were killed 2, 10 and 20 weeks thereafter. The quantitative RT-PCR method was used to estimate the total FN, EIIIA(+)-FN and EIIIB(+)-FN transcription rate. RESULTS: In the UUO model, a significant augmentation of both isoforms was obtained in the kidneys in the first 5-day interval, which was more pronounced at the 21-day interval. In the UN-treated kidneys there appeared only a continuous increase of EIIIA(+)-FN and the splice variant EIIIB(+)-FN failed to show a shift in these animals as compared to the controls. CONCLUSION: Both animal models generated fibrogenic damages of the tubulointerstitium, whereas only the UUO resulted in progressive fibrosis. Absence of EIIIB(+)-FN seems to enhance the progression of fibrogenesis.

Induction of p27KIP1 after unilateral ureteral obstruction is independent of angiotensin II.

BACKGROUND: Unilateral ureteral obstruction (UUO) is characterized by proliferation of tubular and interstitial cells, and infiltration of the renal parenchyma with macrophages/monocytes. These alterations lead ultimately to tubulointerstitial fibrosis and tubular atrophy. Some of these changes are caused by an activated renin-angiotensin system (RAS). We have previously demonstrated that angiotensin II induces the expression of the cell cycle inhibitor p27KIP1 in cultured tubular cells. The current study tested the hypothesis that interference with the RAS may modulate renal expression of p27KIP1 after UUO. METHODS: The ureter of the left kidney of Sprague-Dawley rats was ligated. Sham-operated animals served as controls. Rats were randomized in four groups and received one of the following: no therapy, enalapril, losartan, or triple therapy (hydralazine, reserpine, hydrochlorothiazide). Kidneys were removed and cortical protein lysates were prepared for the detection of p27KIP1 by Western blotting. Immunohistochemistry was performed for p27KIP1, PCNA, ED-1, and alpha-smooth muscle actin. Apoptosis was quantified by TUNEL-staining. RESULTS: p27KIP1 expression as detected by Western blotting reached a maximum 10 days after UUO. Tubular and interstitial cells contributed to this increase in p27KIP1 expression whereas the number of glomerular p27KIP1 positive cell did not change. p27KIP1-positive cells were macrophages/monocytes (positive ED-1 staining) or had the characteristics of myofibroblasts (positive alpha-smooth muscle actin staining). Tubular and interstitial proliferation [proliferating cell nuclear antigen (PCNA)-positive staining] and apoptosis [terminal deoxy transferase uridine triphosphate nick end labeling (TUNEL) staining] also was increased after UUO. However, individual cells stained either positive for p27KIP1 or PCNA, but not both. Although enalapril and losartan reduced the number of macrophages/monocytes and attenuated the degree of tubular and interstitial apoptosis, these drugs did not influence p27KIP1 expression. There was no change in the number of p27KIP1-positive cells in the contralateral kidney undergoing hypertrophy. CONCLUSION: Induction of p27KIP1 in this model represents an endogenous response to likely limit proliferation that is independent of angiotensin II. Since there was no close correlation between apoptosis and p27KIP1 expression, it may be that the overall number of p27KIP1 expressing cells sets a general restriction point for apoptosis rather than defines an individual level of cell fate.