Inflammation and endothelial activation are linked to renal function in long-term kidney transplantation

The aim of this study was to investigate the relationships between inflammation and adhesion molecules in long-term kidney transplantation. We measured serum concentrations of tumor necrosis factor-alpha (TNFalpha) and intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1) in 35 renal transplant recipients (mean age of transplantation 5 +/- 3 years) and in 35 chronic renal insufficiency (CRI) patients; twenty-six healthy subjects were enrolled as controls. Transplanted showed higher values than controls of TNFalpha (P < 0.0001), ICAM-1 (P < 0.0001), and VCAM-1 (P < 0.0001). CRI group as well exhibited higher concentrations than controls of TNFalpha (P < 0.0001), ICAM-1 (P < 0.0001), and VCAM-1 (P < 0.0001). Transplanted and CRI patients had similar blood pressure and renal function levels, and TNFalpha, ICAM-1, and VCAM-1 were not significantly different in the two groups. In transplanted group ICAM-1, VCAM-1, and TNFalpha correlated negatively and independently with glomerular filtration rate (GFR) -P < 0.00001 for all. TNFalpha as well correlated with ICAM-1 and VCAM-1 (P < 0.001, respectively). In CRI group, TNFalpha correlated with serum creatinine, ICAM-1, and VCAM-1 (P = 0.01 for all). In conclusion, in long-term renal transplantation, the level of kidney function and both inflammation and endothelial activation are closely related. In fact, the multiple regression analysis demonstrated that the level of kidney insufficiency and the levels of the studied molecules were independently associated.     

Laminin and transforming growth factor beta-1 in children with vesicoureteric reflux

High-grade vesicoureteric reflux (VUR) promotes the development of renal nephropathy (RN) due to scar formation. This process involves transforming growth factor beta-1 (TGF beta₁), which stimulates production of the extracellular matrix proteins, including laminin (LN). The aim of the study was to assess LN and TGF beta₁ concentration according to VUR grade. The study group (1) consisted of 54 patients aged 6.23 ± 4.15 years with VUR, including: A, 19 with grade II; B, 19 with grade III; and C, 16 with grades IV or V reflux. The control group (2) contained 27 healthy patients aged 6.76 ± 4.02 years. LN and total TGF beta₁ concentrations in serum and urine were determined by the immunoenzymatic (EIA) method. To assess total serum TGF beta₁ levels, we used a solid-phase enzyme-linked immunosorbent assay (ELISA). Both serum and urinary levels of LN and TGF beta₁ in VUR patients were higher compared with controls (p < 0.05). The highest urinary concentration of LN and TGF beta₁ was found in subgroup C. A positive correlation was noted between urinary TGF beta₁ and LN. Increased TGF-beta₁ and LN levels in urine of high-grade VUR children suggests a potential role in fibrogenesis. Further trials are needed to investigate the role of serum and urinary LN level in VUR children.     

Comparison of nutritional status in hemodialysis patients with and without failed renal allografts

Yelken MB, Gorgulu N, Caliskan Y, Yazici H, Turkmen A, Yildiz A, Sever MS. Comparison of nutritional status in hemodialysis patients with and without failed renal allografts
Clin Transplant 2010: 24: 481–487.
© 2009 John Wiley & Sons A/S. Abstract: Background: The survival of patients returning to hemodialysis (HD) following kidney transplant failure is unfavorable. However, the factors responsible for this poor outcome are largely unknown; chronic inflammation due to failed allograft and malnutrition may contribute to morbidity and mortality. We aimed to compare the nutritional status and its relation with inflammation in patients on HD with and without previous kidney transplantation. Methods: Forty‐three patients with failed renal allografts (27 males; mean age 36 ± 9 yr) and 40 never transplanted HD patients (24 males; mean age 39 ± 9 yr) were included in the study. Body weight, triceps (TSF), biceps (BSF), subscapular (SSSF), and suprailiac skinfold thicknesses (SISF); mid‐arm, mid‐arm muscle, hip and waist circumferences; as well as body mass indices (BMIs) were determined as anthropometric parameters. Moreover, biochemical markers of nutritional status, including serum cholesterol and albumin as well as high‐sensitive C‐reactive protein (hs‐CRP), as a marker of inflammation, were measured. Associations among these variables were analyzed. Results: There were no significant differences considering age, gender or duration of renal replacement therapy between the two groups. The TSF (p < 0.0001), BSF (p = 0.005), SSSF (p = 0.001), SISF (p < 0.0001) skinfold thicknesses; mid‐arm (p = 0.003) and mid‐arm muscle circumferences (p = 0.037) and BMIs (p = 0.001) of the patients with failed renal allografts were significantly lower than those of the never transplanted HD patients. Waist circumference was significantly lower as well (p = 0.028). Patients with failed transplants were characterized by lower serum albumin (p < 0.0001) and higher hs‐CRP levels (p = 0.001) as compared with never transplanted HD patients. Conclusions: This study confirms the concept that retained failed allografts may induce chronic inflammation in chronic HD patients which may result in a worse nutritional status.     

Effect of triptolide on expression of thrombospondin-1 and transforming growth factor-β1 in renal tubular cells

Abstract

The objective of our study is to investigate the effect of triptolide on expression of thrombospondin-1 and transforming growth factor β1 in renal tubular cells. Human renal tubular epithelial cells were stimulated by different concentrations of triptolide (0.1, 1, and 10?μg/L) in the presence of angiotensin-II (10^?7?mol/L). Real Time PCR was used to detect the mRNA expression of thrombospondin-1 and transforming growth factor β1. Western blot analysis was used to detect the protein expression. ELISA was used to detect the level of total and active transforming growth factor β1. The mRNA expression of thrombospondin-1 (3.66?±?0.48 vs. 1.33?±?0.26, p?<?0.05) and transforming growth factor β1 (3.58?±?0.59 vs. 1.26?±?0.28, p?<?0.05) were up-regulated obviously when stimulated by angiotensin-II. And the protein expression of thrombospondin-1 (0.5126?±?0.0936 vs. 0.1025?±?0.0761, p?<?0.01) and transforming growth factor β1 (0.5948?±?0.0736 vs. 0.1318?±?0.0614, p?<?0.01) were also up-regulated simultaneously when stimulated by angiotensin-II. The expression of thrombospondin-1 and transforming growth factor β1 induced by angiotensin-II were down-regulated markedly with 1?μg/L and 10?μg/L of triptolide in mRNA and protein levels (p?<?0.05, p?< 0.01). And triptolide (1 and 10?μg/L) could reduce the expression of total and active transforming growth factor β1 (p?<?0.05, p?<?0.01). In conclusion, triptolide can inhibit the expression of thrombospondin-1 and transforming growth factor β1 in mRNA and protein levels and down-regulate the levels of total and active transforming growth factor β1.     

Agonist of peroxisome proliferator-activated receptor-gamma, rosiglitazone, reduces renal injury and dysfunction in a murine sepsis model.

BACKGROUND: Agonists of the peroxisome proliferator-activated receptor-gamma may help to regulate inflammation by modulating the production of inflammatory mediators and adhesion molecules. The purpose of this study was to determine the protective effects of rosiglitazone on renal injury in a sepsis model and to explore the mechanism. METHODS: In lipopolysaccharide (LPS)-induced mouse sepsis, we examined the effect of rosiglitazone on LPS-induced overproduction of inflammatory mediators, on the expression of adhesion molecules in renal tubular epithelial cells and on renal function. The mechanism of the protective effect was investigated in vitro using human renal tubular epithelial cells. RESULTS: Rosiglitazone significantly decreased serum tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta levels during sepsis. The levels of blood urea nitrogen and creatinine were significantly lower in mice pre-treated with rosiglitazone than that in LPS-treated mice. Rosiglitazone reduced the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tubular epithelial cells and interstitium of LPS-treated mice. Pre-treatment with rosiglitazone reduced the infiltration of macrophages/monocytes in renal tissue. In cultured tubular epithelial cells, rosiglitazone significantly decreased the expression of ICAM-1 and VCAM-1 induced by TNF-alpha or IL-1beta, inhibited the degradation of inhibitor kappaBalpha (IkappaBalpha) and blocked the activation of the p65 subunit of nuclear factor (NF)-kappaB. CONCLUSIONS: These results indicate that pre-treatment with rosiglitazone attenuated the production of TNF-alpha and IL-1beta and reduced adhesion molecule expression in renal tubular epithelial cells of LPS-treated mice. Rosiglitazone has an anti-inflammatory effect in renal tubular epithelial cells through the inhibition of NF-kappaB activation.     

Fibrosis and evidence for epithelial-mesenchymal transition in the kidneys of patients with staghorn calculi

Study Type – Therapy (case control) Level of Evidence 2b What’s known on the subject? and What does the study add? It has been shown that patients with nephrolithiasis did not have normal kidney function (retrospective study). Renal inflammation and fibrosis have been shown in kidney biopsies of nephrolithiasis patients, particularly those with brushite and cystine stones. No pathological change in kidney biopsies of patients with idiopathical calcium oxalate stone is noted. Our cross‐sectional study of patients with large kidney stone formation (mostly staghorn stones) shows that the patients have reduced overall kidney function regardless of stone type, and robust signs of inflammation and fibrosis are observed in stone‐containing renal tissues. Reduced kidney function is closely associated with the degree of renal fibrosis. This is the first study demonstrating that renal tubular cells in stone‐baring kidneys are undergoing epithelial‐mesenchymal transition, and TGF‐β₁ is overproduced in these mesenchymalized cells.

OBJECTIVES

? To quantify fibrotic lesions in renal tissues obtained from patients with large calculi and to evaluate association with renal function. ? Presence of epithelial‐mesenchymal transition (EMT) in stone‐containing renal tissues was investigated.

PATIENTS, SUBJECTS AND METHODS

? In all, 50 patients with nephrolithiasis with large calculi and matched healthy controls (37) were recruited. ? Plasma creatinine (Cr) and corrected Cr clearance (CCr) were determined in all subjects. ? Of the 50 patients, 38 had renal tissue available for histological analysis. Fibrosis was assessed by Masson’s trichrome staining. Co‐expression of epithelial cytokeratins and mesenchymal markers [α‐smooth muscle actin (αSMA) and vimentin] in renal tubular cells was detected by dual immunofluorescence staining. ? Expression of fibronectin, transforming growth factor β₁ (TGF‐β₁) and CD68 were investigated.

RESULTS

? Overall, the kidney function of the patients was significantly reduced, indicated by increased plasma Cr and decreased corrected CCr compared with healthy controls. ? Inflammation grading in renal tissues of the patients was correlated with the percentage of the fibrotic area. Renal fibrosis was inversely correlated with renal function. ? Cytokeratins co‐expressed with αSMA and vimentin were found in nephrolithiatic renal tubular cells, and these cells strongly expressed fibronectin and TGF‐β₁. ? Infiltration of CD68‐positive cells was a common finding in the inflamed renal sections.

CONCLUSIONS

? Kidneys of large stone‐forming patients had robust signs of inflammation and fibrosis, and there was a close correlation of renal fibrosis with renal dysfunction. ? This is the first study to show evidence for renal tubular cells showing signs of EMT in large stone‐containing kidneys. Plausibly, TGF‐β₁ triggers EMT, which at least in part contributes to large stone‐induced renal fibrosis.     

Nutritional vitamin D supplementation in haemodialysis: A potential vascular benefit?

Aim: Vitamin D deficiency is highly prevalent in end‐stage renal disease and has been associated with atherosclerosis, endothelial dysfunction and left ventricular hypertrophy. Although activated vitamin D has shown to be cardioprotective, the cardiovascular benefits of nutritional vitamin D (i.e. ergocalciferol or cholecalciferol) have not been explored in the dialysis population. The aim of this investigation was to evaluate the effect of ergocalciferol therapy on vascular adhesion molecules, markers of inflammation and atherosclerosis among haemodialysis patients. Methods: This was a pilot study of matched haemodialysis patients. For every patient enrolled taking ergocalciferol, an age and race matched control was recruited. Predialysis blood samples were collected and assayed for adhesion molecules (soluble vascular cell adhesion molecule‐1 (sVCAM‐1), soluble intercellular adhesion molecule‐1 (sICAM‐1), E‐selectin and P‐selectin), inflammatory cytokines (interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α)), oxLDL‐β₂GPI and IgG anticardiolipin. Results: A total of 40 haemodialysis patients were studied (20 on ergocalciferol therapy, 20 not receiving ergocalciferol therapy). Patients taking ergocalciferol had higher 25‐hydroxyvitamin D levels compared with those not taking ergocalciferol. Even though doxercalciferol usage and dosing was similar between groups, plasma sVCAM‐1, sICAM‐1 and P‐selectin concentrations were lower among ergocalciferol treated patients. No significant differences in E‐selectin, IL‐6, TNF‐α, oxLDL‐β₂GPI or anticardiolipin antibody levels were observed. Conclusion: Patients receiving ergocalciferol had lower plasma levels of vascular adhesion molecules despite equivalent use of activated vitamin D therapy. Future investigations should confirm the role of nutritional vitamin D therapy, in addition to activated D therapy, in haemodialysis patients and the potential vascular benefits of these agents.     

Elevated resistin is related to inflammation and residual renal function in haemodialysed patients

AIM: Resistin is an adipocytokine that recently generated much interest. Because of the fact that inflammation, endothelial cell damage or injury is invariably associated with such clinical conditions as thrombosis, atherosclerosis and their major clinical consequences, that is, cardiovascular disease and resistin play a role in linking inflammation and cardiovascular disease, the aim of the study was to assess resistin in correlation with markers of inflammation, endothelial cell injury and residual renal function in haemodialysed (HD) patients. METHODS: We assessed resistin, markers of coagulation: thrombin-antithrombin complexes (TAT), prothrombin fragments 1+2; fibrinolysis: tPA, plasminogen activator inhibitor type 1, plasmin-antiplasmin complexes (PAP); endothelial function/injury: von Willebrand factor (vWF), thrombomodulin, intracellular adhesion molecule (ICAM); inflammation: high sensitivity C-reactive protein (hsCRP), tumour necrosis factor alpha and interleukin-6 (IL-6). RESULTS: Healthy volunteers and HD patients did not differ significantly regarding age, leucocyte count, serum iron, aspartate and alanine aminotransferases activities, calcium, cholesterol, tPA concentration. Triglycerides, CRP (assessed by high sensitivity method), phosphate, urea, creatinine, IL-6, tumour necrosis factor alpha, vWF, prothrombin fragments 1+2, TAT, PAP, thrombomodulin, ICAM, plasminogen activator inhibitor type 1 and resistin, were elevated in HD patients when compared with the control group. Serum albumin, total protein, haemoglobin and haematocrit were significantly lower in HD patients when compared with the control group. In HD patients with hsCRP 0e; 6 mg/L, resistin, IL-6, vWF and F1+2 were significantly higher, whereas tPA was significantly lower than in patients with hsCRP<6 mg/L. Moreover, HD patients with residual renal function have significantly lower resistin when compared with patients without it. Resistin was significantly higher in diabetics. In HD patients, resistin correlated significantly, in univariate analysis, with calcium, phosphate, PTH, TIBC, vWF residual renal function, urea, hsCRP, IL-6 and tended to correlate with tPA and ferritin. In the healthy volunteers, resistin was related to IL-6 and hsCRP. In multiple regression analysis, resistin was independently related to hsCRP, IL-6, residual renal function in HD patients. CONCLUSION: Elevated resistin related to markers of inflammation may represent a novel link between inflammation and adipocytokines in HD patients. Impaired renal function and inflammation are responsible for elevated resistin in HD patients.     

Urinary kallikrein excretion is related to renal function change and inflammatory status in chronic kidney disease patients receiving angiotensin II receptor blocker treatment

Aim: This study was to evaluate the correlation of urinary kallikrein to renal function, proteinuria and urinary cytokines in chronic kidney disease patients in a longitudinal follow up. Method: We measured urinary kallikrein and cytokines in 50 patients who were followed up for 12 months. Results: Using regression model we found that the kallikrein excretion (estimated by log kallikrein/creatinine) was positively correlated to log estimated glomerular filtration rate in the beginning and the end of follow up (P = 0.049 and 0.006, respectively). No correlation existed between kallikrein excretion and proteinuria. The kallikrein excretion decreased after 12 months of follow up, which was also associated with the decrease of log estimated glomerular filtration rate. There was a significant positive correlation between the log urinary kallikrein and monocyte chemoattractant protein‐1 (MCP‐1) concentration (correlation coefficient = 0.277; P = 0.049). Urinary kallikrein excretion was also positively correlated with serum MCP‐1 level (correlation coefficient = 0.431; P = 0.002). No correlation existed between urinary kallikrein and transforming growth factor β‐1 or tumour necrosis factor‐α concentration. Conclusion: Urinary kallikrein excretion is positively correlated to renal function, serum and urinary inflammatory mediator MCP‐1 in chronic kidney disease patients. These findings indicate that urinary kallikrein excretion may reflect the change of renal function and kidney inflammatory status.     

IL‐10‐specific autoantibodies predict major adverse cardiovascular events in kidney transplanted patients ‐ a retrospective cohort study

End‐stage renal failure is associated with persistent systemic inflammation. The aim of this study was to investigate if systemic inflammation at the time of kidney transplantation is linked to poor graft survival, major adverse cardiovascular events (MACE), and increased mortality, and if these processes are modulated by naturally occurring cytokine‐specific autoantibodies (c‐aAbs), which have been shown to regulate cytokine activity in vitro. Serum levels of cytokines, high‐sensitivity C‐reactive protein (hsCRP) and c‐aAbs specific for interleukin (IL)‐1α, tumor necrosis factor (TNF)‐α, IL‐6, and IL‐10 were measured at the time of transplantation in a retrospective cohort study of 619 kidney transplanted patients with a median follow‐up of 4.9 years (range 1.2–8.2 years). Systemic inflammation was associated with all‐cause mortality in simple and multiple Cox regression analyses. IL‐10‐specific c‐aAbs were associated with MACE after transplantation, suggesting that IL‐10 may be a protective factor. Similarly, patients with a history of MACE before transplantation had lower levels of TNF‐α‐specific c‐aAbs, hence we hypothesized that TNF may be a risk factor of MACE. These findings support that pro‐inflammatory activity before transplantation is a pathological driver of MACE and all‐cause mortality after transplantation. This information adds to pretransplantation risk estimation in renal transplant candidates.     

Urinary angiotensinogen level is increased in renal transplant recipients with masked hypertension and is correlated with left ventricular mass index and albuminuria in these patients

Activation of the local renin‐angiotensin system (RAS) is an independent risk factor for the development of proteinuria and left ventricular hypertrophy (LVH) more commonly seen in masked hypertensives. It has been reported that urinary angiotensinogen (UAGT) level provides a specific index of the intrarenal RAS status. The aim of this study was to evaluate the association between UAGT and left ventricular mass index (LVMI) and urinary albumin‐creatinine ratio (UACR) in renal transplant recipients (RTRs) with masked hypertension (HT). A total of 116 non‐diabetic‐treated hypertensive RTRs were included in this study. The patients were divided into two groups: masked hypertensives and controlled hypertensives. Forty‐two (36.2%) of RTRs had masked HT. Mean UACR and LVMI levels were higher in RTRs with masked HT than in RTRs with controlled HT (P < 0.001). UAGT level was also higher in masked hypertensives compared to controlled hypertensives (P < 0.001). Multivariable regression analysis showed that UAGT was positively correlated with UACR (β = 0.024, P = 0.001) and LVMI (β = 0.082, P = 0.001) in masked hypertensives. Consequently, masked HT was considerably frequent (36.2%) in treated hypertensive RTRs and high UAGT levels accompanied by high albuminuria and LVMI levels were seen in these patients. Overproduction of the UAGT may play a pivotal role in the development of LVH and proteinuria in masked hypertensives.     

Effect of shock wave number on renal oxidative stress and inflammation

What’s known on the subject? and What does the study add? Oxidative stress and inflammation are tissue‐ and cell‐level components of shock wave lithotripsy (SWL)‐induced acute renal injury, which we recently showed to be localized principally to the medulla within the focal zone of the lithotripter. This study reports that the magnitude of the oxidative stress and inflammation observed in the medulla after SWL is dependent on the number of shock waves delivered to the kidney, indicating that this is a sensitive measure of renal injury caused by shock waves. OBJECTIVE To determine if the magnitude of the acute injury response to shock‐wave lithotripsy (SWL) depends on the number of SWs delivered to the kidney, as SWL causes acute renal oxidative stress and inflammation which are most severe in the portion of the kidney within the focal zone of the lithotripter. MATERIALS AND METHODS Pigs (7–8 weeks old) received 500, 1000 or 2000 SWs at 24 kV from a lithotripter to the lower pole calyx of one kidney. At 4 h after treatment the kidneys were removed, and samples of cortex and medulla were frozen for analysis of the cytokine, interleukin‐6, and for the stress response protein, heme oxygenase‐1 (HO‐1). Urine samples taken before and after treatment were analysed for the inflammatory cytokine, tumour necrosis factor‐α. For comparison, we included previously published cytokine data from pigs exposed to sham treatment. RESULTS Treatment with either 1000 or 2000 SWs caused a significant induction of HO‐1 in the renal medulla within the focal zone of the lithotripter (F2, 1000 SWs, P < 0.05; 2000 SWs, P < 0.001). Interleukin‐6 was also significantly elevated in the renal medulla of the pigs that received either 1000 or 2000 SWs (P < 0.05 and <0.001, respectively). Linear dose–response modelling showed a significant correlation between the HO‐1 and interleukin‐6 responses with SW dose (P < 0.001). Urinary excretion of tumour necrosis factor‐α from the lithotripsy‐treated kidney increased only for pigs that received 2000 SWs (P < 0.05). CONCLUSION The magnitude of renal oxidative stress and inflammatory response in the medulla increased with the number of SWs. However, it is not known if the HO‐1 response is beneficial or deleterious; determining that will inform us whether SWL‐induced renal injury can be assessed by quantifying markers of oxidative stress and inflammation.     

Recent advancement of understanding pathogenesis of type 1 diabetes and potential relevance to diabetic nephropathy

Type 1 diabetes mellitus is an autoimmune disease characterized by progressive destruction of pancreatic beta cells by genetic and environmental factors which leads to an absolute dependence of insulin for survival and maintenance of health. Although the majority of mechanisms of beta cell destruction remain unclear, many molecules, including proinflammatory cytokines and chemokines such as tumor necrosis factor alpha and monocyte chemoattractant protein-1, are implicated in the development of beta cell damage. Furthermore, beta cell destruction is enhanced by the Th1 and Th17 subsets of CD4+ T cells. In contrast, there are mechanisms involved in the maintenance of peripheral tolerance by regulatory T cells, the function of which depends on the pleiotropic cytokine transforming growth factor beta. Development and progression of renal injuries in patients with diabetic nephropathy are also associated with several growth factors and proinflammatory cytokines, including tumor necrosis factor alpha, insulin-like growth factor-1, monocyte chemoattractant protein-1, vascular endothelial growth factor, and transforming growth factor beta. Although the pathogenic mechanisms underlying type 1 diabetes and diabetic nephropathy are principally different, i.e., autoimmunity and inflammation, some common factors, including susceptibility genes and proinflammatory cytokines, are involved in both mechanisms, including infiltrating cell recruitment, upregulation of other cytokines and chemokines, or apoptosis.     

Renal PPARγ mRNA expression increases with impairment of renal function in patients with chronic kidney disease

Aim: Peroxisome proliferator‐activated receptor gamma (PPARγ) is generally accepted as renoprotective factor in type 2 diabetes mellitus, and PPARγ agonists have been reported to reduce albuminuria. However, little is known about renal PPARγ expression in chronic kidney disease, and especially human data are scarce. We hypothesized that renal PPARγ expression is associated with extent of proteinuria, kidney function, histological diagnosis and inflammatory mediators. Therefore, we investigated PPARγ mRNA expression in human kidney biopsies. Methods: We quantified PPARγ mRNA as well as the expression of macrophage chemoattractant protein‐1, transforming growth factor beta‐1 and interleukin‐6 in 64 human kidney biopsies from patients with chronic kidney disease and mild‐to‐marked proteinuria of diverse aetiology. We measured renal function, and macrophage invasion was quantified by CD68 and vascularization by CD34 immunostaining. Results: PPARγ mRNA expression correlated inversely with renal function. Higher blood pressure levels were associated with higher PPARγ expression levels. PPARγ mRNA expression correlated significantly (P < 0.001) with macrophage chemoattractant protein‐1 mRNA expression and showed a negative trend with transforming growth factor beta‐1 mRNA expression. No differences in PPARγ expression were detected with regard to extent of proteinuria, histological diagnosis, macrophage invasion, interleukin‐6 expression, and age or body mass index. Conclusions: PPARγ expression increases with loss of renal function and may be an important factor in maintaining normal renal function serving as a key protective mechanism to renal injury.     

A novel murine model of allogeneic vaccination against renal cancer

OBJECTIVES

To develop a murine model for whole‐cell allogeneic vaccination in renal cancer, as such vaccines aim to direct immune responses against patient tumour cells, due to shared antigens between the vaccine and tumour cells.

MATERIALS AND METHODS

A novel murine renal cell line, allogeneic to BALB/c, was developed from a C57BL/6 mouse by primary cell culture (RVIK). It was immortalized by HPV16 E6/E7 and transfected with ras in an attempt to improve its immunogenicity. The cell line was characterized and tested as a vaccine in a BALB/c tumour‐protection model after subsequent tumour challenge with autologous RenCa tumour cells.

RESULTS

RVIK alone, with no ras induced cross‐reactive immunity, providing a valid non‐tumorigenic allogeneic whole‐cell vaccine model for renal cancer. Ras transfection per se did not improve RVIK immunity.

CONCLUSIONS

RVIK is a novel immunogenic murine renal epithelial cell line, which confers protection when used as an allogeneic vaccine. It provides proof of principle for the effectiveness of allogeneic whole‐cell vaccines and may therefore form the basis of a useful model of allogeneic vaccination to further optimize vaccination schedules, formulation and adjuvants for a clinical setting.     

RIPK3‐Mediated Necroptosis Promotes Donor Kidney Inflammatory Injury and Reduces Allograft Survival

Kidney transplant injury occurs with ischemia and alloimmunity. Members of the receptor interacting protein kinase family (RIPK1,3) are key regulators of “necroptosis,” a newly recognized, regulated form of necrosis. Necroptosis and apoptosis death appear to be counterbalanced as caspase‐8 inhibition can divert death from apoptosis to necrosis. Inhibition of necroptosis in donor organs to limit injury has not been studied in transplant models. In this study, necroptosis was triggered in caspase inhibited tubular epithelial cells (TEC) exposed to tumor necrosis factor alpha in vitro, while RIPK1 inhibition with necrostatin‐1 or use of RIPK3^?/? TEC, prevented necroptosis. In vivo, short hairpin RNA silencing of caspase‐8 in donor B6 mouse kidneys increased necroptosis, enhanced high‐mobility group box 1 release, reduced renal function and accelerated rejection when transplanted into BALB/c recipients. Using ethidium homodimer perfusion to assess necrosis in vivo, necrosis was abrogated in RIPK3^?/? kidneys postischemia. Following transplantation, recipients receiving RIPK3^?/? kidneys had longer survival (p = 0.002) and improved renal function (p = 0.03) when compared to controls. In summary, we show for the first time that RIPK3‐mediated necroptosis in donor kidneys can promote inflammatory injury, and has a major impact on renal ischemia–reperfusion injury and transplant survival. We suggest inhibition of necroptosis in donor organs may similarly provide a major clinical benefit.

     

Effects of combined valproic acid and the epidermal growth factor/vascular endothelial growth factor receptor tyrosine kinase inhibitor AEE788 on renal cell carcinoma cell lines in vitro

OBJECTIVE

To evaluate adhesion and growth inhibiting effects of the multiple receptor tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) on renal cell carcinoma (RCC) cells.

MATERIALS AND METHODS

Caki‐1 cells were treated with AEE788 and VPA, either alone or in combination, to investigate RCC cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins. Tumour cell proliferation was examined by MTT dye reduction assay. Effects of drug treatment on cell signalling pathways were determined by Western blotting. The expression levels of integrin α and β subtypes were evaluated by flow cytometry (surface expression) and Western blotting (intracellular protein expression).

RESULTS

RCC cell treatment with AEE788 and VPA in combination resulted in a stronger inhibition of tumour cell proliferation than that caused by either drug alone. There were also additive effects of the combined treatment on tumour cell adhesion to endothelial cells and to immobilized laminin (but not to immobilized fibronectin and collagen). AEE788 alone or combined with VPA reduced Akt expression and histone H3 acetylation. Both compounds altered integrin α and β subtype expression, in particular α1, α3 and β4, and blocked integrin‐dependent integrin‐linked kinase and focal‐adhesion kinase (total and phosphorylated) signalling.

CONCLUSIONS

Both AEE788 and VPA profoundly block the interaction of RCC cells with endothelium and extracellular matrix and reduce tumour growth in vitro. Therefore, this combined regimen warrants further preclinical and possible clinical study for treating advanced RCC.     

Different inflammatory cytokines release after open and endovascular reconstructions influences wound healing

Prodromal signs of a non‐healing wound after revascularisation, which might be strictly linked with impending failure of vascular reconstructions, are associated with an inflammatory response mediated by several circulating adhesion molecules, extracellular endopeptidases, and cytokines. The aim of our study was to investigate the role of selected plasma biomarkers in the prediction of both wound healing and failure of infrapopliteal vein graft or percutaneous trans‐luminal angioplasty (PTA) with selective stent positioning of the superficial femoral artery (SFA) in a population affected with critical limb ischaemia. A total of 68 patients who underwent either surgical or endovascular revascularisation of the inferior limb with autologous saphenous vein infrapopliteal bypass or PTA and selective stenting of the SFA were enrolled in our study. Patients were divided into two groups according to treatment: 41 patients were included in Group 1 (open surgery) and 27 in Group 2 (endovascular procedure). Plasma and blood samples were collected on the morning of surgery and every 6 months thereafter for up to 2 years of follow‐up or until an occlusion occurred of either the vein bypass graft or the vessel treated endovascularly. Fifteen age‐matched healthy male volunteers were considered a reference for biological parameters. Vascular cell adhesion molecule 1 [VCAM‐1]/CD106, inter‐cellular adhesion molecule‐1 [ICAM‐1]/CD54), interleukin‐1 (IL‐1), interleukin‐6 (IL‐6), tumour necrosis factor alpha (TNF‐α), and metalloproteinases (MMP)‐2 and ‐9 plasma levels were measured with enzyme‐linked immunosorbent assay (ELISA) kits. The mean observed time to heal of 54 wounds was 13 ± 4 months, with no statistically significant differences among the groups . The healing failure of the remaining wounds was strictly related to an unsuccessful open (n = 12) or endovascular (n = 8) treatment. The 2‐year primary patency rate was 65% (SE = .09) in Group 1 and 52% (SE = .1) in Group 2. When compared with mean concentration values of Group 1, VCAM‐1 and ICAM‐1 were always significantly higher during follow‐up in patients of Group 2 (P < .05). Furthermore, in the same group, IL‐6 and tumour necrosis factor alpha (TNF‐α) were found to be significantly higher at 6‐ and 12‐month (P < .05) when compared with surgically treated patients. Cox regression analysis showed that elevated plasma levels of VCAM‐1, ICAM‐1, IL‐6, and TNF‐α during follow up were strongly related to impaired wound healing and/or revascularisation failure (P < .05). Elevated plasma levels of inflammatory markers VCAM‐1, ICAM‐1, IL‐6, and TNF‐α may be related to the failure of wound healing and revascularisation procedures. Interestingly, we have observed that endovascular treatments cause a higher level of these inflammation biomarkers when compared with a vein graft, although wound‐healing and patency and limb salvage rates are not influenced.     

Active TGF‐β1 Expression in kidney transplantation: a comparative study of Cyclosporin‐A (CyA) and tacrolimus (FK506)

Abstract Chronic rejection is a major cause of graft dysfunction following kidney transplantation. This fibroproliferative disease may be promoted by overproduction of transforming growth factor beta (TGF‐β). Previous studies have suggested that cyclosporin‐A (CyA) might increase production of this growth factor. The current study was designed to measure the expression of TGF‐β in renal transplant biopsies from patients immunosuppressed with either CyA or tacrolimus. Paraffin‐embedded renal biopsies were sectioned, dewaxed and incubated with primary antibody against active TGF‐β₁ antibody. After washing, the sections were treated with secondary antibody conjugated with fluorescein isothiocyanate (FITC). In each case the sections were assessed by semi‐quantitative scanning laser confocal microscopy. Biopsies from patients receiving CyA expressed significantly more active TGF‐β₁ than biopsies from patients receiving tacrolimus (P < 0.0001, Mann‐Whitney test). The increased level of active TGF‐β₁ expression in renal biopsies of patients receiving CyA may indicate a mechanism of chronic rejection.