Ondřej Viklický

SDZ-RAD PREVENTS MANIFESTATION OF CHRONIC REJECTION IN RAT RENAL ALLOGRAFTS

BACKGROUND Chronic rejection remains the most frequent cause of renal graft loss over the long term. However, effective treatment of this process is not yet available. SDZ-RAD (40-O-[2-hydroxyethyl]-rapamycin) is a new, orally active rapamycin derivative with potent immunosuppressive activity. We have examined the effects of SDZ-RAD in a well-established model of chronic renal allograft rejection in rats. METHODS Kidneys of Fisher (F334) rats were orthotopically transplanted into bilaterally nephrectomized Lewis recipients. To suppress an initial episode of acute rejection, rats were briefly treated with low doses of cyclosporine for the first 10 days. Thereafter they received either SDZ-RAD (0.5 mg/kg(day) or vehicle. At 24 weeks, functional evaluations were performed, kidneys were harvested, and histological, immunohistological, and reverse transcription-polymerase chain reaction evaluations were performed. RESULTS Animals treated with SDZ-RAD developed lower proteinuria and less glomerulosclerosis as compared with controls. Additionally SDZ-RAD reduced the infiltration of macrophages and lymphocytes and the expression of intercellular adhesion molecule-1, laminin, and fibronectin. Furthermore, we observed a reduced expression of growth factor mRNA (transforming growth factor-beta and platelet-derived growth factor-AA) in these animals. CONCLUSION Our results demonstrated that SDZ-RAD effectively ameliorates chronic renal allograft rejection in rats, probably mediated by suppression of growth factors.

Association between heat shock protein 70s and Toll‐like receptor polymorphisms with long‐term renal allograft survival

Long‐term renal allograft survival has not improved significantly in recent years and only a minority of grafts survives for more than 15 years. To evaluate the association between HSPA1A G(190)C, HSPA1B A(1267)G and TLR4 A(299)G polymorphisms and allograft survival we analyzed DNA of patients with long‐term renal graft function over 15 years (Tx15), consecutively transplanted recipients (Tx), patients with acute rejection and healthy controls. HSPA1B (1267)AA was less prevalent in Tx versus Tx15 (P = 0.02) and versus controls (P = 0.004). HSPA1B (1267)GG was more frequent in Tx versus Tx15 (P = 0.005) and versus controls (P = 0.002). HSPA1B (1267)G allele occurred more often in Tx versus Tx15 (P = 0.03), and versus controls (P = 0.02). TLR4 (299)AG genotype prevalence was increased in Tx15 versus Tx (P = 0.02), while TLR4 (299)G allele was more frequent in Tx15 versus Tx (P = 0.02). The increased frequency of HSPA1B (1267)AA and TLR4 (299)AG genotypes in Tx15 group indicates that better cytoprotective functions in HSPA1B (1267)AA and reduced proinflammatory response in TLR4 (299)AG carriers might have improved renal allograft survival.

Pregnancy-associated plasma protein a and soluble receptor for advanced glycation end products after kidney transplantation.

Background: Pregnancy-associated plasma protein A (PAPP-A) and soluble receptor for advanced glycation end products (sRAGE) are new markers related to vascular damage. Methods: Thirty-one patients who had undergone kidney transplantation (TX) in the year 2005 under tacrolimus-based immunosuppression were included in this prospective single-center study. PAPP-A and sRAGE were determined before TX and 2 and 6 weeks and 3 months after TX. The results were correlated with the 3-month protocol kidney graft biopsy findings. Results: Both PAPP-A and sRAGE decreased after TX (mean values in serum: PAPP-A 20.8, 13.7, 12.1, and 10.7 mIU/l, respectively, before and 2 and 6 weeks and 3 months after TX, p < 0.001; sRAGE 4,403.4, 2,512.7, 1,909.0, and 1,817.6 pg/ml, respectively, before and 2 and 6 weeks and 3 months after TX, p < 0.001) and were correlated with the graft function (PAPP-A vs. modification of diet in renal disease formula r = –0.52, p < 0.001; sRAGE vs. modification of diet in renal disease formula r = –0.54, p < 0.001). Additionally, the PAPP-A levels correlated with interstitial inflammation (r = 0.57, p < 0.05) and vascular intimal thickening (r = 0.47, p < 0.05), while sRAGE correlated with arteriolar hyalinosis (r = 0.49, p < 0.05). Conclusions: Our study demonstrates the role of the kidney in the metabolism and/or the removal of PAPP-A and sRAGE. After successful TX, these substances decrease, and, on the contrary, early chronic vascular changes in the kidney TX are associated with elevation of their serum levels.

Genetic variability of major inflammatory mediators has no impact on the outcome of kidney transplantation.

Background. Functionally relevant polymorphisms in genes of the Th1 and Th2-inflammatory pathway influence the susceptibility to acute rejection (AR), chronic allograft nephropathy (CAN), and subclinical rejection (SR) as well as graft survival after renal transplantation. Because these findings have not been validated, we sought confirmatory evidence of these associations in a larger group of renal transplant recipients. Methods. A total of 436 kidney transplant recipients were genotyped for 9 single nucleotide polymorphisms (TNF-&agr;-308G/A, MCP-1-2518A/G, RANTES-403G/A, −109T/C and −28C/G, CCR2+190G/A, IFN-&ggr;+874A/T, TGF-&bgr;+869T/C and +915G/C) and for the 32-bp indel polymorphism in CCR5. The effects of these polymorphisms on the incidence of AR, SR, CAN and graft survival were analyzed in single locus and haplotype models. Results. Single locus analysis revealed that there was no significant difference in the distribution of the genotype frequencies between patients with and without AR, and between patients with CAN or SR, and individuals without CAN. Furthermore, no influence of any of the polymorphisms on the long-term graft survival was observed. Haplotype [TGF-&bgr; +869G; TGF-&bgr; +915C] seemed to be associated with the presence of SR (odds ratio: 3.45, 95% confidence interval: 1.19 – 9.99, P=0.023), but the association was nonsignificant due to the insufficient power. Conclusion. In contrast to previous allelic association studies, neither of the polymorphisms has been associated with the outcome of kidney transplantation in the single locus analysis nor in the haplotype model. Our findings reinforce the need for more rigorous research compliant with the currently accepted standards for polymorphism-disease association studies.

The effect of different immunosuppressive regimens on TGF-β1 expression in kidney transplant patients

Transforming growth factor (TGF)‐β1 is a key profibrogenic cytokine associated with the pathogenesis of chronic allograft nephropathy (CAN). The primary aim of this study was to evaluate TGF‐β1 expression in protocol kidney graft biopsy in patients treated with different immunosuppressive regimens. Protocol kidney graft biopsies were carried out in 77 patients with stable graft function at 1 year after kidney transplantation, treated with a triple‐drug regimen based on cyclosporin A (CyA; n = 49) or tacrolimus (TAC; n = 28). Morphological findings were assessed using the Banff 97 classification. TGF‐β1 expression was analysed using immunochemistry, and semiquantitatively scored in different renal structures (total score 0–18). Clinical data were analysed at the time of biopsy, and 12 months thereafter. No significant relation was found between the used immunosuppressive regimen and the histomorphological picture in the graft. TGF‐β1 expression within graft tissue was significantly higher in patients treated with CyA when compared with TAC (9.94 ± 4.2 vs. 5.0 ± 3.2; P < 0.001). Serum creatinine and glomerular filtration rate (GFR; Cockroft‐Gault calculation) were comparable in both groups but, in the course of the next 12 months, GFR significantly decreased only in the CyA‐treated group (from 1.03 ± 0.33 to 0.96 ± 0.37 ml/s) while not changing in the TAC‐treated group. Patients treated with TAC had significantly lower diastolic blood pressure and serum cholesterol. The significantly lower TGF‐β1 expression in 1‐year protocol kidney graft biopsy in TAC‐treated patients with stable renal function, and the different development of graft function in both groups suggest a possible benefit of TAC for long‐term graft acceptance.

ACE gene polymorphism and long-term renal graft function.

OBJECTIVES The long-term outcome of transplanted kidneys has not changed substantially and only a minority of grafts survives more than 15 yr. The aim of this study was to determine the influence of ACE gene polymorphism on long-term outcome after renal transplantation. DESIGN AND METHODS Using PCR, we evaluated ACE I/D gene polymorphism in a group of patients with long-term graft function (LTF) over 15 yr and compared it with control groups of transplant recipients and population sample. RESULTS The distribution of genotypes in the LTF group differed from transplant controls (p < 0.05). Moreover, DD homozygotes in the LTF group had better creatinine clearance (DD: 1.1 +/- 0.3, ID: 0.96 +/- 0.3, II: 0.76 +/- 0.3 mL/s; p < 0.05). There were no differences in genotype distribution between transplant and population samples. CONCLUSIONS Results of our study have demonstrated a possible connection between the DD variant of ACE I/D gene polymorphism and excellent long-term graft function.

SLCO1B1 polymorphism is not associated with risk of statin-induced myalgia/myopathy in a Czech population.

Background Gene SLCO1B1, encoding solute organic anionic transport polypeptide OATP1B1, belongs to the group of candidates potentially influencing statin treatment safety. OATP1B1 regulates (not only) the hepatic uptake of statins. Its genetic variation was described as an important predictor of statin-associated myopathy in a cohort of patients treated with a maximum dose of simvastatin. However, the impact of SLCO1B1 gene polymorphism on this risk in patients treated with other statins or lower doses of simvastatin needs to be assessed. Therefore, we performed the present study. Material/Methods SLCO1B1 tagging rs4363657 polymorphism was analyzed in 2 groups of patients with dyslipidemia (treated with simvastatin or atorvastatin, 10 or 20 mg per day), subgroup with statin-induced myalgia (N=286), and subgroup (N=707) without myalgia/myopathy, and in 2301 population controls without lipid-lowering treatment. Results Frequency of the individual genotypes in patients with myalgia/myopathy (TT=62.3%, CT=34.5%, CC=2.8%) did not significantly differ (both P values over 0.19) from that in patients without muscle symptoms (TT=61.4%, CT=32.9%, CC=5.7%) or from the population controls (TT=63.9%, CT=32.5%, CC=3.6%). Null results were also obtained for the dominant and recessive models of the analysis. Conclusions In Czech patients treated with low statin doses, there is no association between SLCO1B1 gene polymorphism and risk of myalgia/myopathy.

Mycophenolate Mofetil Ameliorates Accelerated Progressive Nephropathy in Rat

Background: Renal ischemia and hypertension have been suggested to be involved in the progression of renal diseases. Recently, we developed a model of accelerated major histocompatibility complex-independent renal injury, where high-renin hypertension aggravates functional and morphological changes induced by ischemia/reperfusion (I/R). In this model, we evaluated the effect of immunosuppressant mycophenolate mofetil (MMF) to test its capability to slow the progression of accelerated nephropathy. Methods: 34 anesthetized uninephrectomized hypertensive transgenic (mREN2)27 rats (TGR) received a clamp on the renal pedicle for 45 min. Animals were treated with MMF 10 mg/kg/day (n = 10), 20 mg/kg/day (n = 10) or placebo (n = 10) orally via gavage for 12 weeks. Four animals were sham operated and not treated. Proteinuria and blood pressure were evaluated throughout the experiment. At the end of the experiment, kidney function was evaluated and kidneys harvested for morphological analysis and immunohistochemistry (CD4+, CD8+ lymphocytes and specific rat monocyte/macrophage marker ED-1+ cells). Results: At week 12, both MMF-treated groups had lower proteinuria as compared to the placebo group (MMF 10: 22.4 ± 9.8, MMF 20: 20.9 ± 5.6 vs. 126.7 ± 35.8; p < 0.01; sham 28.1 ± 1.4 mg/day) and reduced glomerulosclerosis (MMF 10: 11.4 ± 7.8, MMF 20: 5.2 ± 2.7 vs. 20.9 ± 10.9; p < 0.05; sham 15.7 ± 9.2%). There were no differences in systolic blood pressure among groups. MMF-treated rats had lower CD4+ (MMF 10: 61.2 ± 46.4, MMF 20: 29.3 ± 18.2 vs. 125.3 ± 42.8; p < 0.01, sham 84.9 ± 6.1 cells/field of view) and CD8+ (MMF 10: 13.7 ± 10.2, MMF 20: 10.0 ± 8.1 vs. 37.8 ± 14.3; p < 0.01; sham: 31.8 ± 7.6 cells/field of view) lymphocytes infiltration and ED-1 macrophages infiltration (MMF 10: 5.5 ± 6.4, MMF 20: 2.5 ± 2.8 vs. 16.7 ± 4.1; p < 0.01; sham 12.2 ± 4.6 cells/field of view) than placebo-treated rats. Conclusion: Our results thus support the hypothesis about the key role of immune mechanisms in progression of chronic nephropathies.

RAGE polymorphisms, renal function and histological finding at 12 months after renal transplantation☆

OBJECTIVES Rage (receptor for advanced glycation end products) is involved in pathogenesis of many diseases. The aim of the study was to test whether polymorphisms of RAGE gene are associated with the outcome of kidney transplantation. DESIGN AND METHODS Four polymorphisms of the RAGE gene (-429T/C, -374T/A, Gly82Ser and 2184A/G) were assessed in 145 renal transplant recipients and their relationship to histological changes in 12 months protocol kidney graft biopsy and renal function was examined. RESULTS Genotype frequencies of each polymorphism corresponded to expected frequencies according to Hardy-Weinberg equilibrium. No differences between allelic and genotype frequencies among patients with normal histological findings, chronic allograft nephropathy and subclinical rejection were observed. CONCLUSION This is the first study on polymorphisms of the RAGE gene in patients with the transplanted kidney. No association of RAGE selected gene polymorphisms with 12-months outcome of renal transplants was shown in study.

G-Protein Beta-3-Subunit and eNOS Gene Polymorphism in Transplant Recipients with Long-Term Renal Graft Function

Background: Despite new immunosuppressive drugs, only a minority of graft survive over 15 years. The aim of our study was to determine the influence of gene polymorphisms in the G-protein-β3 subunit (Gβ3) and endothelial nitric oxide synthase (eNOS) on the long-term outcome of kidney grafts. Methods: Using PCR, corresponding genotypes in Gβ3 (C825T) and eNOS (G894T) gene polymorphism were evaluated in patients with preserved graft function over 15 years and in a control group of transplant recipients. Results: There were no differences in allele and genotype distributions of both polymorphisms between groups. In Gβ3 polymorphism, the 825T allele carriers had a significantly lower body mass index while in eNOS polymorphism there were no links between genotypes, renal function and atherosclerosis risk factors. Conclusions: Our data suggest that these gene polymorphisms have only a minor influence on long-term renal graft function.