Ewa Kwiatkowska

The impact of CTLA4 and PTPN22 genes polymorphisms on long-term renal allograft function and transplant outcomes

Abstract Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) downregulates the immune system. Lymphoid tyrosine phosphatase (Lyp)—PTPN22 protein—is suggested to be negative regulator of T-cell reaction. There are several polymorphisms in the CTLA4 and PTPN22 genes, which can influence the immune response and allograft function after kidney transplantation. The aim of this study was to examine the impact of CTLA4 and PTPN22 genes polymorphisms on the long-term renal transplant function and recipients’ outcomes during a 5-year follow-up observation. The study enrolled 268 Caucasian renal transplant recipients. Genotyping of the rs231775 (+49AG) CTLA4 gene polymorphism was performed using real-time PCR and rs2476601 (C1858T) PTPN22 gene polymorphism using PCR-RFLP method. The 5-year graft survival rate was 81.7%. Dialysis was necessary in 22 (8%) patients, 7 (2.6%) patients died and 20 (7.4%) switched to another transplantation center. We found no association between studied polymorphisms and graft loss/dialysis. Comparison of the distribution of the +49AG CTLA4 and C1858T PTPN22 genes polymorphisms genotypes among dead and living patients showed no statistically significant differences. Above results suggest that the rs231775 (+49AG) CTLA4 and rs2476601 (C1858T) PTPN22 genes polymorphisms are not associated with long-term allograft failure, graft loss and mortality after transplantation.

Does Glucose Present in the Dialysate Limit Oxidative Stress in Patients Undergoing Regular Hemodialysis

Background: Decreased glucose concentration in the blood causes the inhibition of the hexose monophosphate (HMP) cycle in the erythrocyte. NADPH, which is the source of the reductive equivalents necessary for the reproduction of glutathione (GSH), is not regenerated. The presence of glucose in dialysate should provide the stability of its concentration in the blood of patients undergoing hemodialysis (HD). The aim of the study was to assess the influence of glucose in the dialysate on the intensity of oxidative stress in patients undergoing regular HD. Methods: The study comprised 43 patients hemodialyzed with dialysate containing (HD-g(+)) or not containing glucose (HD-g(–)). The concentrations of the products of reaction with thiobarbituric acid-reactive substance (TBARS) and GSH as well as the activity of erythrocyte superoxide dismutase were determined. Glucose concentrations in the blood before and immediately after dialysis were also measured. Results: After flow-through dialysis the glucose concentration in the blood decreases both when dialysate does not contain glucose (4.8 vs. 1.6 mmol/l) and when dialysate contains glucose (6.6 vs. 5.8 mmol/l). HD caused changes in the TBARS concentration: in the HD-g(+) group the concentration decreased after HD, whereas in the HD-g(–) group it increased. In both groups of patients studied the GSH concentration changed after HD; in the HD-g(–) group it decreased and in the HD-g(+) group it increased. The results obtained in the groups of patients examined were confirmed by in vitro studies. Conclusions: The presence of glucose in the dialysate guarantees the normal activity of the HMP cycle, which provides the production of reductive equivalents for the regeneration of reduced GSH – free radicals scavenger – and therefore the limitation of oxidative stress.

A Common Profile of Disordered Angiogenic Factor Production and the Exacerbation of Inflammation in Early Preeclampsia, Late Preeclampsia, and Intrauterine Growth Restriction

Preeclampsia and intrauterine growth restriction are two separate disease entities that, according to numerous reports, share the same pathogenesis. In both, angiogenesis disorders and generalized inflammation are the dominant symptoms. In this study, we hypothesized that both diseases demonstrate the same profile in early preeclampsia, late preeclampsia, and intrauterine growth restriction patients, with the only difference being the degree of exacerbation of lesions. One hundred sixty-seven patients were enrolled in the study and divided into four groups: early preeclampsia, late preeclampsia, and intrauterine growth restriction groups, and one control group. Concentrations of the angiogenesis and inflammatory markers soluble fms-like tyrosine kinase receptor 1, placental growth factor, high-sensitivity C-reactive protein, and interleukin-6 were determined, and the behavior of these markers and correlations among them were studied. Higher concentrations of soluble fms-like tyrosine kinase receptor 1, high-sensitivity C-reactive protein, and interleukin-6 and a lower concentration of placental growth factor were observed in the study groups compared with the control group. No differences in concentrations of the studied markers were found among the study groups but significant correlations were observed. The higher values for the angiogenesis and inflammatory markers both in preeclampsia patients and patients with intrauterine growth restriction of placental origin compared with the control group suggest the existence of the same underlying disorders in the development of these pathologies. The observed mutual correlations for disordered angiogenesis and inflammatory markers are suggestive of a mutual relationship between these processes in the development of pathologies evolving secondary to placental ischemia. The same lesion profile was observed for both preeclampsia and ‘placental’ intrauterine growth restriction patients, which could be used in developing common diagnostic criteria for pregnant patients.

Urinary Metalloproteinases-9 and -2 and Their Inhibitors TIMP-1 and TIMP-2 are Markers of Early and Long-Term Graft Function After Renal Transplantation.

Background/Aims: Renal ischemia-reperfusion (I-R) injury (IRI) is an inseparable feature of organ transplantation and may have a negative impact on the graft, its function and survival. Acute tubular necrosis, which is reversible thanks to the regenerative capacity of renal tubular epithelial cells, is the main cause of acute renal failure secondary to IRI. MMP-2 and MMP-9 are proteolytic enzymes involved in digesting proteins that are components of the extracellular matrix (ECM) and the basement membrane of the nephrons. This way post-reperfusion MMP activation allows the inflammatory process to spread. Methods: In our studies, we focused on identifying whether the concentrations of MMP-2 and MMP-9 and their natural inhibitors TIMP-1 and TIMP-2 in urine sample at day 1 and day 30 as well as after 12 months following renal transplantation are markers of early and long-term renal function during meanly five-years observation. Moreover, in urine sampled at months 6 and 12 after renal transplantation, we determined the content of TGF-β as a graft fibrosis indicator. Results: MMP-9 concentration in the early post-transplant period is a major marker of early and long-term function of the transplanted kidney. Its increased concentration was correlated with lesions related to tubular atrophy and fibrosis in renal biopsies performed at months 3 and 12 after transplantation. Its concentration is correlated with TGF-β content in a later period. Conclusions: TIMP-1 and-2 are primarily markers of an early function of the transplanted kidney. Early post-transplant concentration of MMP-2 is a marker of proteinuria in early and long-term post-transplant periods.

Correlation between ICAM1 and VCAM1 gene polymorphisms and histopathological changes in kidney allograft biopsies

Introduction The immunoglobulin-like molecules intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) are responsible for endothelial cell-leukocyte adhesion followed by transmigration of leukocytes through the endothelial cell lining. The aim of this study was to examine the correlation between polymorphisms in ICAM1 and VCAM1 genes and histopathological changes in transplanted kidney biopsies. Material and methods The study enrolled 82 Caucasian renal transplant recipients (48 males, 34 females). Genotyping of the rs5498 ICAM1 and the rs1041163 and rs3170794 VCAM1 gene polymorphisms was performed using real-time polymerase chain reaction (PCR). Biopsies were performed in 82 patients and were reviewed by a renal pathologist and the Banff working classification criteria were used. Results There were no significant associations between VCAM gene polymorphisms and histopathological changes in kidney allograft biopsies. ICAM1 gene polymorphism was associated with the grade of interstitial fibrosis. Interstitial fibrosis was more severe among individuals with the G allele than those with the A allele (AA vs. GG+AG, p = 0.017). There were no statistically significant associations between ICAM1 gene polymorphism and other histopathological changes in kidney allograft biopsies. Conclusions The results of our study suggest that rs5498 ICAM1 gene polymorphism is associated with the grade of interstitial fibrosis in kidney recipients and the changes are more severe in patients with the G allele.

Oxidative Stress Indices in Rats Under Immunosuppression

Immunosuppressants lead to generation of reactive oxygen species (ROS). Oxidative stress (OxS) can initiate chronic allograft nephropathy (CAN). The most active antioxidant enzymes, superoxide dysmutase (SOD) and catalase (CAT), are present in erythrocytes. Glutathione peroxidase (GPx) is produced in the proximal tubules of nephrons. Malonyldialdehyde (MDA) concentrations are a marker of OxS intensity in plasma. In vitro and animal model studies have shown increased or decreased OxS during treatment with tacrolimus (Tac) or cyclosporine (CyA). Results obtained in humans after solid organ transplantation have been contradictory, because of confounding factors such as ischemia-reperfusion injury, donor and recipient ages, endothelial injury, and comorbidity. The aim of this study was to assess the intensity of OxS among rats under chronic immunosuppression (IS) without a transplantation. We examined 49 male Wistar rats. IS started at 12 weeks of age was continued for 6 months: group I were controls (n=7); group II, Tac+sirolimus (Rapamycin [Rapa])+corticosteroids (CS; n=6); group III, CyA+Rapa+CS (n=4 of which 2 died); group IV, Rapa+mycophenolate mofetil (MMF)+CS (n=6); group V, CyA+MMF+CS (n=6); group VI, CsA+MMF+CS for 3 months followed by conversion to Rapa (n=6); group VII, Tac+MMF+CS (n=6 rats); and group VIII, Tac+MMF+CS for 3 months followed by conversion to Rapa (n=6). The drug doses were as follows: Tac 4 mg/kg/d; MMF 20 mg/kg/d; CyA 5mg/kg/d; Rapa 0.5 mg/kg/d; and CS 4 mg/kg/d. Multiple regression analysis revealed that all IS drugs decreased GPx activity (P<.001) except CS, which increased it (P<.0001). Multiple regression analysis showed that CsA and Tac decreased plasma MDA concentrations (P<.01), whereas CS increased them (P<.05). In conclusion, all IS drugs except CS damage proximal tubules of nephrons.

Effect of Hemodialysis on the Content of Fatty Acids in Monolayers of Erythrocyte Membranes in Patients with Chronic Renal Failure

Lipid metabolism disorders are found in patients with chronic renal failure (CRF). Changes in the content of fatty acids of the phospholipid fraction of erythrocyte membranes can lead to changes in the rheological properties. The objective of our study was to assess the effect of hemodialysis on the composition of fatty acids in two fractions of phospholipids: sphingomyelin (SPH, representative of the external monolayer) and phosphatidylethanolamine (PE, representative of the internal monolayer). Venous blood was drawn from patient with CRF before and after the HD procedure. Lipids from the erythrocyte stroma were extracted using the Rose and Oklander method and then were separated into phospholipid fractions using thin-layer chromatography (TLC). PE and SPH fractions were extracted, and the fatty acid profile was determined using gas chromatography (Perkin Elmer 8400; RTx 2330 column; length: 105 m). In the phospholipid fractions tested, a high content of saturated FA with a medium carbon chain (C 16:0 to C 18:2) and a long carbon chain such as C 24:0, C 24:1; C 22:6; and C 26:0 was found. The HD procedure affected the FA profile in the fractions tested. The proportion of saturated and unsaturated long-chain FA (above 18 C) increased in PE. However, the content of medium-chain FA C 16:0 to C 18:1 decreased. A significant decrease in the content of the majority of long-chain FA could be noted in SPH. The ratio of unsaturated (U) to saturated (S) fatty acids in the SPH fraction increased. Hemodialysis has a significant effect on the content of fatty acids in the PE and SPH fractions of erythrocyte membranes in patients with CRF.

Influence of Glucose in Dialyzing Fluid on Purine Concentrations in Hemodialyzed Patients with Chronic Renal Failure

Background: In chronic renal failure the accumulation of some purine nucleotides (in erythrocytes) develops both in patients undergoing conservative treatment and in hemodialyzed patients. The aim of the study was: (1) To find if hemodialysis (HD) sessions using dialyzing fluid containing glucose leads to an increase in ATP concentration and changes in the concentration of other nucleotides, nucleosides and oxypurines in erythrocytes. The potential consequence of such purine concentration changes is the increase of 2,3-DPG concentration and an improved transportation of oxygen in erythrocytes which are more resistant to hemolysis. (2) To compare blood concentrations of purine nucleotides, nucleosides and oxypurines in patients undergoing chronic HD with dialyzing fluid containing or lacking glucose. Significant differences could suggest the long-term influence of glucose in dialyzing fluid on erythrocyte energetic state. Methods: Whole blood nucleotide concentrations were evaluated with the use of a high-performance liquid chromatography technique. Results: Before the HD session the patients in the ‘plus glucose’ group had significantly higher concentrations of ATP, ADP, AMP, TAN, NAD, NADP, GTP + GDP, GMP, Urd and HYP than patients in the ‘no glucose’ group. After the HD the patients in the ‘plus glucose’ group had significantly higher concentrations of ADP, AMP, TAN, NAD, NADP, Urd and HYP than in the ‘no glucose’ group. Both before and after the HD session, the uric acid concentrations and AEC were significantly lower in the ‘plus glucose’ group than in the ‘no glucose’ group. A significant decrease in the whole blood hypoxanthine (p < 0.05) and uric acid (p < 0.001) concentrations after HD was found in the ‘no glucose’ group while a significant increase in ADP concentration (p < 0.05) was detected in the patients’ erythrocytes in the ‘plus glucose’ group. In this group a significant decrease of GTP + GDP and GMP (p < 0.05), uric acid concentration (p < 0.001) and adenylate energy charge (p < 0.05) were observed after the dialysis. However, no significant differences in nucleotide concentrations before and after the HD were found in the ‘no glucose’ group. Conclusion: The presence of glucose in the dialyzing fluid causes a significant modification of the energetic state of cells which is reflected by the purines’ and their metabolites’ concentrations in the erythrocytes. Higher ATP concentrations in patients with renal failure who have been dialyzed with the fluid containing glucose can be considered as an organism adaptation to a decreased amount of RBC and hemoglobin concentration.

Copper modifies the activity of sodium-transporting systems in erythrocyte membrane in patients with essential hypertension.

The aim of the study was to verify the hypothesis if copper could influence the activity of sodium-transporting systems in erythrocyte membrane that could be related to essential hypertension. The examined group of patients consisted of 15 men with hypertension. The control group was 11 healthy male volunteers. The Na+/H+ exchanger (NHE) activity in erythrocytes was determined according to Orlov et al. The activity of transporting systems (ATP-Na+/K+; co-Na+/K+/Cl−; ex-Na+/Li+; free Na+ and K+ outflow [Na+, K+-outflow]) was determined according to Garays method. The concentration of copper in plasma was assessed using atomic absorption spectrometry. The activity of ATP-Na+/K+ (μmol/L red blood cells [RBCs]/h) in hypertensive patients was 2231.5±657.6 vs 1750.5±291 in the control (p<0.05), the activity of co-Na+/K+/Cl− (μmol/L RBCs/h) in hypertensives was 171.3±77.9 vs 150.7±53.9 in the control (NS). Na+-outflow (μmol/L RBCs/h) in hypertensives was 118.3±51.6 vs 113.3±24.4 in the control (NS). The K+-outflow (μmol/L RBCs/h) in hypertensives was 1361.7±545.4 vs 1035.6±188.3 in the control (NS). The activity of ex-Na+/Li+ (μmol/L RBCs/h) in hypertensive patients was 266.1±76.1 vs 204.1±71.6 in the control (p<0.05). NHE activity (mmol/L RBCs/h) in hypertensives was 9.7±2.96 vs 7.7±1.33 in the control (p<0.05). In hypertensive patients, negative correlation was found between the activity of Na+/K+/Cl− co-transport and plasma copper concentration (Rs=−0.579, p <0.05) and between the activity of ex-Na+/Li+ and plasma copper concentration (Rs=−0.508, p<0.05). Plasma copper concentration significantly influences the activity of sodium transporting systems in erythrocyte membrane. Copper supplementation could be expected to provide therapeutic benefits for hypertensive patients.

Maternal endothelial damage as a disorder shared by early preeclampsia, late preeclampsia and intrauterine growth restriction

Abstract Introduction: Preeclampsia (PE) and intrauterine growth restriction (IUGR) are separate disease entities that have frequently been reported as sharing the same pathogenesis. In both of them, angiogenesis disorders and generalized endothelial damage with an accompanying inflammation are the dominant symptoms. In this study, we attempted to prove that both these processes demonstrate the same profile in early PE, late PE and IUGR patients, while the only difference is in the degree of exacerbation of the lesions. Patients, materials and methods: In 167 patients divided into four groups, three of those with early PE, late PE and IUGR and one control group, fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), high sensitive c-reactive protein (hsCRP) and fibronectin were determined. The behavior of these parameters in each of the groups was studied, and correlations between them were sought for. Results: Higher concentrations of sFlt-1, hsCRP and fibronectin and a lower concentration of PlGF were found in the study groups compared to the control group. Significant correlations were observed between the factors concerned. Conclusions: The higher values of disordered angiogenesis markers, endothelial damage markers and inflammatory markers both in the PE and the intrauterine growth restriction (IUGR) groups suggest the existence of shared disorders in the development of these pathologies. The correlations between disordered angiogenesis markers and endothelial damage markers argue in favor of a mutual relationship between these two processes in the development of pathologies evolving as secondary to placental ischemia. The results obtained confirm that the lesion profiles are the same in both PE and IUGR patients, which can be utilized in developing common diagnostic criteria.