Improved renal function after kidney transplantation is associated with heme oxygenase-1 polymorphism

Heme oxygenase-1 (HO-1) has a microsatellite polymorphism based on the number of guanosine-thymidine nucleotide repeats (GT) repeats that regulates expression levels and could have an impact on organ survival post-injury. We correlated HO-1 polymorphism with renal graft function. The HO-1 gene was sequenced (N = 181), and the allelic repeats were divided into subclasses: short repeats (S) (< 27 repeats) and long repeats (L) (>= 27 repeats). A total of 47.5% of the donors carried the S allele. The allograft function was statistically improved six months, two and three yr after transplantation in patients receiving kidneys from donors with an S allele. For the recipients carrying the S allele (50.3%), the allograft function was also better throughout the follow-up, but reached statistical significance only three yr after transplantation (p = 0.04). Considering only those patients who had chronic allograft nephropathy (CAN; 74 of 181), allograft function was also better in donors and in recipients carrying the S allele, two and three yr after transplantation (p = 0.03). Recipients of kidney transplantation from donors carrying the S allele presented better function even in the presence of CAN.

Toll-like receptors-related genes in kidney transplant patients with chronic allograft nephropathy and acute rejection

Introduction: Toll-like receptors (TLR) comprehend an emerging family of receptors that recognize pathogen-associated molecular patterns and promote the activation of leukocytes. Surgical trauma and ischemia-reperfusion injury are likely to provide exposure to endogenous ligands for TLR in virtually all kidney transplant recipients. Methods: Macroarray (GEArray OHS-018.2 Series-Superarray) analyses of 128 genes involved in TLR signaling pathway were performed in nephrectomy samples of patients with chronic allograft nephropathy (CAN) and acute rejection (AR, vascular and non vascular). The analysis of each membrane was performed by GEArray Expression Analysis Suite 2.0. Results: Macroarray profile identified a gene expression signature that could discriminate CAN and AR. Three genes were significantly expressed between CAN and vascular AR: Pellino 2; IL 8 and UBE2V1. In relation to vascular and non-vascular AR, there were only two genes with statistical significance: IL-6 and IRAK-3. Conclusion: Vascular and non-vascular AR and CAN showed different expression of a few genes in TLR pathway. The analysis of nephrectomy showed that activation of TLR pathway is present in AR and CAN. (C) 2008 Elsevier B.V. All rights reserved.

Clinical correlations of human cytomegalovirus strains and viral load in kidney transplant recipients

Little is known about clinical differences associated with cytomegalovirus (CMV) infection by distinct strains in renal transplant patients. Different clinical pictures may be associated with specific viral genotypes. viral load, as well as host factors. The objective of this study was to identify CMV strains to determine viral load (antigenemia), and their correlation with clinical data in renal transplant recipients. Seventy-one patients were enrolled, comprising 91 samples. After selection, polymorphonuclear cells were used to amplify and sequence the gB region of CMV DNA. The sequences were analyzed to ascertain the frequency of different genotypes. Additionally, the results of this Study showed that the gB coding gene presents a great variability, revealing a variety of patterns: classical gB (1.4%), gB1V (46.4%), classical gB2 (35.2%), gB2V (2.8%), gB3 (1.4%), classical gB4 (4.9%) and gB4V (4.9%). The mean viral load in kidney transplant patient was 75.1 positive cells (1-1000). A higher viral load was observed in patients with genotype 4 infection. Statistically significant differences were detected between gB1 and gB4 (p=0.010), and between gB2 and gB4 (p=0.021). The average numbers of positive cells in relation to clinical presentation were: 34.5 in asymptomatic, 49.5 in CMV associated syndrome and 120.7 in patients with invasive disease (p=0.048). As a group, gB1 was the most frequent strain and revealed a potential risk for developing invasive disease. Viral load also seemed to be important as a marker associated with clinical presentation of the disease. (C) 2008 Elsevier B.V. All rights reserved.

Vimentin Expression and Myofibroblast Infiltration Are Early Markers of Renal Dysfunction in Kidney Transplantation: An Early Stage of Chronic Allograft Dysfunction?

Introduction. The objective of this study was to show the morphologic characteristics of allograft renal biopsies in renal transplant patients with stable renal function, which can potentially be early markers of allograft dysfunction, after 5 years of follow-up. Methods. Forty-nine renal transplant patients with stable renal function were submitted to renal biopsies and simultaneous measurement of serum creatinine (Cr). Histology was evaluated using Banff scores, determination of interstitial fibrosis by Sirius red staining and immunohistochemical study of proximal tubule and interstitial compartment (using cytokeratin, vimentin, and myofibroblasts as markers). Biopsies were evaluated according to the presence or absence of the epitheliomesenchymal transition (EMT). The interstitial presence of myofibroblasts and tubular presence of vimentin was also analyzed simultaneously. Renal function was measured over the follow-up period to estimate the reduction of graft function. Results. Median posttransplant time at enrollment was 105 days. Patients were followed for 64.3 +/- 8.5 months. The mean Cr at biopsy time was 1.44 +/- 0.33 mg/dL, and after the follow-up it was 1.29 +/- 0.27 mg/dL. Nine patients (19%) had a reduction of their graft function. Eleven biopsies (22%) had tubulointerstitial alterations according to Banff score. Seventeen biopsies (34%) presented EMT. Fifteen biopsies (32%) had high interstitial expression of myofibroblasts and tubular vimentin. Using Cox multivariate analysis, HLA and high expression of interstitial myofibroblasts and tubular vimentin were associated with reduction of graft function, yielding a risk of 3.3 (P = .033) and 9.8 (P = .015), respectively. Conclusion. Fibrogenesis mechanisms occur very early after transplantation and are risk factors for long-term renal function deterioration.

N-acetylcysteine protects against renal injury following bilateral ureteral obstruction

Background. Obstructive nephropathy decreases renal blood flow (RBF) and glomerular filtration rate (GFR), causing tubular abnormalities, such as urinary concentrating defect, as well as increasing oxidative stress. This study aimed to evaluate the effects of N-acetylcysteine (NAC) on renal function, as well as on the protein expression of aquaporin 2 (AQP2) and endothelial nitric oxide synthase (eNOS), after the relief of bilateral ureteral obstruction (BUO). Methods. Adult male Wistar rats were divided into four groups: sham (sham operated); sham operated + 440 mg/kg body weight (BW) of NAC daily in drinking water, started 2 days before and maintained until 48 h after the surgery; BUO (24-h BUO only); BUO + NAC-pre (24-h BUO plus 440 mg/kg BW of NAC daily in drinking water started 2 days before BUO); and BUO + NAC-post (24-h BUO plus 440 mg/kg BW of NAC daily in drinking water started on the day of BUO relief). Experiments were conducted 48 h after BUO relief. Results. Serum levels of thiobarbituric reactive substances, which are markers of lipid peroxidation, were significantly lower in NAC-treated rats than in the BUO group rats. The administration of NAC provided significant protection against post-BUO GFR drops and reductions in RBF. Renal cortices and BUO rats presented decreased eNOS protein expression of eNOS in the renal cortex of BUO group rats, whereas it was partially recovered in BUO + NAC-pre group rats. Urine osmolality was significantly lower in BUO rats than in sham group rats or NAC-treated rats, the last also presenting less interstitial fibrosis. Post-BUO downregulation of AQP2 protein expression was averted in the BUO + NAC-pre group rats. Conclusions. This study demonstrates that NAC administration ameliorates the renal function impairment observed 48 h after the relief of 24-h BUO. Oxidative stress is important for the suppression of GFR, RBF, tissue AQP2 and eNOS in the polyuric phase after the release of BUO.

Factors associated with health-related quality of life after successful kidney transplantation: a population-based study

Kidney transplantation improves the quality of life of end-stage renal disease patients. The quality of life benefits, however, pertain to patients on average, not to all transplant recipients. The aim of this study was to identify factors associated with health-related quality of life after kidney transplantation. Population-based study with a cross-sectional design was carried out and quality of life was assessed by SF-36 Health Survey Version 1. A multivariate linear regression model was constructed with sociodemographic, clinical and laboratory data as independent variables. Two hundred and seventy-two kidney recipients with a functioning graft were analyzed. Hypertension, diabetes, higher serum creatinine and lower hematocrit were independently and significantly associated with lower scores for the SF-36 oblique physical component summary (PCSc). The final regression model explained 11% of the PCSc variance. The scores of oblique mental component summary (MCSc) were worse for females, patients with a lower income, unemployed and patients with a higher serum creatinine. The regression model explained 9% of the MCSc variance. Among the studied variables, comorbidity and graft function were the main factors associated with the PCSc, and sociodemographic variables and graft function were the main determinants of MCSc. Despite comprehensive, the final regression models explained only a little part of the heath-related quality of life variance. Additional factors, such as personal, environmental and clinical ones might influence quality of life perceived by the patients after kidney transplantation.

Expression of TLR-4 and -2 in peripheral mononuclear cells in renal transplant patients with TLR-4 gene polymorphism

Introduction: TLR-4 has also been identified as a receptor for endogenous alarmins, which are increased post transplantation. TLR-4 has also been associated with a polymorphism that could impact graft outcome. Objective: To assess the expression of TLR-4 in kidney transplant patients carrying or not a polymorphism. Methods: TLR-4 polymorphism (A299G/T399I) was studied in 200 renal transplant patients. Healthy volunteers were also enrolled as control group. The polymorphism analysis was performed using restriction enzymes technique (RFLP). Functionality of TLR-4 polymorphism was assessed in samples from controls by quantification of TNF-alpha after LPS stimulus. TLR-4 and -2 expressions were also analyzed by flow cytometry. Results: TLR-4 polymorphism was present in 8.5% of renal transplant patients. This polymorphism was associated with impairment in TNF-alpha secretion. In general, in renal transplant patients, TLR-4 expression in monocytes and in neutrophils was lower than in health volunteers. TLR-2 and TLR-4 expressions in healthy volunteers with A299G/T399I TLR-4 polymorphism was higher than in wild-type genotype healthy volunteers (p<0.01 and p<0.05, respectively), and also higher than A299G/T399I TLR-4 polymorphism renal transplant patients (p<0.05). TLR-2 expression on neutrophils in wild-type genotype renal transplant patients was higher compared to wild-type genotype healthy volunteers, and was also higher in relation to A299G/T399I kidney transplanted patients (p<0.01). Conclusion: Stable renal transplant patients with TLR-4 polymorphism have a lower expression of TLR-4 and TLR-2 receptors in peripheral mononuclear cells, which ultimately indicate a less responsiveness for alarmins. (C) 2010 Elsevier B.V. All rights reserved.

Functional and morphologic evaluation of kidney proximal tubuli and correlation with renal allograft prognosis

P>Renal transplant patients with stable graft function and proximal tubular dysfunction (PTD) have an increased risk for chronic allograft nephropathy (CAN). In this study, we investigated the histologic pattern associated with PTD and its correlation with graft outcome. Forty-nine transplant patients with stable graft function were submitted to a biopsy. Simultaneously, urinary retinol-binding protein (uRBP) was measured and creatinine clearance was also determined. Banff`s score and semi-quantitative histologic analyses were performed to assess tubulointerstitial alterations. Patients were followed for 24.0 +/- 7.8 months. At biopsy time, mean serum creatinine was 1.43 +/- 0.33 mg/dl. Twelve patients (24.5%) had uRBP >= 1 mg/l, indicating PTD and 67% of biopsies had some degree of tubulointerstitial injury. At the end of the study period, 18 (36.7%) patients had lost renal function. uRBP levels were not associated with morphologic findings of interstitial fibrosis and tubular atrophy (IF/TA), interstitial fibrosis measured by Sirius red or tubulointerstitial damage. However, in multivariate analysis, the only variable associated with the loss of renal function was uRBP level >= 1 mg/l, determining a risk of 5.290 of loss of renal function (P = 0.003). Renal transplant patients who present PTD have functional alteration, which is not associated with morphologic alteration. This functional alteration is associated to progressive decrease in renal function.

A role for regulatory T cells in renal acute kidney injury

Ischemia reperfusion injury (IRI) is a potential contributor for the development of chronic allograft nephropathy. T cells are important mediators of injury, even in the absence of alloantigens. We performed a depletion of TCD4(+)CTLA4(+)Foxp3(+) cells with anti-CD25(PC61), a treatment with anti-GITR (DTA-1) and rat-IgG, followed by 45 min of ischemia and 24/72 h of reperfusion, and then analyzed blood urea, kidney histopathology and gene expression in kidneys by QReal Time PCR. After 24 h of reperfusion, depletion of TCD4(+)CTLA4(+)Foxp3(+) cells reached 30.3%(spleen) and 67.8%(lymph nodes). 72 h after reperfusion depletion reached 43.1%(spleen) and 90.22%(lymph nodes) and depleted animals presented with significantly poorer renal function, while DTA-1 (anti-GITR)-treated ones showed a significant protection, all compared to serum urea from control group (IgG: 150.10 +/- 50.04; PC61: 187.23 +/- 31.38; DTA-1: 64.53 +/- 25.65, mg/dL, p<0.05). These data were corroborated by histopathology. We observed an increase of HO-1 expression in animals treated with DTA-1 at 72 h of reperfusion with significant differences. Thus, our results suggest that PC61 (anti-CD25) mAb treatment is deleterious, while DTA-1 (anti-GITR) mAb treatment presents a protective role in the renal IRI, indicating that some regulatory populations of T cells might have a role in IRI. (C) 2009 Elsevier B.V. All rights reserved.

Administration of Neural Precursor Cells Ameliorates Renal Ischemia-Reperfusion Injury

In this study we evaluated whether administration of stem cells of neural origin (neural precursor cells, NPCs) could be protective against renal ischemia-reperfusion injury (IRI). We hypothesized that stem cell outcomes are not tissue-specific and that NPCs can improve tissue damage through paracrine mechanisms, especially due to immunomodulation. To this end, Wistar rats (200-250 g) were submitted to 1-hour ischemia and treated with NPCs (4 x 10(6) cells/animal) at 4 h of reperfusion. To serve as controls, ischemic animals were treated with cerebellum homogenate harvested from adult rat brain. All groups were sacrificed at 24 h of reperfusion. NPCs were isolated from rat fetus telencephalon and cultured until neurosphere formation (7 days). Before administration, NPCs were labeled with carboxyfluorescein diacetate succinimydylester (CFSE). Kidneys were harvested for analysis of cytokine profile and macrophage infiltration. At 24 h, NPC treatment resulted in a significant reduction in serum creatinine (IRI + NPC 1.21 + 0.18 vs. IRI 3.33 + 0.14 and IRI + cerebellum 2.95 + 0.78mg/dl, p < 0.05) and acute tubular necrosis (IRI + NPC 46.0 + 2.4% vs. IRI 79.7 + 14.2%, p < 0.05). NPC-CFSE and glial fibrillary acidic protein (GFAP)-positive cells (astrocyte marker) were found exclusively in renal parenchyma, which also presented GFAP and SOX-2 (an embryonic neural stem cell marker) mRNA expression. NPC treatment resulted in lower renal proinflammatory IL1-beta and TNF-alpha expression and higher anti-inflammatory IL-4 and IL-10 transcription. NPC-treated animals also had less macrophage infiltration and decreased serum proinflammatory cytokines (IL-1 beta, TNF-alpha and INF-gamma). Our data suggested that NPC therapy improved renal function by influencing immunological responses. Copyright (C) 2009 S. Karger AG, Basel

A Role for galectin-3 in renal tissue damage triggered by ischemia and reperfusion injury

Ischemic-reperfusion injury (IRI) triggers an inflammatory response involving neutrophils/macrophages, lymphocytes and endothelial cells. Galectin-3 is a multi-functional lectin with a broad range of action such as promotion of neutrophil adhesion, induction of oxidative stress, mastocyte migration and degranulation, and production of pro-inflammatory cytokines. The aim of this study was evaluate the role of galectin-3 in the inflammation triggered by IRI. Galectin-3 knockout (KO) and wild type (wt) mice were subjected to 45 min of renal pedicle occlusion. Blood and kidney samples were collected at 6, 24, 48 and 120 h. Blood urea was analyzed enzymatically, while MCP-1, IL-6 and IL-1 beta were studied by real-time PCR. Reactive oxygen species (ROS) was investigated by flow cytometry. Morphometric analyses were performed at 6, 24, 48 and 120 h after reperfusion. Urea peaked at 24 h, being significantly lower in knockout animals (wt = 264.4 +/- 85.21 mg/dl vs. gal-3 KO = 123.74 +/- 29.64 mg/dl, P = 0.001). Galectin-3 knockout animals presented less acute tubular necrosis and a more prominent tubular regeneration when compared with controls concurrently with lower expression of MCP-1, IL-6, IL-1 beta, less macrophage infiltration and lower ROS production at early time points. Galectin-3 seems to play a role in renal IRI involving the secretion of macrophage-related chemokine, pro-inflammatory cytokines and ROS production.

Renal ischemia in rats: Mitochondria function and laser autofluorescence

Ischemia-reperfusion injury is the major cause of organ dysfunction or even nonfunction following transplantation. It can attenuate the long-term survival of transplanted organs. To evaluate the severity of renal ischemia injury determined by histology, we applied laser(442 nm and 532 nm) induced fluorescence (LIF), mitochondria respiration, and membrane swelling to evaluate 28 Wistar rats that underwent left kidney warm ischemia for 20, 40, 60, or 80 minutes. LIF performed before ischemia (control) was repeated at 20, 40, 60, and 80 minutes thereafter. We harvested left kidney tissue samples immediately after LIF determination for histology and mitochondrial analyses: state 3 and 4 respiration, respiration control rate (RCR), and membrane swelling. The association of optic spectroscopy with histological damage showed: LIF, 442 nm (r(2) = 0.39, P < .001) and 532 nm, (r(2) = 0.18, P = .003); reflecting laser/fluorescence-induced, 442 nm (r(2) = 0.20, P = .002) and 532 nm (r(2) = 0.004, P = .67). The associations between mitochondria function and tissue damage were: state 3 respiration (r(2) = 0.43, P = .0004), state 4 respiration (r(2) = 0.03, P = 0.38), RCR (r(2) = 0.28, P = .007), and membrane swelling (r(2) = 0.02, P = .43). The intensity of fluorescence emitted by tissue excited by laser, especially at a wave length of 442 nm, was determined in real time. Mitochondrial state 3 respiration and respiratory control ratio also exhibited good correlations with the grade of ischemic tissue damage.

Functional analysis of a novel missense NBC1 mutation and of other mutations causing proximal renal tubular acidosis

Mutations in the Na+-HCO3- cotransporter NBC1 cause severe proximal tubular acidosis (pRTA) associated with ocular abnormalities. Recent studies have suggested that at least some NBC1 mutants show abnormal trafficking in the polarized cells. This study identified a new homozygous NBC1 mutation (G486R) in a patient with severe pRTA. Functional analysis in Xenopus oocytes failed to detect the G486R activity due to poor surface expression. In ECV304 cells, however, G486R showed the efficient membrane expression, and its transport activity corresponded to approximately 50% of wild-type (WT) activity. In Madin-Darby canine kidney (MDCK) cells, G486R was predominantly expressed in the basolateral membrane domain as observed for WT. Among the previously identified NBC1 mutants that showed poor surface expression in oocytes, T485S showed the predominant basolateral expression in MDCK cells. On the other hand, L522P was exclusively retained in the cytoplasm in ECV304 and MDCK cells, and functional analysis in ECV304 cells failed to detect its transport activity. These results indicate that G486R, like T485S, is a partial loss of function mutation without major trafficking abnormalities, while L522P causes the clinical phenotypes mainly through its inability to reach the plasma membranes. Multiple experimental approaches would be required to elucidate potential disease mechanism by NBC1 mutations.

Lineage-Negative Bone Marrow Cells Protect Against Chronic Renal Failure

Progressive renal failure continues to be a challenge. The use of bone marrow cells represents a means of meeting that challenge. We used lineage-negative (Lin(-)) cells to test the hypothesis that Lin(-) cell treatment decreases renal injury. Syngeneic Fischer 344 rats were divided into four groups: sham ( laparotomy only, untreated); Nx (five-sixth nephrectomy and untreated); NxLC1 (five-sixth nephrectomy and receiving 2 x 10(6) Lin(-) cells on postnephrectomy day 15); and NxLC3 (five-sixth nephrectomy and receiving 2 x 10(6) Lin(-) cells on postnephrectomy days 15, 30, and 45). On postoperative day 16, renal mRNA expression of interleukin (IL)-1 beta, tumor necrosis factor-alpha, and IL-6 was lower in NxLC rats than in Nx rats. On postnephrectomy day 60, NxLC rats presented less proteinuria, glomerulosclerosis, anemia, renal infiltration of immune cells, and protein expression of monocyte chemoattractant protein-1, as well as decreased interstitial area. Immunostaining for proliferating cell nuclear antigen showed that, in comparison with sham rats, Nx rats presented greater cell proliferation, whereas NxLC1 rats and NxLC3 rats presented less cell proliferation than did Nx rats. Protein expression of the cyclin-dependent kinase inhibitor p21 and of vascular endothelial growth factor increased after nephrectomy and decreased after Lin(-) cell treatment. On postnephrectomy day 120, renal function (inulin clearance) was significantly better in Lin(-) cell-treated rats than in untreated rats. Lin(-) cell treatment significantly improved survival. These data suggest that Lin(-) cell treatment protects against chronic renal failure. STEM CELLS 2009; 27: 682-692

Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na(+)/K(+)-ATPase

Background: Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na(+)/K(+)-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na(+)/K(+)-ATPase expression and activity in rats injected with Bothrops alternatus snake venom. Methods: Male Wistar rats were injected with venom (0.8 mg/kg, iv.) and renal function was assessed 6.24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na(+)/K(+)-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively. Results: Venom caused lobulation of the capillary tufts, dilation of Bowman`s capsular space. F-actin disruption in Bowman`s capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na(+)/K(+)-ATPase alpha(1) subunit were increased 6 h post-venom, whereas Na(+)/K(+)-ATPase activity increased 6 h and 24 h post-venom. Conclusions: Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na(+)/K(+)-ATPase expression and activity in the early phase of renal damage. General significance: Enhanced Na(+)/K(+)-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage. (C) 2011 Elsevier B.V. All rights reserved.