5-Aminoisoquinolinone reduces renal injury and dysfunction caused by experimental ischemia/reperf... more 5-Aminoisoquinolinone reduces renal injury and dysfunction caused by experimental ischemia/reperfusion. Background. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the development of ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of a water-soluble and potent PARP inhibitor, 5-aminoisoquinolinone (5-AIQ), on the renal injury and dysfunction caused by oxidative stress of the rat kidney in vitro and in vivo. Methods. Primary cultures of rat renal proximal tubular cells, subjected to oxidative stress caused by hydrogen peroxide (H 2 O 2), were incubated with increasing concentrations of 5-AIQ (0.01 to 1 mmol/L) after which PARP activation, cellular injury, and cell death were measured. In in vivo experiments, anesthetized male Wistar rats were subjected to renal bilateral ischemia (45 minutes) followed by reperfusion (6 hours) in the absence or presence of 5-AIQ (0.3 mg/kg) after which renal dysfunction, injury and PARP activation were assessed. Results. Incubation of proximal tubular cells with H 2 O 2 caused a substantial increase in PARP activity, cellular injury, and cell death, which were all significantly reduced in a concentration-dependent by 5-AIQ [inhibitory concentration 50 (IC 50) ∼ 0.03 mmol/L]. In vivo, renal I/R resulted in renal dysfunction, injury, and PARP activation, primarily in the proximal tubules of the kidney. Administration of 5-AIQ significantly reduced the biochemical and histologic signs of renal dysfunction and injury and markedly reduced PARP activation caused by I/R.
respectively. GW274150 abolished the rise in the plasma levels GW274150, a potent and highly sele... more respectively. GW274150 abolished the rise in the plasma levels GW274150, a potent and highly selective inhibitor of iNOS, of nitrate (indicating reduced NO production). GW274150 also reduces experimental renal ischemia/reperfusion injury. reduced the renal dysfunction in wild-type mice to levels similar Background. Generation of nitric oxide (NO) by inducible to that observed in iNOSϪ/Ϫ mice subjected to I/R. Renal nitric oxide synthase (iNOS) may contribute to renal ischemia/ MPO activity and MDA levels were significantly reduced in reperfusion (I/R) injury. The aim of this study was to investigate wild-type mice administered GW274150 and iNOSϪ/Ϫ mice the effects of GW274150, a novel, highly selective, potent and subjected to renal I/R, indicating reduced polymorphonuclear long-acting inhibitor of iNOS activity in rat and mouse models leukocyte (PMN) infiltration and lipid peroxidation. of renal I/R. Conclusions. These results suggest that (1) an enhanced for-Methods. Rats were administered GW274150 (5 mg/kg inmation of NO by iNOS contributes to the pathophysiology of travenous bolus administered 30 minutes prior to I/R) and renal I/R injury and (2) GW274150 reduces I/R injury of the subjected to bilateral renal ischemia (45 minutes) followed by kidney. We propose that selective inhibitors of iNOS activity reperfusion (6 hours). Serum and urinary indicators of renal may be useful against renal dysfunction and injury associated dysfunction, tubular and reperfusion injury were measured, with I/R of the kidney. specifically, serum urea, creatinine, aspartate aminotransferase (AST) and N-acetyl--d-glucosaminidase (NAG) enzymuria. In addition, renal sections were used for histologic scoring of renal injury and for immunologic evidence of nitrotyrosine for-Nitric oxide (NO), generated by any one of the three mation and poly [adenosine diphosphate (ADP)-ribose] (PAR). isoforms of nitric oxide synthase (NOS) plays an impor-Nitrate levels were measured in rat plasma using the Griess tant role in renal function, both under normal and pathoassay. Mice (wild-type, administered 5 mg/kg GW274150, and iNOSϪ/Ϫ) were subjected to bilateral renal ischemia (30 minphysiologic conditions [1, 2]. Although NO appears to utes) followed by reperfusion (24 hours) after which renal play an important functional role within the proximal dysfunction (serum urea, creatinine), renal myeloperoxidase tubule, it can also contribute to pathophysiology of this (MPO) activity and malondialdehyde (MDA) levels were meapart of the kidney [3, 4]. All three isoforms of NOS sured. have been located in the kidney; the constitutive isoform Results. GW274150, administered prior to I/R, significantly reduced serum urea, serum creatinine, AST, and NAG indicat-[endothelial NOS (eNOS)] and neuronal NOS (nNOS)] ing reduction of renal dysfunction and injury caused by I/R. isoforms have been identified in the renal vasculature GW274150 reduced histologic evidence of tubular injury and and macula densa, respectively [1, 2] and inducible NOS markedly reduced immunohistochemical evidence of nitrotyro-(iNOS) can be located and induced in several renal cell sine and PAR formation, indicating reduced peroxynitrite fortypes (e.g., in glomerular mesangial and proximal tubular mation and poly (ADP-ribose) polymerase (PARP) activation, cells) by cytokines, lipopolysaccharide (LPS), and during renal ischemia/reperfusion (I/R) [2-6], leading to renal
We examined the expression of both the natriuretic peptides and natriuretic peptide receptors (NP... more We examined the expression of both the natriuretic peptides and natriuretic peptide receptors (NPR) in primary cultures of rat proximal tubular (RPT) cells using Northern blot assay for peptides and receptors and radioimmunoassay and immunohistochemical analysis for atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide. Freshly isolated cells expressed mRNA coding for ANF, BNP, and the NPR-C. The presence of ANF and BNP in freshly isolated cells was confirmed by immunocytochemical staining. As cells approached confluence, there was a marked increase in mRNA expression for ANF and BNP. Immunocytochemical analysis and radioimmunoassay confirmed that both these peptides were co-localised in RPT cells and present in the cell supernatant. These changes in peptide expression were associated with a concurrent decrease in the expression of the NPR-C and the appearance of the NPR-A and-B. These results confirm that freshly isolated RPT cells possess the components of an autocrine natriuretic peptide system and that growth in primary culture is associated with changes in both peptide system and that growth in primary culture is associated with changes in both peptide and receptor subtype expression, raising the possibility that the endogenous production of ANF and BNP may be involved in the control of control cell growth.
High-density lipoproteins (HDL) have been shown to reduce organ injury and mortality in animal mo... more High-density lipoproteins (HDL) have been shown to reduce organ injury and mortality in animal models of shock via modulation of the expression of adhesion molecules and pro-inflammatory enzymes. As renal inflammation plays an important role in the development of ischemia/reperfusion (I/R) injury of the kidney, the aim of this study was to investigate the ability of HDL to alleviate renal dysfunction and injury in a rat model of renal I/R. HDL (80 mg/kg, intravenous) was administered to male Wistar rats 30 min before bilateral renal ischemia for 45 min followed by reperfusion for up to 48 h. After 6-h reperfusion, HDL significantly reduced (1) renal and tubular dysfunction, (2) tubular and reperfusion-injury, and (3) histologic evidence of renal injury. HDL also improved renal function (after 24-h and 48-h reperfusion) and reduced histologic signs of renal injury (after 48-h reperfusion). Administration of HDL significantly reduced the numbers of polymorphonuclear leukocytes (PMN) i...
Results: The optimal signature contained 28 genes. There was a statistically significant correlat... more Results: The optimal signature contained 28 genes. There was a statistically significant correlation between actual versus predicted time to recurrence for blind data (rho = 0.975; p < 0.0001).
The presence of cytotoxic HLA antibodies (Ab1) against donor lymphocytes in pretransplant sera is... more The presence of cytotoxic HLA antibodies (Ab1) against donor lymphocytes in pretransplant sera is almost always associated with rapid rejection of the renal transplant. We have investigated the possibility that antiidiotypic antibodies (Ab2) to cytotoxic HLA antibodies might modulate the immune response and favorably influence renal allograft outcome. The role of antibodies (Ab3) which potentiate the cytotoxic effect of Ab1 was also studied. Pretransplant sera from 63 patients were tested for inhibitory or potentiating activity in the short antiidiotypic assay. Inhibitory activity was detected in 30 patients and in 28 the transplant survived more than a year. Of patients without antibody activity 11 of 17 had grafts surviving more than one year, and of those showing potentiating activity 11 of 16 were functioning at a year. The difference in transplant survival between the first group and the other two groups was statistically significant (P less than 0.05). There was no significant difference in survival rates between the latter two groups. Potentiating activity is therefore not an independent predictor of transplant failure, whereas the presence of antiidiotypic antibody activity did correlate with improved allograft survival.
Methods in molecular biology (Clifton, N.J.), 2012
Culture of isolated kidney glomerular cells has been employed for almost four decades as a tool t... more Culture of isolated kidney glomerular cells has been employed for almost four decades as a tool to dissect pathophysiological effects of individual cell types in renal disease. This chapter aims to highlight in detail the available techniques to isolate, culture, and characterize human glomerular epithelial and mesangial cells. To establish primary culture of these cells, glomeruli are isolated from the cortex of kidney by differential sieving and cellular outgrowths from cultured glomeruli further subcultured in appropriately coated tissue culture plates/flasks. Methods used for characterization of isolated glomerular mesangial and epithelial cells (podocytes) are described as are the phenotypic markers useful for identification. Other sources of isolated glomerular cells such as immortalized cell lines are briefly discussed.
We have previously shown that the presence in pretransplant recipient sera of Fc receptor blockin... more We have previously shown that the presence in pretransplant recipient sera of Fc receptor blocking antibodies detected by the EA inhibition assay is correlated with improved allograft survival. Twenty-four such sera were assessed for the presence of autoantibodies by the EA inhibition and lymphocytotoxicity assays. No autolymphocytotoxic antibodies were found, and autologous EA inhibition was noted in only one case. EA-inhibiting alloantibodies did occur, and their presence was correlated with improved allograft survival. Sera from 37 dialysis patients were also studied, and neither autologous EA inhibiting nor autologous lymphocytotoxic antibodies were present. Thus Fc receptor blocking alloantibodies that were correlated with improved renal transplant survival were not autoantibodies.
Primary and secondary alloantibody responses were monitored in (AOxPVG)F1 hybrid rats after three... more Primary and secondary alloantibody responses were monitored in (AOxPVG)F1 hybrid rats after three transfusions of DA blood; the initial transfusion was either untreated or pretreated with monoclonal antibody directed to class I antigens or other cell surface markers. Mean antibody activity in recipient sera against class I DA antigens was significantly decreased by pretreatment with the monoclonal antibodies. The most marked suppression was associated with pretreatment by antibodies to the four major nonoverlapping epitopes of the RT1Aa antigen. Subsequent transfusions of DA blood failed to stimulate a secondary response. Crossreactivity of the alloantibody reactivity with BDIX antigens was diminished by pretreating the transfusions with rat anti-RT1A antibodies and, to a lesser extent, with a mouse monoclonal antibody (OX-18) to a common class I determinant. Monoclonal antibody pretreatment had no effect on the humoral response to class II DA antigens. These studies indicate that blood transfusions pretreated with monoclonal antibodies induce a less-potent cytotoxic humoral immune response and that reactivity is most effectively suppressed by completely masking the class I antigen. This technique may prove of clinical value in preventing the sensitization caused by blood transfusions in potential transplant recipients.
Cisplatin exhibits dose-limiting nephrotoxicity in rodents and man. This study investigates the m... more Cisplatin exhibits dose-limiting nephrotoxicity in rodents and man. This study investigates the mechanism of cisplatin nephrotoxicity in vivo and in an in vitro model system. Nephrotoxicity was induced in rats (6 mg/kg cisplatin i.p.) and mice (10 mg/kg cisplatin i.p.). Cisplatin administration significantly elevated blood urea nitrogen (BUN) and serum creatinine in male Sprague Dawley rats day 5 post-treatment (BUN Delta+28+/-5 micromol/ml; serum creatinine Delta+108+/-4 nmol/ml, P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05) and in male C57BL6 mice day 4 post-treatment (BUN Delta+21+/-4 micromol/ml; serum creatinine Delta+81+/-5 nmol/ml, P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). Nephrotoxicity was confirmed by histological analysis that revealed significant damage to the proximal tubules of cisplatin- versus saline vehicle-treated animals. Inhibition of gamma glutamyltranspeptidase prevented cisplatin nephrotoxicity in Sprague Dawley rats (day 5 BUN Delta+1+/-2 micromol/ml; serum creatinine Delta+8+/-4 nmol/ml) and C57BL6 mice (day 4 BUN Delta+1+/-0.8 micromol/ml; serum creatinine Delta-1+/-2 nmol/ml), but not cellular toxicity in rat proximal tubular (RPT) or human proximal tubular (HPT) cultures. Inhibition of aminopeptidase N (AP-N) or renal dipeptidase (RDP) in male Sprague Dawley rats, or in RPT and HPT cell cultures, did not reduce cisplatin toxicity. In contrast to published findings inhibition of C-S lyase did not prevent the nephrotoxicity of cisplatin in vivo or cellular toxicity in vitro. These data demonstrate that the biotransformation enzymes AP-N, RDP and C-S lyase are not implicated in the metabolism of cisplatin to a nephrotoxic metabolite as has been previously hypothesised. Instead, our data demonstrate that gamma glutamyltranspeptidase is a key enzyme involved in mediating cisplatin nephrotoxicity, which potentially acts to cleave cisplatin-GSH conjugates to a toxic metabolite.
Age-related and disease-induced glomerulosclerosis (GS) in rats have both been well defined in a ... more Age-related and disease-induced glomerulosclerosis (GS) in rats have both been well defined in a number of strains and experimental models, but the inter-relationship between the two is not clear. The present study was undertaken to compare the pattern of glomerular injury in these two types of GS. One- and two-shot Thy1 glomerulonephritis (GN) was induced at 2 months of age and followed for 12 months. At 12 months histological injury in proteinuric rats was characterized by segmental hyaline lesions. Two-shot Thy1 GN resulted in accelerated, but morphologically identical injury at 8 months. Histological lesions predictive of subsequent accelerated GS were evaluated at 1, 2, 4 and 6 months. In this regard, glomerular hypercellularity, rather than hypertrophy or matrix increase, was the most consistent histological index of later accelerated disease. The profibrotic cytokines transforming growth factor (TGF)-beta(1) and -beta(3) were localized distinctly to segmental hyaline lesions, but not to areas of matrix increase within the glomerular tuft. This study reveals that GS after Thy1 GN represents acceleration of an age-related disease, presents evidence for use of prolonged glomerular hypercellularity as the best histological index of future disease progression, and correlates the key lesion of GS in these animals, the segmental hyaline lesion, with the presence of TGF-beta peptides.
Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. Inflammatory activ... more Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. Inflammatory activity within the stroma of invasive colorectal tumours is known to be a key predictor of disease activity with type, density and location of immune cells impacting on patient prognosis. To date, there has been no report of inflammatory phenotype within pre-malignant human colonic adenomas. Assessing the stromal microenvironment and particularly, inflammatory activity within colorectal neoplastic lesions is central to understanding early colorectal carcinogenesis. Inflammatory cell infiltrate was assessed by immunohistochemistry in paired colonic adenoma and adjacent normal colonic mucosa samples, and adenomas exhibiting increasing degrees of epithelial cell dysplasia. Macrophage phenotype was assessed using double stain immunohistochemistry incorporating expression of an intracellular enzyme of function. A targeted array of inflammatory cytokine and receptor genes, validated by RT-PCR, was used to assess inflammatory gene expression. Inflammatory cell infiltrates are a key feature of sporadic adenomatous colonic polyps with increased macrophage, neutrophil and T cell (specifically helper and activated subsets) infiltration in adenomatous colonic polyps, that increases in association with characteristics of high malignant potential, namely, increasing degree of cell dysplasia and adenoma size. Macrophages within adenomas express iNOS, suggestive of a pro-inflammatory phenotype. Several inflammatory cytokine genes (CXCL1, CXCL2, CXCL3, CCL20, IL8, CCL23, CCL19, CCL21, CCL5) are dysregulated in adenomas. This study has provided evidence of increased inflammation within pre-malignant colonic adenomas. This may allow potential mechanistic pathways in the initiation and promotion of early colorectal carcinogenesis to be identified.
Macrophages are intimately involved in the development of immune-mediated inflammation, including... more Macrophages are intimately involved in the development of immune-mediated inflammation, including glomerulonephritis. We have transduced primary cultures of macrophages to express IL-10 and tested the ability of these cells to control rat nephrotoxic nephritis (NTN), a model of human glomerulonephritis. Ad-IL-10-transduced bone-marrow-derived macrophages (BMDM) produced large amounts of IL-10 in culture, and their TNF-alpha production was decreased in response to interferon-gamma and LPS. Transduced macrophages were injected into the renal artery of rats, 6 h after the induction of NTN, where they localized efficiently to inflamed rat glomeruli. Delivery of IL-10-expressing macrophages to nephritic rats produced a marked reduction in albuminuria compared with unmodified NTN or injection of Ad-null-transduced BMDM. IL-10 treatment decreased the number of glomerular ED1- and ED3-positive cells, MHC class II expression, and the number of fibrinoid lesions. Interestingly, anti-inflammatory changes in the Ad-IL-10-injected kidney were mirrored by changes in the contralateral kidney. These results highlight that Ad-IL-10-transduced macrophages infiltrate inflamed glomeruli and reduce the severity of glomerular inflammation, emphasizing the value of local delivery of genetically modified macrophages in the manipulation of inflammatory disease.
The generation of endogenous hydrogen sulfide may either limit or contribute to the degree of tis... more The generation of endogenous hydrogen sulfide may either limit or contribute to the degree of tissue injury caused by ischemia/reperfusion. A total of 74 male Wistar rats were used to investigate the effects of endogenous and exogenous hydrogen sulfide in renal ischemia/reperfusion. Administration of the irreversible cystathionine g-lyase (CSE) inhibitor, dL-propargylglycine, prevented the recovery of renal function after 45 min ischemia and 72 h reperfusion. The hydrogen sulfide donor sodium hydrosulfide attenuated the (renal, tubular, and glomerular) dysfunction and injury caused by 45 min ischemia and 6 h reperfusion. Western blot analysis of kidneys taken at 30 min reperfusion showed that sodium hydrosulfide significantly attenuated phosphorylation of mitogen-activated protein kinases (p-38, c-JUN N-terminal protein kinase 1/2, and extracellular signal-regulated kinase 1/2) and activation of nuclear factor-kB. At 6 h reperfusion, sodium hydrosulfide significantly attenuated the histological score for acute tubular necrosis, the activation of caspase-3 and Bid, the decline in the expression of anti-apoptotic Bcl-2, and the expression of nuclear factor-kB-dependent proteins (inducible nitric oxide synthase, cyclo-oxygenase-2, and intercellular adhesion molecule-1). These findings suggest that (1) the synthesis of endogenous hydrogen sulfide by CSE is essential to protect the kidney against ischemia/reperfusion injury and dysfunction and aids in the recovery of renal function following ischemia/reperfusion, (2) hydrogen sulfide generated by sodium hydrosulfide reduces ischemia/reperfusion injury and dysfunction, and morphological changes of the kidney, and (3) the observed protective effects of hydrogen sulfide are due to both anti-apoptotic and anti-inflammatory effects.
Renal transplantation: Cyclosporin A and antibody development after donor-specific transfusion. T... more Renal transplantation: Cyclosporin A and antibody development after donor-specific transfusion. The survival of a one haplotype, mismatched living-related renal allograft is improved by donor specific transfusion (DST) before transplantation although the mechanism is unclear. The major risk of DST is sensitization of the recipient to donor lymphocytes precluding transplantation. Fifty prospective recipients of a living related transplant received either DST with cyclosporin A (group I) or DST alone (group II). Persistent donor sensitization precluding transplantation occurred in no patients in group I but in six in group II (P < 0.05). Ten of 14 of those who developed donor cytotoxicity had previously been pregnant or received 10 third party transfusions compared with II of 36 without such a history (P < 0.05). Alloantibodies detected by a cellular ELISA developed following DST in 29% patients and antiidiotypic antibodies detected by the short antiidiotypic assay (SAA) in 36%; antiidiotypic activity occurred more frequently in those given cyclosporin A (P < 0.02). Potentiating activity in the SAA which occurred in sera from six patients after DST had no influence on transplant outcome. Persistent sensitization, particularly in potential transplant recipients who have been pregnant or received many transfusions, can be prevented by giving cyclosporin A with DST; the mechanisms of this effect may be the induction of antiidiotypic antibodies. Both alloantibodies and antiidiotypic antibodies are induced by DST and may protect a subsequent renal allograft from the specific donor.
Background. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in D... more Background. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the development of ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of a water-soluble and potent PARP inhibitor, 5-aminoisoquinolinone (5-AIQ), on the renal injury and dysfunction caused by oxidative stress of the rat kidney in vitro and in vivo.
5-Aminoisoquinolinone reduces renal injury and dysfunction caused by experimental ischemia/reperf... more 5-Aminoisoquinolinone reduces renal injury and dysfunction caused by experimental ischemia/reperfusion. Background. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the development of ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of a water-soluble and potent PARP inhibitor, 5-aminoisoquinolinone (5-AIQ), on the renal injury and dysfunction caused by oxidative stress of the rat kidney in vitro and in vivo. Methods. Primary cultures of rat renal proximal tubular cells, subjected to oxidative stress caused by hydrogen peroxide (H 2 O 2), were incubated with increasing concentrations of 5-AIQ (0.01 to 1 mmol/L) after which PARP activation, cellular injury, and cell death were measured. In in vivo experiments, anesthetized male Wistar rats were subjected to renal bilateral ischemia (45 minutes) followed by reperfusion (6 hours) in the absence or presence of 5-AIQ (0.3 mg/kg) after which renal dysfunction, injury and PARP activation were assessed. Results. Incubation of proximal tubular cells with H 2 O 2 caused a substantial increase in PARP activity, cellular injury, and cell death, which were all significantly reduced in a concentration-dependent by 5-AIQ [inhibitory concentration 50 (IC 50) ∼ 0.03 mmol/L]. In vivo, renal I/R resulted in renal dysfunction, injury, and PARP activation, primarily in the proximal tubules of the kidney. Administration of 5-AIQ significantly reduced the biochemical and histologic signs of renal dysfunction and injury and markedly reduced PARP activation caused by I/R.
respectively. GW274150 abolished the rise in the plasma levels GW274150, a potent and highly sele... more respectively. GW274150 abolished the rise in the plasma levels GW274150, a potent and highly selective inhibitor of iNOS, of nitrate (indicating reduced NO production). GW274150 also reduces experimental renal ischemia/reperfusion injury. reduced the renal dysfunction in wild-type mice to levels similar Background. Generation of nitric oxide (NO) by inducible to that observed in iNOSϪ/Ϫ mice subjected to I/R. Renal nitric oxide synthase (iNOS) may contribute to renal ischemia/ MPO activity and MDA levels were significantly reduced in reperfusion (I/R) injury. The aim of this study was to investigate wild-type mice administered GW274150 and iNOSϪ/Ϫ mice the effects of GW274150, a novel, highly selective, potent and subjected to renal I/R, indicating reduced polymorphonuclear long-acting inhibitor of iNOS activity in rat and mouse models leukocyte (PMN) infiltration and lipid peroxidation. of renal I/R. Conclusions. These results suggest that (1) an enhanced for-Methods. Rats were administered GW274150 (5 mg/kg inmation of NO by iNOS contributes to the pathophysiology of travenous bolus administered 30 minutes prior to I/R) and renal I/R injury and (2) GW274150 reduces I/R injury of the subjected to bilateral renal ischemia (45 minutes) followed by kidney. We propose that selective inhibitors of iNOS activity reperfusion (6 hours). Serum and urinary indicators of renal may be useful against renal dysfunction and injury associated dysfunction, tubular and reperfusion injury were measured, with I/R of the kidney. specifically, serum urea, creatinine, aspartate aminotransferase (AST) and N-acetyl--d-glucosaminidase (NAG) enzymuria. In addition, renal sections were used for histologic scoring of renal injury and for immunologic evidence of nitrotyrosine for-Nitric oxide (NO), generated by any one of the three mation and poly [adenosine diphosphate (ADP)-ribose] (PAR). isoforms of nitric oxide synthase (NOS) plays an impor-Nitrate levels were measured in rat plasma using the Griess tant role in renal function, both under normal and pathoassay. Mice (wild-type, administered 5 mg/kg GW274150, and iNOSϪ/Ϫ) were subjected to bilateral renal ischemia (30 minphysiologic conditions [1, 2]. Although NO appears to utes) followed by reperfusion (24 hours) after which renal play an important functional role within the proximal dysfunction (serum urea, creatinine), renal myeloperoxidase tubule, it can also contribute to pathophysiology of this (MPO) activity and malondialdehyde (MDA) levels were meapart of the kidney [3, 4]. All three isoforms of NOS sured. have been located in the kidney; the constitutive isoform Results. GW274150, administered prior to I/R, significantly reduced serum urea, serum creatinine, AST, and NAG indicat-[endothelial NOS (eNOS)] and neuronal NOS (nNOS)] ing reduction of renal dysfunction and injury caused by I/R. isoforms have been identified in the renal vasculature GW274150 reduced histologic evidence of tubular injury and and macula densa, respectively [1, 2] and inducible NOS markedly reduced immunohistochemical evidence of nitrotyro-(iNOS) can be located and induced in several renal cell sine and PAR formation, indicating reduced peroxynitrite fortypes (e.g., in glomerular mesangial and proximal tubular mation and poly (ADP-ribose) polymerase (PARP) activation, cells) by cytokines, lipopolysaccharide (LPS), and during renal ischemia/reperfusion (I/R) [2-6], leading to renal
We examined the expression of both the natriuretic peptides and natriuretic peptide receptors (NP... more We examined the expression of both the natriuretic peptides and natriuretic peptide receptors (NPR) in primary cultures of rat proximal tubular (RPT) cells using Northern blot assay for peptides and receptors and radioimmunoassay and immunohistochemical analysis for atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide. Freshly isolated cells expressed mRNA coding for ANF, BNP, and the NPR-C. The presence of ANF and BNP in freshly isolated cells was confirmed by immunocytochemical staining. As cells approached confluence, there was a marked increase in mRNA expression for ANF and BNP. Immunocytochemical analysis and radioimmunoassay confirmed that both these peptides were co-localised in RPT cells and present in the cell supernatant. These changes in peptide expression were associated with a concurrent decrease in the expression of the NPR-C and the appearance of the NPR-A and-B. These results confirm that freshly isolated RPT cells possess the components of an autocrine natriuretic peptide system and that growth in primary culture is associated with changes in both peptide system and that growth in primary culture is associated with changes in both peptide and receptor subtype expression, raising the possibility that the endogenous production of ANF and BNP may be involved in the control of control cell growth.
High-density lipoproteins (HDL) have been shown to reduce organ injury and mortality in animal mo... more High-density lipoproteins (HDL) have been shown to reduce organ injury and mortality in animal models of shock via modulation of the expression of adhesion molecules and pro-inflammatory enzymes. As renal inflammation plays an important role in the development of ischemia/reperfusion (I/R) injury of the kidney, the aim of this study was to investigate the ability of HDL to alleviate renal dysfunction and injury in a rat model of renal I/R. HDL (80 mg/kg, intravenous) was administered to male Wistar rats 30 min before bilateral renal ischemia for 45 min followed by reperfusion for up to 48 h. After 6-h reperfusion, HDL significantly reduced (1) renal and tubular dysfunction, (2) tubular and reperfusion-injury, and (3) histologic evidence of renal injury. HDL also improved renal function (after 24-h and 48-h reperfusion) and reduced histologic signs of renal injury (after 48-h reperfusion). Administration of HDL significantly reduced the numbers of polymorphonuclear leukocytes (PMN) i...
Results: The optimal signature contained 28 genes. There was a statistically significant correlat... more Results: The optimal signature contained 28 genes. There was a statistically significant correlation between actual versus predicted time to recurrence for blind data (rho = 0.975; p < 0.0001).
The presence of cytotoxic HLA antibodies (Ab1) against donor lymphocytes in pretransplant sera is... more The presence of cytotoxic HLA antibodies (Ab1) against donor lymphocytes in pretransplant sera is almost always associated with rapid rejection of the renal transplant. We have investigated the possibility that antiidiotypic antibodies (Ab2) to cytotoxic HLA antibodies might modulate the immune response and favorably influence renal allograft outcome. The role of antibodies (Ab3) which potentiate the cytotoxic effect of Ab1 was also studied. Pretransplant sera from 63 patients were tested for inhibitory or potentiating activity in the short antiidiotypic assay. Inhibitory activity was detected in 30 patients and in 28 the transplant survived more than a year. Of patients without antibody activity 11 of 17 had grafts surviving more than one year, and of those showing potentiating activity 11 of 16 were functioning at a year. The difference in transplant survival between the first group and the other two groups was statistically significant (P less than 0.05). There was no significant difference in survival rates between the latter two groups. Potentiating activity is therefore not an independent predictor of transplant failure, whereas the presence of antiidiotypic antibody activity did correlate with improved allograft survival.
Methods in molecular biology (Clifton, N.J.), 2012
Culture of isolated kidney glomerular cells has been employed for almost four decades as a tool t... more Culture of isolated kidney glomerular cells has been employed for almost four decades as a tool to dissect pathophysiological effects of individual cell types in renal disease. This chapter aims to highlight in detail the available techniques to isolate, culture, and characterize human glomerular epithelial and mesangial cells. To establish primary culture of these cells, glomeruli are isolated from the cortex of kidney by differential sieving and cellular outgrowths from cultured glomeruli further subcultured in appropriately coated tissue culture plates/flasks. Methods used for characterization of isolated glomerular mesangial and epithelial cells (podocytes) are described as are the phenotypic markers useful for identification. Other sources of isolated glomerular cells such as immortalized cell lines are briefly discussed.
We have previously shown that the presence in pretransplant recipient sera of Fc receptor blockin... more We have previously shown that the presence in pretransplant recipient sera of Fc receptor blocking antibodies detected by the EA inhibition assay is correlated with improved allograft survival. Twenty-four such sera were assessed for the presence of autoantibodies by the EA inhibition and lymphocytotoxicity assays. No autolymphocytotoxic antibodies were found, and autologous EA inhibition was noted in only one case. EA-inhibiting alloantibodies did occur, and their presence was correlated with improved allograft survival. Sera from 37 dialysis patients were also studied, and neither autologous EA inhibiting nor autologous lymphocytotoxic antibodies were present. Thus Fc receptor blocking alloantibodies that were correlated with improved renal transplant survival were not autoantibodies.
Primary and secondary alloantibody responses were monitored in (AOxPVG)F1 hybrid rats after three... more Primary and secondary alloantibody responses were monitored in (AOxPVG)F1 hybrid rats after three transfusions of DA blood; the initial transfusion was either untreated or pretreated with monoclonal antibody directed to class I antigens or other cell surface markers. Mean antibody activity in recipient sera against class I DA antigens was significantly decreased by pretreatment with the monoclonal antibodies. The most marked suppression was associated with pretreatment by antibodies to the four major nonoverlapping epitopes of the RT1Aa antigen. Subsequent transfusions of DA blood failed to stimulate a secondary response. Crossreactivity of the alloantibody reactivity with BDIX antigens was diminished by pretreating the transfusions with rat anti-RT1A antibodies and, to a lesser extent, with a mouse monoclonal antibody (OX-18) to a common class I determinant. Monoclonal antibody pretreatment had no effect on the humoral response to class II DA antigens. These studies indicate that blood transfusions pretreated with monoclonal antibodies induce a less-potent cytotoxic humoral immune response and that reactivity is most effectively suppressed by completely masking the class I antigen. This technique may prove of clinical value in preventing the sensitization caused by blood transfusions in potential transplant recipients.
Cisplatin exhibits dose-limiting nephrotoxicity in rodents and man. This study investigates the m... more Cisplatin exhibits dose-limiting nephrotoxicity in rodents and man. This study investigates the mechanism of cisplatin nephrotoxicity in vivo and in an in vitro model system. Nephrotoxicity was induced in rats (6 mg/kg cisplatin i.p.) and mice (10 mg/kg cisplatin i.p.). Cisplatin administration significantly elevated blood urea nitrogen (BUN) and serum creatinine in male Sprague Dawley rats day 5 post-treatment (BUN Delta+28+/-5 micromol/ml; serum creatinine Delta+108+/-4 nmol/ml, P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05) and in male C57BL6 mice day 4 post-treatment (BUN Delta+21+/-4 micromol/ml; serum creatinine Delta+81+/-5 nmol/ml, P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). Nephrotoxicity was confirmed by histological analysis that revealed significant damage to the proximal tubules of cisplatin- versus saline vehicle-treated animals. Inhibition of gamma glutamyltranspeptidase prevented cisplatin nephrotoxicity in Sprague Dawley rats (day 5 BUN Delta+1+/-2 micromol/ml; serum creatinine Delta+8+/-4 nmol/ml) and C57BL6 mice (day 4 BUN Delta+1+/-0.8 micromol/ml; serum creatinine Delta-1+/-2 nmol/ml), but not cellular toxicity in rat proximal tubular (RPT) or human proximal tubular (HPT) cultures. Inhibition of aminopeptidase N (AP-N) or renal dipeptidase (RDP) in male Sprague Dawley rats, or in RPT and HPT cell cultures, did not reduce cisplatin toxicity. In contrast to published findings inhibition of C-S lyase did not prevent the nephrotoxicity of cisplatin in vivo or cellular toxicity in vitro. These data demonstrate that the biotransformation enzymes AP-N, RDP and C-S lyase are not implicated in the metabolism of cisplatin to a nephrotoxic metabolite as has been previously hypothesised. Instead, our data demonstrate that gamma glutamyltranspeptidase is a key enzyme involved in mediating cisplatin nephrotoxicity, which potentially acts to cleave cisplatin-GSH conjugates to a toxic metabolite.
Age-related and disease-induced glomerulosclerosis (GS) in rats have both been well defined in a ... more Age-related and disease-induced glomerulosclerosis (GS) in rats have both been well defined in a number of strains and experimental models, but the inter-relationship between the two is not clear. The present study was undertaken to compare the pattern of glomerular injury in these two types of GS. One- and two-shot Thy1 glomerulonephritis (GN) was induced at 2 months of age and followed for 12 months. At 12 months histological injury in proteinuric rats was characterized by segmental hyaline lesions. Two-shot Thy1 GN resulted in accelerated, but morphologically identical injury at 8 months. Histological lesions predictive of subsequent accelerated GS were evaluated at 1, 2, 4 and 6 months. In this regard, glomerular hypercellularity, rather than hypertrophy or matrix increase, was the most consistent histological index of later accelerated disease. The profibrotic cytokines transforming growth factor (TGF)-beta(1) and -beta(3) were localized distinctly to segmental hyaline lesions, but not to areas of matrix increase within the glomerular tuft. This study reveals that GS after Thy1 GN represents acceleration of an age-related disease, presents evidence for use of prolonged glomerular hypercellularity as the best histological index of future disease progression, and correlates the key lesion of GS in these animals, the segmental hyaline lesion, with the presence of TGF-beta peptides.
Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. Inflammatory activ... more Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. Inflammatory activity within the stroma of invasive colorectal tumours is known to be a key predictor of disease activity with type, density and location of immune cells impacting on patient prognosis. To date, there has been no report of inflammatory phenotype within pre-malignant human colonic adenomas. Assessing the stromal microenvironment and particularly, inflammatory activity within colorectal neoplastic lesions is central to understanding early colorectal carcinogenesis. Inflammatory cell infiltrate was assessed by immunohistochemistry in paired colonic adenoma and adjacent normal colonic mucosa samples, and adenomas exhibiting increasing degrees of epithelial cell dysplasia. Macrophage phenotype was assessed using double stain immunohistochemistry incorporating expression of an intracellular enzyme of function. A targeted array of inflammatory cytokine and receptor genes, validated by RT-PCR, was used to assess inflammatory gene expression. Inflammatory cell infiltrates are a key feature of sporadic adenomatous colonic polyps with increased macrophage, neutrophil and T cell (specifically helper and activated subsets) infiltration in adenomatous colonic polyps, that increases in association with characteristics of high malignant potential, namely, increasing degree of cell dysplasia and adenoma size. Macrophages within adenomas express iNOS, suggestive of a pro-inflammatory phenotype. Several inflammatory cytokine genes (CXCL1, CXCL2, CXCL3, CCL20, IL8, CCL23, CCL19, CCL21, CCL5) are dysregulated in adenomas. This study has provided evidence of increased inflammation within pre-malignant colonic adenomas. This may allow potential mechanistic pathways in the initiation and promotion of early colorectal carcinogenesis to be identified.
Macrophages are intimately involved in the development of immune-mediated inflammation, including... more Macrophages are intimately involved in the development of immune-mediated inflammation, including glomerulonephritis. We have transduced primary cultures of macrophages to express IL-10 and tested the ability of these cells to control rat nephrotoxic nephritis (NTN), a model of human glomerulonephritis. Ad-IL-10-transduced bone-marrow-derived macrophages (BMDM) produced large amounts of IL-10 in culture, and their TNF-alpha production was decreased in response to interferon-gamma and LPS. Transduced macrophages were injected into the renal artery of rats, 6 h after the induction of NTN, where they localized efficiently to inflamed rat glomeruli. Delivery of IL-10-expressing macrophages to nephritic rats produced a marked reduction in albuminuria compared with unmodified NTN or injection of Ad-null-transduced BMDM. IL-10 treatment decreased the number of glomerular ED1- and ED3-positive cells, MHC class II expression, and the number of fibrinoid lesions. Interestingly, anti-inflammatory changes in the Ad-IL-10-injected kidney were mirrored by changes in the contralateral kidney. These results highlight that Ad-IL-10-transduced macrophages infiltrate inflamed glomeruli and reduce the severity of glomerular inflammation, emphasizing the value of local delivery of genetically modified macrophages in the manipulation of inflammatory disease.
The generation of endogenous hydrogen sulfide may either limit or contribute to the degree of tis... more The generation of endogenous hydrogen sulfide may either limit or contribute to the degree of tissue injury caused by ischemia/reperfusion. A total of 74 male Wistar rats were used to investigate the effects of endogenous and exogenous hydrogen sulfide in renal ischemia/reperfusion. Administration of the irreversible cystathionine g-lyase (CSE) inhibitor, dL-propargylglycine, prevented the recovery of renal function after 45 min ischemia and 72 h reperfusion. The hydrogen sulfide donor sodium hydrosulfide attenuated the (renal, tubular, and glomerular) dysfunction and injury caused by 45 min ischemia and 6 h reperfusion. Western blot analysis of kidneys taken at 30 min reperfusion showed that sodium hydrosulfide significantly attenuated phosphorylation of mitogen-activated protein kinases (p-38, c-JUN N-terminal protein kinase 1/2, and extracellular signal-regulated kinase 1/2) and activation of nuclear factor-kB. At 6 h reperfusion, sodium hydrosulfide significantly attenuated the histological score for acute tubular necrosis, the activation of caspase-3 and Bid, the decline in the expression of anti-apoptotic Bcl-2, and the expression of nuclear factor-kB-dependent proteins (inducible nitric oxide synthase, cyclo-oxygenase-2, and intercellular adhesion molecule-1). These findings suggest that (1) the synthesis of endogenous hydrogen sulfide by CSE is essential to protect the kidney against ischemia/reperfusion injury and dysfunction and aids in the recovery of renal function following ischemia/reperfusion, (2) hydrogen sulfide generated by sodium hydrosulfide reduces ischemia/reperfusion injury and dysfunction, and morphological changes of the kidney, and (3) the observed protective effects of hydrogen sulfide are due to both anti-apoptotic and anti-inflammatory effects.
Renal transplantation: Cyclosporin A and antibody development after donor-specific transfusion. T... more Renal transplantation: Cyclosporin A and antibody development after donor-specific transfusion. The survival of a one haplotype, mismatched living-related renal allograft is improved by donor specific transfusion (DST) before transplantation although the mechanism is unclear. The major risk of DST is sensitization of the recipient to donor lymphocytes precluding transplantation. Fifty prospective recipients of a living related transplant received either DST with cyclosporin A (group I) or DST alone (group II). Persistent donor sensitization precluding transplantation occurred in no patients in group I but in six in group II (P < 0.05). Ten of 14 of those who developed donor cytotoxicity had previously been pregnant or received 10 third party transfusions compared with II of 36 without such a history (P < 0.05). Alloantibodies detected by a cellular ELISA developed following DST in 29% patients and antiidiotypic antibodies detected by the short antiidiotypic assay (SAA) in 36%; antiidiotypic activity occurred more frequently in those given cyclosporin A (P < 0.02). Potentiating activity in the SAA which occurred in sera from six patients after DST had no influence on transplant outcome. Persistent sensitization, particularly in potential transplant recipients who have been pregnant or received many transfusions, can be prevented by giving cyclosporin A with DST; the mechanisms of this effect may be the induction of antiidiotypic antibodies. Both alloantibodies and antiidiotypic antibodies are induced by DST and may protect a subsequent renal allograft from the specific donor.
Background. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in D... more Background. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the development of ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of a water-soluble and potent PARP inhibitor, 5-aminoisoquinolinone (5-AIQ), on the renal injury and dysfunction caused by oxidative stress of the rat kidney in vitro and in vivo.
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