A population pharmacokinetic model to predict the individual starting dose of tacrolimus in adult renal transplant recipients.

AIMS: The aims of this study were to describe the pharmacokinetics of tacrolimus immediately after kidney transplantation, and to develop a clinical tool for selecting the best starting dose for each patient. METHODS: Data on tacrolimus exposure were collected for the first 3 months following renal transplantation. A population pharmacokinetic analysis was conducted using nonlinear mixed-effects modelling. Demographic, clinical and genetic parameters were evaluated as covariates. RESULTS: A total of 4527 tacrolimus blood samples collected from 337 kidney transplant recipients were available. Data were best described using a two-compartment model. The mean absorption rate was 3.6 h-1 , clearance was 23.0 l h-1 (39% interindividual variability, IIV), central volume of distribution was 692 l (49% IIV) and the peripheral volume of distribution 5340 l (53% IIV). Interoccasion variability was added to clearance (14%). Higher body surface area (BSA), lower serum creatinine, younger age, higher albumin and lower haematocrit levels were identified as covariates enhancing tacrolimus clearance. Cytochrome P450 (CYP) 3A5 expressers had a significantly higher tacrolimus clearance (160%), whereas CYP3A4*22 carriers had a significantly lower clearance (80%). From these significant covariates, age, BSA, CYP3A4 and CYP3A5 genotype were incorporated in a second model to individualize the tacrolimus starting dose: [Formula: see text] Both models were successfully internally and externally validated. A clinical trial was simulated to demonstrate the added value of the starting dose model. CONCLUSIONS: For a good prediction of tacrolimus pharmacokinetics, age, BSA, CYP3A4 and CYP3A5 genotype are important covariates. These covariates explained 30% of the variability in CL/F. The model proved effective in calculating the optimal tacrolimus dose based on these parameters and can be used to individualize the tacrolimus dose in the early period after transplantation.

Data on genotypic distribution and linkage disequilibrium of several ANRIL polymorphisms in hemodialysis patients.

A long non-coding RNA called ANRIL located on chromosome 9p21.3 has been identified as a novel genetic factor associated with cardiovascular disease. Investigation of several single nucleotide polymorphisms (SNPs) of Noncoding Antisense RNA in the INK4 Locus (ANRIL) gene are of particular interest. This article reports data related to the research article entitled: "Association of ANRIL gene polymorphisms with major adverse cardiovascular events in hemodialysis patients" (Arbiol-Roca et al. [1]). Data presented show the genotypic distribution of four selected ANRIL SNPs: rs10757278, rs4977574, rs10757274 and rs6475606 in a cohort constituted by 284 hemodialysis patients. This article analyzes the Hardy-Weinberg disequilibrium of each studied SNP, and the linkage disequilibrium between them.

Association of ANRIL gene polymorphisms with major adverse cardiovascular events in hemodialysis patients.

BACKGROUND: Cardiovascular disease is the leading cause of death in patients with chronic kidney disease (CKD). Single nucleotide polymorphisms (SNPs) in ANRIL gene have been associated with higher cardiovascular morbidity and mortality in general population. The main objective was to ascertain whether ANRIL polymorphisms could identify risk of major adverse cardiovascular event (MACE) in patients starting on hemodialysis (HD). METHODS: This was a prospective observational cohort study. 284 CKD patients starting on HD were included in the study and followed until achievement of the primary end-point (MACE) or end of the study. All patients were genotyped for four ANRIL SNPs (rs10757278, rs4977574, rs10757274 and rs6475606). Kaplan-Meier curves and multivariate Cox survival analyses, together with multiple logistic regression were used to analyze the association between ANRIL SNPs and MACE. RESULTS: We found that ANRIL SNP rs10757278 was a representative SNP of a strong linkage disequilibrium block and showed significant genotypic associations with MACE in hemodialysis patients. Homozygous patients for the risk allele (GG) showed 2.17 (1.05-4.49) fold increased risk of MACE during hemodialysis than carriers of the protective allele (AA or AG). Diabetes mellitus was a strong enhancer of this effect. CONCLUSIONS: Our results indicate that ANRIL polymorphisms may confer risk to development of MACE in incident patients on hemodialysis.

Contribution of Population Pharmacokinetics to Dose Optimization of Ganciclovir-Valganciclovir in Solid-Organ Transplant Patients.

Treatment of solid-organ transplant (SOT) patients with ganciclovir (GCV)-valganciclovir (VGCV) according to the manufacturer's recommendations may result in over- or underexposure. Bayesian prediction based on a population pharmacokinetics model may optimize GCV-VGCV dosing, achieving the area under the curve (AUC) therapeutic target. We conducted a two-arm, randomized, open-label, 40% superiority trial in adult SOT patients receiving GCV-VGCV as prophylaxis or treatment of cytomegalovirus infection. Group A was treated according to the manufacturer's recommendations. For group B, the dosing was adjusted based on target exposures using a Bayesian prediction model (NONMEM). Fifty-three patients were recruited (27 in group A and 26 in group B). About 88.6% of patients in group B and 22.2% in group A reached target AUC, achieving the 40% superiority margin (P< 0.001; 95% confidence interval [CI] difference, 47 to 86%). The time to reach target AUC was significantly longer in group A than in group B (55.9 ± 8.2 versus 15.8 ± 2.3 days,P< 0.001). A shorter time to viral clearance was observed in group B than in group A (12.5 versus 17.6 days;P= 0.125). The incidences of relapse (group A, 66.67%, and group B, 9.01%) and late-onset infection (group A, 36.7%, and group B, 7.7%) were higher in group A. Neutropenia and anemia were related to GCV overexposure. GCV-VCGV dose adjustment based on a population pharmacokinetics Bayesian prediction model optimizes GCV-VGCV exposure. (This study has been registered at ClinicalTrials.gov under registration no. NCT01446445.).

Dendritic cells phenotype fitting under hypoxia or lipopolysaccharide; adenosine 5'-triphosphate-binding cassette transporters far beyond an efflux pump.

This study examines adenosine 5'-triphosphate-binding cassette (ABC) transporters as a potential therapeutic target in dendritic cell (DC) modulation under hypoxia and lipopolysaccharide (LPS). Functional capacity of dendritic cells (DCs) (mixed lymphocyte reaction: MLR) and maturation of iDCs were evaluated in the presence or absence of specific ABC-transporter inhibitors. Monocyte-derived DCs were cultured in the presence of interleukin (IL)-4/granulocyte-macrophage colony-stimulating factor (GM-CSF). Their maturation under hypoxia or LPS conditions was evaluated by assessing the expression of maturation phenotypes using flow cytometry. The effect of ABC transporters on DC maturation was determined using specific inhibitors for multi-drug resistance (MDR1) and multi-drug resistance proteins (MRPs). Depending on their maturation status to elicit T cell alloresponses, the functional capacity of DCs was studied by MLR. Mature DCs showed higher P-glycoprotein (Pgp) expression with confocal microscopy. Up-regulation of maturation markers was observed in hypoxia and LPS-DC, defining two different DC subpopulation profiles, plasmacytoid versus conventional-like, respectively, and different cytokine release T helper type 2 (Th2) versus Th1, depending on the stimuli. Furthermore, hypoxia-DCs induced more B lymphocyte proliferation than control-iDC (56% versus 9%), while LPS-DCs induced more CD8-lymphocyte proliferation (67% versus 16%). ABC transporter-inhibitors strongly abrogated DC maturation [half maximal inhibitory concentration (IC50 ): P-glycoprotein inhibition using valspodar (PSC833) 5 µM, CAS 115104-28-4 (MK571) 50 µM and probenecid 2·5 µM], induced significantly less lymphocyte proliferation and reduced cytokine release compared with stimulated-DCs without inhibitors. We conclude that diverse stimuli, hypoxia or LPS induce different profiles in the maturation and functionality of DC. Pgp appears to play a role in these DC events. Thus, ABC-transporters emerge as potential targets in immunosuppressive therapies interfering with DCs maturation, thereby abrogating innate immune response when it is activated after ischaemia.

Hepatocyte growth factor gene therapy enhances infiltration of macrophages and may induce kidney repair in db/db mice as a model of diabetes.

AIMS/HYPOTHESIS: We previously demonstrated hepatocyte growth factor (HGF) gene therapy was able to induce regression of glomerulosclerosis in diabetic nephropathy through local reparative mechanisms. The aim of this study was to test whether bone-marrow-derived cells are also involved in this HGF-induced reparative process. METHODS: We have created chimeric db/db mice as a model of diabetes that produce enhanced green fluorescent protein (EGFP) in bone marrow cells. We performed treatment with HGF gene therapy either alone or in combination with granulocyte-colony stimulating factor, in order to induce mobilisation of haematopoietic stem cells in these diabetic and chimeric animals. RESULTS: We find HGF gene therapy enhances renal expression of stromal-cell-derived factor-1 and is subsequently associated with an increased number of bone-marrow-derived cells getting into the injured kidneys. These cells are mainly monocyte-derived macrophages, which may contribute to the renal tissue repair and regeneration consistently observed in our model. Finally, HGF gene therapy is associated with the presence of a small number of Bowman's capsule parietal epithelial cells producing EGFP, suggesting they are fused with bone-marrow-derived cells and are contributing to podocyte repopulation. CONCLUSIONS/INTERPRETATION: Altogether, our findings provide new evidence about the therapeutic role of HGF and open new opportunities for inducing renal regeneration in diabetic nephropathy.

Determination of ganciclovir in human plasma by ultra performance liquid chromatography-UV detection.

OBJECTIVES: Implement a sensitive UHPLC method for the assay of ganciclovir in human plasma. DESIGN AND METHODS: We developed and validated a chromatographic method coupled to ultraviolet detection for quantification of ganciclovir, with a short run time using a small volume of human plasma. Comparison of system performance was made with respect to analysis time, efficiency and sensitivity. RESULTS: Correlation coefficients (r) of the calibration curves ranged from 0.999744 to 0.999784. Within-day and between-day imprecision and inaccuracy, specificity and recovery were also evaluated for validation. The method was precise and accurate and the retention time was 0.7 min. The calibration curves were linear between 0.5 and 30 µg/mL. There was a good correlation between HPLC and UHPLC techniques. CONCLUSIONS: We developed a method that is currently applied in a clinical study assessing GCV plasma concentration variability after ganciclovir and valganciclovir administration.

Tubular epithelial cells transfected with hHGF counteracts monocyte chemotactic protein-1 up-regulation after hypoxia/reoxygenation insult.

Acute kidney injury (AKI) which is mainly produced by nephrotoxic or ischemic insults is correlated with a high mortality and morbidity. Proximal tubular epithelial cells (PTEC) play a major role. They are the main target of ischemia/reperfusion injury. PTECs have also been proposed as the effectors of AKI reversibility, but also as the creator of the inflammatory milieu: cytokine, chemokine, and complement expression. An important chemokine implicated in this process is monocyte chemotactic protein-1 (MCP-1) due to its ability to recruit and activate monocytes. Hepatocyte growth factor (HGF) is a pleiotropic factor with mitogenic, anti-apoptotic, and proliferative effects which has recently been studied for its anti-inflammatory and antifibrogenic effects. Our aim was to evaluate the potential inflammatory effect of hypoxia and reoxygenation on rat PTECs. We created a stable human HGF (hHGF) expressing PTEC line that emulated in vivo transfection and analyzed the role of this cell type in the induction and reversibility of AKI. Our results showed the efficiency of transfection with the hHGF gene to promote sustained expression of the protein in the medium (7627.13 +/- 1144.078 to 8211.3 +/- 795.37 pg/mL). When rat PTECs were under a hypoxia/reoxygenation insult, MCP-1 was highly overexpressed (4479.3 +/- 154.3 pg/mL of protein and 5.099 +/- 1.23 times control gene expression). Transfected cells abrogated this effect (288.7 +/- 13.5 pg/mL and 1.169 +/- 0.0759 times control). In conclusion, we observed that the hypoxia/reoxygenation insult stimulated MCP-1 protein secretion in PTECs and that PTECs which were stably transfected and overexpressing hHGF abrogated the inflammatory reaction mediated by hypoxia/reoxygenation, being a suitable model for later studies.

Bone marrow transplantation induces normoglycemia in a type 2 diabetes mellitus murine model.

OBJECTIVE: To study the cellular mechanisms involved in the regression of diabetic nephropathy, bone marrow-derived cells must be identified. The aim of this study was to obtain a diabetic chimeric model with bone marrow cells expressing enhanced green fluorecence protein (EGFP), without modifying the course of diabetic nephropathy. MATERIALS AND METHODS: Bone marrow transplantation (BMT) was performed in an obese type 2 diabetic murine model (db/db) owing to a mutation in the leptin receptor gene. Whole bone marrow from female donor C57BL/6 EGFP+ mice was transplanted into 8-week-old C57BL/6 mice and into 8- and 24-week-old female C57BLKS (db/db) EGFP- mice. Recipient mice received total body irradiation (TBI) followed by bone marrow (BM) cell infusion. We tested various irradiation doses (Gy) and numbers of BM cells. RESULTS: When a low TBI dose and a small number of BM cells were administered, only syngeneic C57BL/6 mice became chimeric, whereas allogeneic db/db mice showed rejection. When Gy dose and BM cells were increased, db/db mice became chimeric. However, 8-week-old db/db mice lost the obese phenotype and became normoglycemic, probably due to peripheral BM cell infiltration. Conversely, 24-week-old db/db mice remained obese showing similar blood glucose values, body weights, albuminuria, and glomerular lesions at nontransplanted db/db mice. CONCLUSIONS: Recipient age greatly influenced the peripheral repopulation after BMT in db/db mice. Only the adult chimeric db/db mice seemed to be a good model to study the cellular mechanisms involved in the regression of diabetic nephropathy.

Population pharmacokinetics of ganciclovir after intravenous ganciclovir and oral valganciclovir administration in solid organ transplant patients infected with cytomegalovirus.

A population pharmacokinetics analysis was performed after intravenous ganciclovir and oral valganciclovir in solid organ transplant patients with cytomegalovirus. Patients received ganciclovir at 5 mg/kg of body weight (5 days) and then 900 mg of valganciclovir (16 days), both twice daily with dose adjustment for renal function. A total of 382 serum concentrations from days 5 and 15 were analyzed with NONMEM VI. Renal function given by creatinine clearance (CL(CR)) was the most influential covariate in CL. The final pharmacokinetic parameters were as follows: ganciclovir clearance (CL) was 7.49.(CL(CR)/57) liter/h (57 was the mean population value of CL(CR)); the central and peripheral distribution volumes were 31.9 liters and 32.0 liters, respectively; intercompartmental clearance was 10.2 liter/h; the first-order absorption rate constant was 0.895 h(-1); bioavailability was 0.825; and lag time was 0.382 h. The CL(CR) was the best predictor of CL, making dose adjustment by this covariate important to achieve the most efficacious ganciclovir exposure.

Hypoxia stimulus: An adaptive immune response during dendritic cell maturation.

The 'injury hypothesis' in organ transplantation suggests that ischemia-reperfusion injury is involved in the adaptative alloimmune response. We previously found that a strong immune/inflammatory response was induced by ischemia during kidney transplantation in rats. We show here that immature dendritic cells (DCs) undergo hypoxia-mediated differentiation comparable to allogeneic stimulation. Hypoxia-differentiated DCs overexpress hypoxia inducible factor-1alpha (HIF-1alpha) and its downstream target genes, such as vascular endothelial growth factor or glucose transporter-1. Rapamycin attenuated DC differentiation, HIF-1alpha expression, and its target gene expression in a dose-dependent manner along with downregulated interleukin-10 secretion. Coculture of hypoxia-differentiated DCs with CD3 lymphocytes induced proliferation of lymphocytes, a process also neutralized by rapamycin. Furthermore, in vivo examination of ischemia-reperfusion-injured mouse kidneys showed a clear maturation of resident DCs that was blunted by rapamycin pretreatment. Our results suggest that hypoxia is a central part of the 'injury hypothesis' triggering DC differentiation under hypoxic conditions. Rapamycin attenuates the hypoxic immune-inflammatory response through inhibition of the HIF-1alpha pathway.

New immunosuppresor strategies in the treatment of murine lupus nephritis.

Renal involvement in systemic lupus erythematosus is a common complication that significantly worsens morbidity and mortality. Although treatment with corticosteroids and cytotoxic drugs may be useful in many cases, morbidity associated with these drugs and the relapsing nature of the disease make it necessary to develop new treatment strategies. Five-month old female NZB/W F1 mice were divided into the following groups: CYP group (n = 10), cyclophosphamide (CYP) 50 mg/kg intraperitoneally every 10 days; RAPA 1 group (n = 10) oral daily sirolimus (SRL), 1 mg/kg; RAPA 12 group (n = 13), oral daily SRL, 12mg/kg; FTY group (n = 10), oral fingolimod (FTY720), 2 mg/kg three times per week. An additional group of 13 non-treated mice were used as a control (control group). Follow-up was performed over four months. Animal survival, body weight, anti-DNA antibodies and proteinuria were determined. Kidneys were processed for conventional histology and immunofluorescence for IgG and complement. Total histological score (HS) was the sum of mesangial expansion, endocapillary proliferation glomerular deposits, extracapillary proliferation, interstitial infiltrates, tubular atrophy and interstitial fibrosis. All treated groups had lower proteinuria at the end of the follow-up with respect to the control group (P < 0.0001). Serum anti-DNA antibodies were appropriately controlled in RAPA 1 and CYP groups, but not in FTY or RAPA 12 groups. SRL and CYP arrested, and perhaps reversed almost all histological lesions. FTY720 ameliorated histological lesions but did not control mesangial expansion or interstitial infiltrates. SRL produces great improvement in murine lupus nephritis, while FTY720 seems a promising alternative if used in appropriate doses.

HGF gene therapy attenuates renal allograft scarring by preventing the profibrotic inflammatory-induced mechanisms.

Inflammatory processes and tissue scarring are characteristic features of chronic allograft nephropathy. Hepatocyte growth factor (HGF) has beneficial effects on renal fibrosis and it also ameliorates renal interstitial inflammation as it has been recently described. Contrarily to protein administration, intramuscular gene electrotransfer allows sustained release of HGF. So, here we hypothesized that gene therapy with human HGF would diminish the characteristic scarring of chronic allograft nephropathy either by antagonizing tissue fibrosis mechanisms or by reducing inflammation. Lewis rats transplanted with cold preserved Fischer kidneys received vehicle (NoHGF) or intramuscular plasmid DNA encoding HGF plus electroporation either before transplantation (IniHGF, early post-transplant cytoprotection of tubular cells) or 8/10 weeks after transplantation (DelHGF, delayed prevention of chronic mechanisms). Serum creatinine and proteinuria were measured every 4 weeks for 24 weeks. Grafts at 12 or 24 weeks were evaluated for glomerulosclerosis, fibrosis inflammatory cells and mediators, cell regeneration and tubulo-interstitial damage. Nontreated animals developed renal insufficiency, progressive proteinuria and fibrosis among other characteristic histological features of chronic allograft nephropathy. Treatment with human HGF, especially when delayed until the onset of fibrogenic mechanisms, reduced renal failure and mortality, diminished tubule-interstitial damage, induced cell regeneration, decreased inflammation, NF-kappaB activation, and profibrotic markers at 12 weeks and prevented late interstitial fibrosis and glomerulosclerosis. The effectiveness of HGF-gene therapy in the prevention of renal allograft scarring is related with the halt of profibrotic inflammatory-induced mechanisms.

Direct electrotransfer of hHGF gene into kidney ameliorates ischemic acute renal failure.

In the early phase of kidney transplantation, the transplanted kidney is exposed to insults like ischemia/reperfusion, which is a leading cause of acute renal failure (ARF). ARF in the context of renal transplantation predisposes the graft to developing chronic damage and to long-term graft loss. Hepatocyte growth factor (HGF) has been suggested to support the intrinsic ability of the kidney to regenerate in response to injury by its morphogenic, mitogenic, motogenic and antiapoptotic activities. In the present paper, we examine whether human HGF (hHGF) gene electrotransfer helps in the recovery from ARF in a model of rat renal warm ischemia. We also assess the advantages of this form of gene therapy by direct electroporation of the kidney, given that transplantation offers the possibility of manipulating the organ in vivo. We have compared the therapeutic efficiency of two electroporation methodologies in a rat ARF model. Although they both targeted the same organ, the two methods were applied to different parts of the animal: muscle and kidney. Kidney direct electrotransfer was shown to be more efficient not only in pharmacokinetic but also in therapeutic terms, so it may become a clinically practical alternative in renal transplantation.

Role of cold ischemia in acute rejection: characterization of a humoral-like acute rejection in experimental renal transplantation.

The aim of the study was to characterize the role of cold ischemia in the process of acute rejection using an experimental renal transplant model. Syngeneic renal transplants were performed between Wistar Agouti rats and allogeneic grafts using Wistar-Agouti rats as recipients of Brown-Norway kidneys. For cold ischemia (CI), kidneys were preserved in Euro-Collins (4 degrees C/ 2.5 hours). Rats were bilaterally nephrectomized at the moment of renal transplant and did not receive any immunosuppressant. The groups were NoAR (n = 6): immediate syngeneic transplant; CI-NoAR (n = 6): syngeneic transplant with CI; AR (n = 13): immediate allogeneic graft; CI-AR (n = 6): allogeneic graft with CI. Allogeneic rats were followed for the survival study. Syngeneic rats, with mean survival time beyond 6 months, were sacrificed on the day 7 to compare grafts with those in the allogeneic groups. H&E- and PAS-stained grafts were evaluated using the Banff criteria. Tissue INF-gamma and TNF-alpha were quantified by RT-real time-PCR on the kidney grafts. Renal insufficiency did not appear in the NoAR group, but it did from the posttransplant day 5 in both acute rejection groups. While NoAR kidneys showed well-conserved renal architecture, then AR group displayed variable degrees of tubular necrosis with scarce cellular infiltration, interstitial hemorrhage, vascular damage with fibrinoid necrosis, perivascular edema, and nuclear disruption. Cold ischemia in rejecting animals increased the mortality rate due to renal insufficiency and accelerated acute rejection. Independently of CI, the proinflammatory cytokines TNF-alpha and INF-gamma were increased in both rejection groups. In conclusion, addition of CI overactivates the acute rejection process via a humoral component.

The enhancement of endogenous cAMP with pituitary adenylate cyclase-activating polypeptide protects rat kidney against ischemia through the modulation of inflammatory response.

BACKGROUND: Cyclic nucleotide analogue administration improves ischemia-reperfusion damage in several organs. The neuropeptide pituitary adenylate cyclase-activating polypeptide, PACAP-38, is a potent stimulus to enhance cellular cAMP levels. This study tested the protective effect of enhancing endogenous cAMP levels by PACAP-38 in a model of warm renal ischemia. METHODS: Sprague-Dawley rats underwent 40 min of bilateral warm renal ischemia. PACAP-38 continuous infusion began either before ischemia or at 6 hr or 18 hr after ischemia. A mini-osmotic pump infused PACAP-38 throughout 7 days of follow-up. Groups were constructed with sham, ischemic control, and dibutyryl cAMP treated animals, and four PACAP-38 treatment groups, using 16 pmol/hr or 160 pmol/hr of the compound, or delaying its administration by 6 hr or 18 hr after ischemia. Renal function was assessed by means of serum creatinine levels on days 1, 2, 3, and 7 after ischemia. Conventional histology was performed on day 7. Renal myeloperoxidase (MPO) activity, infiltrating CD45+ cells, plasma and tissue cAMP, and serum IL-6 were measured. RESULTS: Continuous administration of the high concentration of PACAP-38 ameliorated renal function and morphologic abnormalities induced by warm ischemia. Treatment with dibutyryl cAMP produced morphologic protection but only partial functional effect on the ischemic kidney. A 6-hour delay in the administration of the compound after ischemia offered similar protective effect, whereas an 18-hr delay did not. The neuropeptide clearly increased circulating cAMP after ischemia but not cAMP in renal tissue. PACAP-38 increased circulating IL-6, and minimized renal inflammatory cell infiltration induced by ischemia-reperfusion injury, as evidenced by a reduction of MPO activity and the number of CD45+ cells in ischemic renal tissue. CONCLUSIONS: Enhancement of endogenous circulating cAMP with PACAP-38 modulates postischemic inflammatory response and strongly protects from ischemic acute renal failure, even when administration is delayed for 6 hr after injury.

Protective effect of UR-12670 on chronic nephropathy induced by warm ischaemia in ageing uninephrectomized rats.

BACKGROUND: In young animals, renal ischaemia/reperfusion injury and mass reduction are associated with chronic lesions that mimic those found in chronic rejection. We have shown that the phospholipid platelet-activating factor (PAF) participates in young animals in such chronic nephropathy. Here we examine the long-term effects of the orally active PAF antagonist, UR-12670 in ageing uninephrectomized rats exposed to prolonged warm ischaemia. METHODS: Fifteen- to eighteen-month-old uninephrectomized male Sprague-Dawley rats were allocated into three groups and followed for 16 weeks: UNx, rats without ischaemia; UNxISC, ischaemic kidney (60 min), and UNxISC+UR, ischaemic kidney and UR-12670 from day 0 to the 16th week. Serum creatinine and proteinuria were monitored every 4 weeks. At the end of the study, conventional histology was performed and monocyte-macrophages were identified with the specific monoclonal antibody ED-1. RESULTS: The UNxISC group had severe acute renal failure with a high mortality rate, which was associated with incomplete restoration of renal function. Renal insufficiency in this group was sustained throughout the follow-up. Both UNx and UNxISC groups developed progressive proteinuria from the 12th week. Though UNxISC+UR group showed similar acute renal failure and mortality rate to the ischaemic non-treated group, serum creatinine decreased to levels similar to UNx group, which were maintained until the end of the study. Treatment of ischaemic kidneys with UR-12670 produced a slight decrease in 24-h proteinuria and a reduction in glomerulosclerosis, the mean tubulointerstitial score and number of monocyte-macrophages to values similar to UNx group. CONCLUSIONS: The chronic administration of the PAF antagonist UR-12670 attenuates the long-term effects of ischaemia-reperfusion injury in uninephrectomized ageing rats. The beneficial effect of this agent suggests that PAF contributes to the progression to late renal damage in this model.

Influence of nephron mass in development of chronic renal failure after prolonged warm renal ischemia.

The present study examined the long-term consequences of warm renal ischemia (WRI) with or without renal ablation. Male Sprague-Dawley rats (250-300 g) were subjected to 60 min of complete WRI by pedicle clamping and then followed for 52 wk. Animals were organized into four groups: rats in which both kidneys were subjected to warm ischemia (2WIK); rats with left WRI and right nephrectomy (1WIK); uninephrectomized rats with a left nonischemic kidney (1NK); and sham-operated rats (2NK). Additional animals were studied at 24 h, 7 days, and 16 and 32 wk. In the first week after WRI, rats from the 2WIK and 1WIK groups displayed a similar degree of acute renal damage. After recovering from acute renal failure, 1WIK rats developed progressive and severe proteinuria, whereas it was mild in the 2WIK group, as well as in the 1NK and 2NK groups. Only animals from the 1WIK group developed severe chronic renal failure, glomerulosclerosis, interstitial fibrosis, and upregulation of transforming growth factor-beta(1) (TGF-beta(1)) gene, which was associated with increased TGF-beta(1) protein expression in tubular epithelial cells, arterioles, and in areas of mononuclear interstitial cell infiltrate. On the contrary, long-term renal TGF-beta(1) expression, function, and histology were similar in 2WIK and 2NK rats. The present study shows that prolonged bilateral WRI, when both kidneys are retained in place, induces very mild long-term renal lesions as opposed to the severe renal scarring observed when WRI is combined with contralateral nephrectomy.