Mayaro virus pathogenesis and immunity in rhesus macaques.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes debilitating and persistent arthritogenic disease. While MAYV was previously reported to infect non-human primates (NHP), characterization of MAYV pathogenesis is currently lacking. Therefore, in this study we characterized MAYV infection and immunity in rhesus macaques. To inform the selection of a viral strain for NHP experiments, we evaluated five MAYV strains in C57BL/6 mice and showed that MAYV strain BeAr505411 induced robust tissue dissemination and disease. Three male rhesus macaques were subcutaneously challenged with 105 plaque-forming units of this strain into the arms. Peak plasma viremia occurred at 2 days post-infection (dpi). NHPs were taken to necropsy at 10 dpi to assess viral dissemination, which included the muscles and joints, lymphoid tissues, major organs, male reproductive tissues, as well as peripheral and central nervous system tissues. Histological examination demonstrated that MAYV infection was associated with appendicular joint and muscle inflammation as well as presence of perivascular inflammation in a wide variety of tissues. One animal developed a maculopapular rash and two NHP had viral RNA detected in upper torso skin samples, which was associated with the presence of perivascular and perifollicular lymphocytic aggregation. Analysis of longitudinal peripheral blood samples indicated a robust innate and adaptive immune activation, including the presence of anti-MAYV neutralizing antibodies with activity against related Una virus and chikungunya virus. Inflammatory cytokines and monocyte activation also peaked coincident with viremia, which was well supported by our transcriptomic analysis highlighting enrichment of interferon signaling and other antiviral processes at 2 days post MAYV infection. The rhesus macaque model of MAYV infection recapitulates many of the aspects of human infection and is poised to facilitate the evaluation of novel therapies and vaccines targeting this re-emerging virus.

Development of a next-generation chikungunya virus vaccine based on the HydroVax platform.

Chikungunya virus (CHIKV) is an emerging/re-emerging mosquito-borne pathogen responsible for explosive epidemics of febrile illness characterized by debilitating polyarthralgia and the risk of lethal infection among the most severe cases. Despite the public health risk posed by CHIKV, no vaccine is currently available. Using a site-directed hydrogen peroxide-based inactivation approach, we developed a new CHIKV vaccine, HydroVax-CHIKV. This vaccine technology was compared to other common virus inactivation approaches including ß-propiolactone (BPL), formaldehyde, heat, and ultraviolet (UV) irradiation. Heat, UV, and BPL were efficient at inactivating CHIKV-181/25 but caused substantial damage to neutralizing epitopes and failed to induce high-titer neutralizing antibodies in vaccinated mice. HydroVax-CHIKV and formaldehyde-inactivated CHIKV retained intact neutralizing epitopes similar to live virus controls but the HydroVax-CHIKV approach demonstrated a more rapid rate of virus inactivation. HydroVax-CHIKV vaccination induced high neutralizing responses to homologous and heterologous CHIKV clades as well as to other alphaviruses including Mayaro virus, O'nyong'nyong virus, and Una virus. Following heterologous infection with CHIKV-SL15649, HydroVax-CHIKV-immunized mice were protected against viremia, CHIKV-associated arthritic disease, and lethal CHIKV infection by an antibody-dependent mechanism. In contrast, animals vaccinated with Heat- or UV-inactivated virus showed no protection against viremia in addition to demonstrating significantly exacerbated CD4+ T cell-mediated footpad swelling after CHIKV infection. Together, these results demonstrate the risks associated with using suboptimal inactivation methods that fail to elicit protective neutralizing antibody responses and show that HydroVax-CHIKV represents a promising new vaccine candidate for prevention of CHIKV-associated disease.

Blocking the IL-1 receptor reduces cardiac transplant ischemia and reperfusion injury and mitigates CMV-accelerated chronic rejection.

Ischemia-reperfusion injury (IRI) is an important risk factor for accelerated cardiac allograft rejection and graft dysfunction . Utilizing a rat heart isogeneic transplant model, we identified inflammatory pathways involved in IRI in order to identify therapeutic targets involved in disease. Pathway analyses identified several relevant targets, including cytokine signaling by the IL-1 receptor (IL-1R) pathway and inflammasome activation. To investigate the role of IL-1R signaling pathways during IRI, we treated syngeneic cardiac transplant recipients at 1-hour posttransplant with Anakinra, a US Food and Drug Administration (FDA)-approved IL-1R antagonist; or parthenolide, a caspase-1 and nuclear factor kappa-light-chain-enhancer of activated B cells inhibitor that blocks IL-1ß maturation. Both Anakinra and parthenolide significantly reduced graft inflammation and cellular recruitment in the treated recipients relative to nontreated controls. Anakinra treatment administered at 1-hour posttransplant to recipients of cardiac allografts from CMV-infected donors significantly increased the time to rejection and reduced viral loads at rejection. Our results indicate that reducing IRI by blocking IL-1Rsignaling pathways with Anakinra or inflammasome activity with parthenolide provides a promising approach for extending survival of cardiac allografts from CMV-infected donors.

Macrophage depletion of CMV latently infected donor hearts ameliorates recipient accelerated chronic rejection.

Cytomegalovirus (CMV) infection is linked to acceleration of solid organ transplant vascular sclerosis (TVS) and chronic rejection (CR). Donor latent CMV infection increases cardiac-resident macrophages and T cells leading to increased inflammation, promoting allograft rejection. To investigate the role of cardiac-resident passenger macrophages in CMV-mediated TVS/CR, macrophages were depleted from latently ratCMV (RCMV)-infected donor allografts prior to transplantation. Latently RCMV-infected donor F344 rats were treated with clodronate, PBS, or control liposomes 3 days prior to cardiac transplant into RCMV-naïve Lewis recipients. Clodronate treatment significantly increased graft survival from post-operative day (POD)61 to POD84 and decreased TVS at rejection. To determine the kinetics of the effect of clodronate treatment's effect, a time study revealed that clodronate treatment significantly decreased macrophage infiltration into allograft tissues as early as POD14; altered allograft cytokine/chemokine protein levels, fibrosis development, and inflammatory gene expression profiles. These findings support our hypothesis that increased graft survival as a result of allograft passenger macrophage depletion was in part a result of altered immune response kinetics. Depletion of donor macrophages prior to transplant is a strategy to modulate allograft rejection and reduce TVS in the setting of CMV + donors transplanted into CMV naïve recipients.

Rat Cytomegalovirus Virion-Associated Proteins R131 and R129 Are Necessary for Infection of Macrophages and Dendritic Cells.

Cytomegalovirus (CMV) establishes persistent, latent infection in hosts, causing diseases in immunocompromised patients, transplant recipients, and neonates. CMV infection modifies the host chemokine axis by modulating chemokine and chemokine receptor expression and by encoding putative chemokine and chemokine receptor homologues. The viral proteins have roles in cellular signaling, migration, and transformation, as well as viral dissemination, tropism, latency and reactivation. Herein, we review the contribution of CMV-encoded chemokines and chemokine receptors to these processes, and further elucidate the viral tropism role of rat CMV (RCMV) R129 and R131. These homologues of the human CMV (HCMV)-encoded chemokines UL128 and UL130 are of particular interest because of their dual role as chemokines and members of the pentameric entry complex, which is required for entry into cell types that are essential for viral transmission and dissemination. The contributions of UL128 and UL130 to acceleration of solid organ transplant chronic rejection are poorly understood, and are in need of an effective in vivo model system to elucidate the phenomenon. We demonstrated similar molecular entry requirements for R129 and R131 in the rat cells, as observed for HCMV, and provided evidence that R129 and R131 are part of the viral entry complex required for entry into macrophages, dendritic cells, and bone marrow cells.

A neonatal nonhuman primate model of gestational Zika virus infection with evidence of microencephaly, seizures and cardiomyopathy.

Zika virus infection during pregnancy is associated with miscarriage and with a broad spectrum of fetal and neonatal developmental abnormalities collectively known as congenital Zika syndrome (CZS). Symptomology of CZS includes malformations of the brain and skull, neurodevelopmental delay, seizures, joint contractures, hearing loss and visual impairment. Previous studies of Zika virus in pregnant rhesus macaques (Macaca mulatta) have described injury to the developing fetus and pregnancy loss, but neonatal outcomes following fetal Zika virus exposure have yet to be characterized in nonhuman primates. Herein we describe the presentation of rhesus macaque neonates with a spectrum of clinical outcomes, including one infant with CZS-like symptoms including cardiomyopathy, motor delay and seizure activity following maternal infection with Zika virus during the first trimester of pregnancy. Further characterization of this neonatal nonhuman primate model of gestational Zika virus infection will provide opportunities to evaluate the efficacy of pre- and postnatal therapeutics for gestational Zika virus infection and CZS.

Cytomegalovirus microRNA expression is tissue specific and is associated with persistence.

MicroRNAs (miRNAs) are a class of small noncoding RNAs involved in posttranscriptional regulation. miRNAs are utilized in organisms ranging from plants to higher mammals, and data have shown that DNA viruses also use this method for host and viral gene regulation. Here, we report the sequencing of the small RNAs in rat cytomegalovirus (RCMV)-infected fibroblasts and persistently infected salivary glands. We identified 24 unique miRNAs that mapped to hairpin structures found within the viral genome. While most miRNAs were detected in both samples, four were detected exclusively in the infected fibroblasts and two were specific for the infected salivary glands. The RCMV miRNAs are distributed across the viral genome on both the positive and negative strands, with clusters of miRNAs at a number of locations, including near viral genes r1 and r111. The RCMV miRNAs have a genomic positional orientation similar to that of the miRNAs described for mouse cytomegalovirus, but they do not share any substantial sequence conservation. Similar to other reported miRNAs, the RCMV miRNAs had considerable variation at their 3' and 5' ends. Interestingly, we found a number of specific examples of differential isoform usage between the fibroblast and salivary gland samples. We determined by real-time PCR that expression of the RCMV miRNA miR-r111.1-2 is highly expressed in the salivary glands and that miR-R87-1 is expressed in most tissues during the acute infection phase. Our study identified the miRNAs expressed by RCMV in vitro and in vivo and demonstrated that expression is tissue specific and associated with a stage of viral infection.

Role of cellular cholesterol in pharmacologic preconditioning with cyclosporine in experimental kidney transplantation.

BACKGROUND/AIMS: Ischemia reperfusion injury in the early posttransplant period affects immediate graft function and late allograft dysfunction. Recently, we showed that pharmacologic preconditioning with a calcineurin inhibitor improved transplant outcomes in rat syngeneic kidney transplantation. There is also evidence that cellular cholesterol content increases after many types of renal injury. METHODS: In this study, we looked at the effect of cyclosporine (CsA) on the donor kidney free cholesterol (FC) content in this model. Donor rats were pretreated with one dose of CsA 10 mg/kg administered 24 h or 7 days before being subjected to 2 h cold ischemia and then transplanted. RESULTS: Pharmacologic preconditioning with CsA significantly improved renal function and histology and increased donor kidney FC content. On the other hand, fluvastatin co-administration with CsA abrogated that beneficial effect in association with a decrease in donor kidney FC content. CONCLUSION: CsA preconditioning leads to better outcomes in kidney transplantation and is associated with up-regulation of renal FC content. The latter may then contribute to acquired cytoresistance, possibly by stabilizing the plasma membrane. Thus, use of statins around the time of transplantation may need to be evaluated until further studies are conducted to determine the clinical relevance of this observation.

Donor preconditioning with a calcineurin inhibitor improves outcome in rat syngeneic kidney transplantation.

Ischemia-reperfusion injury (IRI) in the early posttransplant period affects immediate graft function and late allograft dysfunction. This study determines the influence of pharmacologic preconditioning with a calcineurin inhibitor on IRI in a syngeneic F344 rat kidney transplant model. Donor rats were pretreated with one dose of cyclosporine (10 mg/kg) or tacrolimus (1 mg/kg) administered at 24 hr or 7 days before being subjected to 2 hr of cold ischemia and then transplanted. Pharmacologic preconditioning significantly improved renal function, as assessed by serum creatinine and inulin clearance, and histologic score versus vehicle-treated rats. There were no differences between cyclosporine and tacrolimus in the measured outcomes. This renoprotective effect, although not complete, was seen with only one dose of calcineurin inhibitor, and the effect was sustained for at least 7 days before IRI. This approach may represent a viable pharmacologic intervention to decrease IRI at the time of organ transplantation.

Effect of cyclosporine and sirolimus on the expression of connective tissue growth factor in rat experimental chronic nephrotoxicity.

BACKGROUND/AIMS: Connective tissue growth factor (CTGF) is a pro-fibrotic growth factor that acts downstream of transforming growth factor (TGF)-beta. However, CTGF regulation remains unknown. We tried to determine the effect of two commonly used immunosuppressants, cyclosporine (CsA) and sirolimus (SRL), on CTGF expression in a model of chronic nephrotoxicity. METHODS: Adult Sprague-Dawley rats kept on a low-salt diet were treated daily for 4 weeks with vehicle (VH), SRL (0.3 mg/kg), CsA5 (5 mg/kg), CsA10 (10 mg/kg) or both CsA5 and SRL. CTGF and TGF-beta1 expressions were evaluated by Northern blot. Functional and histologic parameters in addition to number of apoptotic cells were determined. RESULTS: At 28 days, both CsA doses were capable of inhibiting CTGF mRNA expression to levels similar to control. On the other hand, SRL increased CTGF expression by 3.5-fold. However, addition of CsA to SRL completely reversed that trend and returned levels to control. The results were different for TGF-beta1, which was increased by both CsA and SRL and to a greater extent by the drug combination. CONCLUSION: Unlike TGF-beta, CTGF does not seem to play an important role in CsA-induced chronic nephrotoxicity. In addition, calcineurin-dependent pathways are likely involved in CTGF regulation.

Effect of pirfenidone on apoptosis-regulatory genes in chronic cyclosporine nephrotoxicity.

BACKGROUND: : Apoptosis was shown to play a role in the progression of fibrosis in a chronic cyclosporine A (CsA) nephrotoxicity animal model. In addition, the antifibrotic molecule pirfenidone (PFD) was shown to ameliorate fibrosis in this model. We evaluated the role of PFD on the expression of apoptosis-regulatory genes in the kidneys of CsA-treated rats. METHODS: : Rats were administered CsA 7.5 mg/kg per day, CsA+PFD (250 mg/kg/day), vehicle (VH), or VH+PFD, and sacrificed at 28 days. Physiologic and histologic changes were studied, and apoptosis was detected by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling stain. The mRNA expression of pro-apoptotic genes p53 and Fas-ligand was evaluated by quantitative polymerase chain reaction, and that of Bcl-xL, an anti-apoptotic gene, was evaluated by Northern blot analysis. In addition to mRNA expression, immunohistochemical studies of caspase 3 were performed. RESULT: : PFD administration to CsA-treated rats significantly ameliorated nephrotoxicity. Apoptosis-positive cells were increased by CsA but significantly reduced by PFD treatment (68+/-19 vs. 3+/-1, P<0.01). In addition, PFD down-regulated the mRNA expression of CsA-induced p53 and Fas-ligand (P<0.01) and increased that of Bcl-xL, previously reduced by CsA (P<0.01). Finally, PFD significantly down-regulated caspase 3 expression, present mostly on renal tubular epithelial cells. None of these changes were observed in VH-treated rats. CONCLUSION: : Whereas CsA favored the expression of pro-apoptotic genes, that effect was ameliorated by PFD. Because apoptosis can partly explain the loss of cells associated with fibrosis, the influence of PFD on apoptosis-regulatory genes in a manner that reduces apoptosis may explain some of its antifibrotic properties.

Sirolimus increases transforming growth factor-beta1 expression and potentiates chronic cyclosporine nephrotoxicity.

BACKGROUND: Sirolimus (SRL) is increasingly being used to decrease cyclosporine (CsA) exposure. SRL is not known to be nephrotoxic and has a mechanism of action distinct from CsA. We investigated the effect of combining CsA and SRL on renal structure and function and on transforming growth factor-beta1 (TGF-beta1) and extracellular matrix (ECM) proteins in a model of chronic CsA nephrotoxicity. METHODS: Rats treated with vehicle, SRL 0.3 mg/kg/day, CsA 5 or 10 mg/kg/day, or CsA5+SRL were sacrificed at 7 or 28 days. Physiologic and histologic changes were studied in addition to TGF-beta1 mRNA and protein expressions, and mRNA expression of plasminogen activator inhibitor-1 (PAI-1) and ECM proteins biglycan and types I and IV collagen. RESULTS: While SRL alone did not alter renal function and structure, it potentiated the nephrotoxic actions of CsA when used in combination with low-dose CsA5 and resulted in significant changes similar to high-dose CsA10. In addition, SRL alone increased TGF-beta1 by 44% to 49% (P < 0.05 vs. VH). When used in combination with low-dose CsA5, SRL potentiated TGF-beta1 mRNA and protein by 121% and 176%, respectively (P < 0.05 vs. VH and CsA5), to levels achieved with high-dose CsA10. The expression of the ECM proteins followed that of TGF-beta1; a similar effect was observed with PAI-1, suggesting a decrease in ECM degradation. CONCLUSION: Because SRL augments nephrotoxicity, caution should be exercised when it is used in combination with CsA. More studies are needed to determine the long-term clinical impact of SRL on nephrotoxicity and allograft function.

Cyclosporin induces renal proto-oncogene RNA message and increased transforming growth factor-beta prior to renal fibrosis: Modification by calcium channel blockade in the salt replete rat.

BACKGROUND: Chronic cyclosporin (CsA) administration has been shown to result in the replacement of epithelial cells in the kidney with fibrous tissue. These changes are kidney-specific, as they do not occur in any other organ. RESULTS: Cyclosporin exposure increases c-fos and c-jun mRNA in the rat kidney but not in the liver. Furthermore, chronic CsA exposure causes a further increase in c-fos and c-jun mRNA and increases the renal expression of transforming growth factor-beta (TGF-beta) mRNA. These changes precede the development of fibrosis. The combined insult of ischaemia and CsA resulted in synergistic increases in c-fos, suggesting that CsA recruited a pathway for c-fos activation different from ischaemia. The calcium channel blocker, verapamil, blocked CsA-induced expression of c-fos and c-jun mRNA, and reduced the amount of TGF-beta expression. CONCLUSION: These data are consistent with the notion that CsA induces protooncogenes, which may be, at least partially, responsible for long-term CsA nephrotoxicity.

Combination therapy with sirolimus and mycophenolate mofetil: effects on the kidney and on transforming growth factor-beta1.

Sirolimus (SRL) is not nephrotoxic, but it has been shown to increase transforming growth factor (TGF)-beta. We investigated the effect of combining mycophenolate mofetil (MMF) with SRL on renal structure and function and on TGF-beta1. Rats treated with vehicle (VH), MMF 10 mg/kg/d, SRL 0.3 mg/kg/d, or SRL+MMF were killed at 28 days. The physiologic and histologic changes and expression of TGF-beta1, plasminogen activator inhibitor-1, and extracellular matrix proteins were studied. Although SRL alone did not alter renal function and structure, it increased TGF-beta1 mRNA by 44% and protein by 48% (P <0.05 vs. VH). Treatment with MMF did not affect TGF-beta1. When combined with SRL, MMF decreased TGF-beta1 expression to VH levels. A similar trend was observed with plasminogen activator inhibitor-1 and extracellular matrix proteins. The long-term consequence of increased TGF-beta in SRL-treated kidneys remains unknown. However, because MMF can reverse this trend, SRL and MMF combination therapy may be protective.

Mycophenolate mofetil ameliorates arteriolopathy and decreases transforming growth factor-beta1 in chronic cyclosporine nephrotoxicity.

Afferent arteriolopathy is the most characteristic lesion of chronic cyclosporine (CsA) nephrotoxicity. We investigated the effect of therapeutic doses of mycophenolate mofetil (MMF) in a model of chronic CsA nephrotoxicity where transforming growth factor-beta (TGF-beta) was shown to play a central role. Rats treated with vehicle, MMF 10 mg/kg/day, CsA 10 mg/kg/day or CsA + MMF were sacrificed at 7 or 28 days. Physiologic and histologic changes were studied in addition to TGF-beta1 mRNA and protein expressions, and mRNA expression of plasminogen activator inhibitor-1 (PAI-1) and the extracellular matrix (ECM) proteins biglycan and types I and IV collagen. While MMF markedly ameliorated afferent arteriolopathy, it had no significant effect on interstitial fibrosis and tubular atrophy. In addition, MMF treatment reduced both TGF-beta1 mRNA and protein levels by 39% and 32%, respectively (p < 0.05 vs. CsA only). The expression of the ECM proteins followed that of TGF-beta1 and was significantly decreased with MMF; a similar effect was observed with PAI-1, suggesting an increase in ECM degradation. These results suggest that MMF exerts a beneficial effect on CsA arteriolopathy and that it decreases TGF-beta1. While this drug combination may be useful clinically, long-term studies are needed to determine if MMF has a lasting benefit.

Cyclosporine nephrotoxicity.

After more than 20 years of cyclosporine use its nephrotoxicity remains a significant clinical problem. Cyclosporine-induced renal injury has been described in solid organs recipients and in patients treated for autoimmune diseases. It is manifested in 2 distinct and well characterized forms, acute nephrotoxicity and chronic nephrotoxicity. This communication reviews the current literature analyzing the available data about the pathogenesis and mechanisms of acute and chronic cyclosporine-induced nephrotoxicity. A working hypothesis for the possible mechanisms of chronic cyclosporine nephrotoxicity will be provided.

Nitric oxide modulates vascular endothelial growth factor and receptors in chronic cyclosporine nephrotoxicity.

BACKGROUND: Vascular endothelial growth factor (VEGF) is involved in angiogenesis, wound healing, and inflammation and exerts its effect via tyrosine kinase receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase (Flk-1 or KDR). We have previously shown that VEGF is up-regulated in a model of chronic cyclosporine (CsA) nephrotoxicity and that l-arginine (l-Arg) improved while N-nitro-l-arginine-methyl ester (L-NAME) worsened fibrosis. We examined the role of nitric oxide modulation on VEGF in this model. METHODS: Pair-fed salt-depleted rats were administered CsA, CsA + L-NAME, CsA +l-Arg, vehicle (VH), VH + L-NAME or VH +l-Arg and were sacrificed at 7 or 28 days. Physiologic and histologic changes were studied in addition to the mRNA expression of VEGF and its receptors Flt-1 and KDR/Flk-1 by Northern blot and the protein expression of VEGF by Western blot and immunohistochemical staining. RESULTS: While L-NAME worsened renal function and histology, l-Arg had the opposite beneficial effect in CsA-treated rats. VEGF mRNA and protein expressions increased with CsA, further increased with L-NAME and became significantly reduced with L-Arg. Flt-1 expression was similar in all groups. On the other hand, KDR/Flk-1 mRNA expression was modulated in a fashion similar to VEGF. Also, nitric oxide modulation did not have an effect on VH-treated rats. CONCLUSIONS: VEGF expression in chronic CsA nephrotoxicity is increased by nitric oxide blockade and decreased by nitric oxide enhancement. Moreover, VEGF probably exerted its effect via the KDR/Flk-1 receptor. The actions of VEGF in this model remain speculative, but it is probable that VEGF plays a role, either independently or through nitric oxide, in CsA-induced fibrosis.

Angiotensin II regulation of vascular endothelial growth factor and receptors Flt-1 and KDR/Flk-1 in cyclosporine nephrotoxicity.

BACKGROUND: Vascular endothelial growth factor (VEGF) is involved in angiogenesis, wound healing and inflammation. VEGF exerts its effect via the tyrosine kinase receptors Flt-1 and KDR/Flk-1. We have previously shown that VEGF is up-regulated in a chronic cyclosporine (CsA) nephrotoxicity model. Our current study examined the role of angiotensin II (Ang II) blockade with enalapril (E) or losartan (L) on VEGF in this model. METHODS: Pair-fed salt-depleted rats were administered vehicle, CsA, CsA + nilvadipine, CsA + hydralazine/hydrochlorthiazide (HCTZ), CsA + E or CsA + L, and were sacrificed at 7 or 28 days. Physiologic and histologic changes were studied in addition to the mRNA expression of VEGF and its receptors Flt-1 and KDR/Flk-1 by Northern blot, and the protein expression of VEGF by Western blot. RESULTS: While all groups achieved similar blood pressures and creatinine clearances, the amelioration in nephrotoxicity was observed only with Ang II blockade. VEGF mRNA and protein expressions increased with CsA and became significantly reduced with Ang II blockade. Flt-1 expression was similar in all groups; it decreased early and remained low. On the other hand, KDR/Flk-1 mRNA expression was higher at seven days in all groups, except in the +E and +L groups where it was significantly lower, and then became further down-regulated at 28 days. CONCLUSIONS: The increased VEGF expression in chronic CsA nephrotoxicity seems to be related to up-regulation of Ang II. In addition, VEGF probably exerted its effect via the KDR/Flk-1 receptor. The actions of VEGF in this model remain speculative, but may be related to its effect on macrophage infiltration or matrix deposition.

Expression of apoptosis-related genes in chronic cyclosporine nephrotoxicity in mice.

To define the mechanism of cyclosporine (CsA)-induced apoptosis, we investigated the expression of apoptosis-related genes in experimental chronic CsA nephrotoxicity. Mice on a low-salt (0.01%) diet were given vehicle (VH, olive oil, 1 mg/kg/day), or CsA (30 mg/kg/day), and sacrificed at 1 and 4 weeks. Apoptosis was detected with deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain, and the expressions of apoptosis-related genes were evaluated by reverse transcription-polymerase chain reaction, immunoblot or immunohistochemistry. The activity of caspase 1 and 3 was also evaluated. The CsA group showed increases in apoptotic cells compared with the VH group (54 +/- 41 vs. 3 +/- 3, p < 0.05), and the number of apoptotic cells correlated well with interstitial fibrosis scores (r = 0.83, p < 0.01). The CsA group showed a significant increase in Fas-ligand mRNA (0.20 vs. 0.02 amol/microgram total RNA, p < 0.05) and Fas protein expression (146% vs. 95%, p < 0.05), compared with the VH group. The CsA group showed significant increases in ICE mRNA (0.21 vs. 0.03 amol/microgram total RNA at 4 weeks, p < 0.05) and CPP32 mRNA (0.18 vs. 0.03 amol/microgram total RNA at 4 weeks, p < 0.05), compared with the VH group. The enzymatic activity of ICE (16.6 vs. 7.9 rho mol/microgram/h, p < 0.05) and CPP32 protease (15.6 vs. 2.7 rho mol/microgram/h, p < 0.05) proteases were increased in the CsA group, compared with the VH group. The ratio between bax and bcl-2 protein increased significantly in the CsA group (5.3-fold), compared with the VH group. Levels of p53 protein also increased in the CsA group. Immunohistochemical detection of Fas, Fas-ligand, ICE and CPP32 revealed strong immunoreactivity in renal tubular cells in areas of structural injury. These findings suggest that local activation of the apoptosis-related genes is associated with CsA-induced apoptotic cell death.

Pirfenidone treatment decreases transforming growth factor-beta1 and matrix proteins and ameliorates fibrosis in chronic cyclosporine nephrotoxicity.

Chronic cyclosporine (CsA) nephrotoxicity is characterized by tubulointerstitial fibrosis. Pirfenidone (PFD) is a novel antifibrotic compound that was shown to prevent and even reverse fibrosis. The mechanism of action of PFD is unclear but involves inhibition of transforming growth factor-beta (TGF-beta). Salt-depleted rats were administered CsA, CsA + PFD, vehicle (VH) or VH + PFD and sacrificed at 28days. Physiologic and histologic changes were studied in addition to TGF-beta1, plasminogen activator inhibitor-1 (PAI-1) and biglycan mRNA expressions by Northern blot. TGF-beta1 immunohistochemistry was also performed. Treatment with PFD ameliorated CsA-induced fibrosis by about 50% (p < 0.05). CsA-induced decrease in creatinine clearance improved with PFD but the difference was not significant. TGF-beta1, PAI-1 and biglycan mRNA expressions increased with CsA (p < 0.05 vs. VH) but strikingly improved with PFD treatment (p < 0.05 vs. CsA), which brought the levels down to VH levels. PFD treatment also decreased TGF-beta1 protein expression by 80%. These results demonstrate that PFD can attenuate renal fibrosis in this model. PFD was associated with a decrease in TGF-beta1 expression, which, in turn, was associated with a decrease in matrix deposition. These experiments suggest that PFD can be clinically useful for preventing chronic CsA nephrotoxicity and may prove to be helpful in other progressive renal diseases.