PDGF signal transduction inhibition ameliorates experimental mesangial proliferative glomerulonephritis

BACKGROUND: Platelet-derived growth factor (PDGF) has been consistently implicated in the cell proliferation and extracellular matrix accumulation, which characterize progressive glomerular disease. In the present study, the effects of a potent and selective inhibitor of PDGF receptor tyrosine kinase, STI 571, were examined in vitro and in vivo. METHODS: Cultured mesangial cells were incubated with PDGF (50 ng/mL) and fibroblast growth factor-2 (FGF-2; 50 ng/mL) and treated with STI 571 (0.13 to 2.0 micromol/L). Experimental mesangial proliferative glomerulonephritis was induced in male Wistar rats with monoclonal OX-7, anti-rat Thy-1.1 antibody with rats randomized to receive either STI 571 (50 mg/kg intraperitoneally daily) or vehicle. Animals were examined six days later. RESULTS: In vitro, both PDGF and FGF-2 induced a threefold increase in mesangial cell 3H-thymidine incorporation. STI 571 reduced PDGF but not FGF-2-stimulated mesangial cell proliferation in a dose-dependent manner, with complete abolition at 0.4 micromol/L. In animals with Thy-1.1 glomerulonephritis, PDGF receptor tyrosine kinase blockade was associated with significant reductions in mesangial cell proliferation (P < 0.001), the number of activated (alpha-smooth muscle positive) mesangial cells, and glomerular type IV collagen deposition (P < 0.001). CONCLUSION: The amelioration of the pathological findings of experimental mesangial proliferative glomerulonephritis by blockade of PDGF receptor activity suggests the potential clinical utility of this approach as a therapeutic strategy in glomerular disease.     

Electroporation-mediated gene transfer that targets glomeruli

Electroporation has been applied to introducing DNA into several organs; however, gene expression was localized around the injected area. Examined was the efficiency of intrarenal injection of DNA followed by in vivo electroporation, using FITC-labeled oligodeoxynucleotides (FITC-ODN) and plasmid DNA expressing beta-galactosidase or luciferase. FITC-ODN or expression vectors were injected into the left renal artery; thereafter, the left kidney was electroporated between a pair of tweezer-type electrodes. FITC-ODN were transferred into all glomeruli, and transfected cells were identified as mesangial cells. Four d after transfection of the pCAGGS-LacZ gene, beta-galactosidase expression was observed in 75% of glomeruli. To compare the transfection efficacy by electroporation with that by the hemagglutinating virus of Japan (HVJ) liposome method, a luciferase reporter gene, pActLuc, was transferred into glomeruli by either electroporation or the HVJ liposome method. On day 4, electroporation resulted in higher glomerular luciferase activity than did the HVJ liposome method. We also observed that co-transfection of pcEBNA, an expression vector for Epstein-Barr virus nuclear antigen, and poriP-cLuc, oriP-harboring vector, resulted in an eightfold higher luciferase gene expression than simple poriP-cLUC: No histologic damages were seen in glomeruli or tubular epithelial cells. In conclusion, gene transfer into renal artery followed by electroporation was an effective and simple strategy for gene transfer that targets glomerular mesangial cells.     

Electroporation-mediated HGF gene transfer ameliorated cyclosporine nephrotoxicity

BACKGROUND: The clinical utility of cyclosporine A (CsA) has been limited by its nephrotoxicity, which is characterized by tubular atrophy, interstitial fibrosis, and progressive renal impairment. Hepatocyte growth factor (HGF) has been reported to protect and salvage from renal injury as a renotropic and antifibrotic factor. Here, we investigated protective effects of HGF gene therapy on rat CsA-induced nephrotoxicity using electroporation-mediated gene transfer. METHOD: CsA was subcutaneously administered daily under low sodium diet, and HGF gene was transferred into skeletal muscle by electroporation on days 7 and 14. We also examined the antiapoptotic mechanism of HGF using human proximal tubular epithelial cells. RESULTS: HGF gene transfer rescued CsA-induced initial tubular injury and suppressed interstitial infiltration of ED-1-positive macrophages in CsA-induced nephrotoxicity. In addition, HGF significantly inhibited tubular cell apoptosis, and increased the number of proliferating tubular epithelial cells. In vitro studies suggest that HGF executes the antiapoptotic function by enhancing the phosphorylation of Akt and Bcl-2. Northern blot analysis demonstrated that HGF gene transfer suppressed cortical mRNA levels of transforming growth factor-beta (TGF-beta). Consequently, HGF gene transfer significantly reduced a striped interstitial phenotypic alteration and fibrosis. CONCLUSION: We demonstrated that HGF gene transfer reduced CsA-induced tubular cell apoptosis and interstitial fibrosis. HGF gene transfer could be a potential strategy for preventing renal fibrosis.     

Autonomic renal denervation ameliorates experimental glomerulonephritis

Increasing evidence indicates that inflammation of visceral organs is significantly affected by the autonomic nervous system. Such neuroimmune interactions have not been studied in the kidney. Here, we show that the rat kidney is innervated by both tyrosine hydroxylase-positive sympathetic efferent nerve fibers and calcitonin gene-related peptide-positive primary afferent nerve fibers, both of which are found in proximity to macrophages and dendritic cells. Complete surgical bilateral renal denervation was performed 2 d before glomerulonephritis was induced by injecting the monoclonal anti-Thy-1.1 antibody OX-7. Denervation significantly reduced albuminuria, mesangiolysis, formation of microaneurysms, deposition of glomerular collagen IV, and expression of TGF-beta compared with sham-operated controls. Accordingly, inflammation, identified by accumulation of interstitial macrophages and renal expression of TNF-alpha, and mesangial cell proliferation were significantly reduced. These findings indicate that autonomic renal denervation ameliorates and, by inference, innervation exacerbates acute inflammation in the kidney; therefore, neurotransmitters or neuropeptides and their receptors might represent novel targets for the treatment of acute glomerulonephritis.     

Specific antagonism of PDGF prevents renal scarring in experimental glomerulonephritis

Glomerular mesangial cell proliferation and/or mesangial matrix accumulation characterizes many progressive renal diseases. Rats with progressive mesangioproliferative glomerulonephritis were treated from day 3 to day 7 after disease induction with a high-affinity oligonucleotide aptamer antagonist against platelet-derived growth factor-B chain (PDGF-B). In comparison with nephritic rats that received vehicle or a scrambled aptamer, treatment with the PDGF-B aptamer led to a significant reduction of mesangioproliferative changes, glomerular hypertrophy, podocyte damage, and glomerular macrophage influx on day 8. Both nephritic control groups subsequently developed progressive proteinuria and decreased renal function. On day 100, glomerulosclerosis, tubulointerstitial damage, glomerular and interstitial accumulation of types III and IV collagen, and overexpression of transforming growth factor-beta were widespread. All of these chronic changes were prevented in rats that received the PDGF-B aptamer, and their functional and morphologic parameters on day 100 were largely indistinguishable from non-nephritic rats. These data provide the first evidence for a causal role of PDGF in the pathogenesis of renal scarring and point to a new, highly effective therapeutic approach to progressive, in particular mesangioproliferative, renal disease.     

Anti-perforin antibody treatment ameliorates experimental crescentic glomerulonephritis in WKY rats

The depletion of CD8+ cells has been shown to prevent the initiation and progression of antiglomerular basement membrane (GBM) crescentic glomerulonephritis (GN) in Wistar-Kyoto (WKY) rats. In this study, we asked whether CD8+ cells produce their effects by perforin/granzyme-mediated or by Fas ligand (FasL)-mediated pathways. The glomerular mRNA expression of perforin and granzyme B corresponded with the number of CD8+ cells, whereas that of granzyme A, Fas, and FasL did not. The enhanced mRNA level of perforin and granzyme B was not evident in CD8+-depleted rats. The number of apoptotic cells in the glomeruli was significantly increased at day 3. Perforin mRNA was found in cells infiltrating the glomerulus by in situ hybridization and by using dual-staining immunohistochemistry perforin protein was found in glomerular CD8+ cells. We found that perforin was readily visualized at the inner surface of the glomerular capillaries by immunoelectron microscopy. Based on these results, we treated animals with a perforin antibody in vivo and found that it significantly reduced the amount of proteinuria, frequency of crescentic glomeruli, and the number of glomerular monocytes and macrophages, although the number of glomerular CD8+ cells was not changed. Our results suggest that CD8+ cells play a role in glomerular injury as effector cells in part through a perforin/granzyme-mediated pathway in the anti-GBM WKY rat model of crescentic GN.     

PDGF-D inhibition by CR002 ameliorates tubulointerstitial fibrosis following experimental glomerulonephritis.

BACKGROUND: Arresting or regressing kidney scarring is of major clinical relevance. Platelet-derived growth factor D (PDGF-D) is widely expressed in fibrotic kidneys. Administration of the PDGF-D neutralizing fully human monoclonal antibody CR002 in the acute phase of progressive anti-Thy 1.1 glomerulonephritis reduced glomerular and secondary tubulointerstitial damage. METHODS: Using this model, we now assessed the effects of CR002 (n=15) vs irrelevant control IgG (n=17) administered on days 17, 28 and 35 after disease induction, i.e. after acute glomerular damage had subsided. RESULTS: In vitro, CR002 inhibited the PDGF-D- but not the PDGF-B-induced proliferation of rat renal fibroblasts. Following the first CR002 injection on day 17, exposure to therapeutic levels was maintained until day 49. Proteinuria in the CR002-treated group was transiently reduced between days 49 and 77 (-19 to -23% in comparison with the controls; P<0.05). On day 100, CR002 treatment reduced the number of rats that had doubled their serum creatinine (CR002: 40 vs controls: 71%; P<0.05). Compared with controls, the CR002 animals, on day 100, significantly lowered glomerular expression of vimentin and collagens as well as tubulointerstitial damage scores, interstitial fibrosis, vimentin and cortical PDGF-D mRNA levels. CONCLUSIONS: PDGF-D antagonism, even after the phase of acute glomerular damage, exerts beneficial effects on the course of tubulointerstitial damage, i.e. the final common pathway of most renal diseases.     

Smad7 gene therapy ameliorates an autoimmune crescentic glomerulonephritis in mice

Autoimmune crescentic glomerulonephritis is characterized by severe immune response with glomerular crescentic formation and fibrosis in the kidney. Recent studies indicate that overexpression of renal Smad7 attenuates both renal fibrosis and inflammation in rat remnant kidney. However, little attention has been paid to the potential role of TGF-beta/Smad signaling in autoimmune kidney disease. This study tested the hypothesis that blocking TGF-beta signaling by overexpression of Smad7 may have a therapeutic effect in a mouse model of autoimmune crescentic glomerulonephritis that was induced in C57BL/6 x DBA/2J F1 hybrid mice by giving DBA/2J donor lymphocytes. Smad7 gene was transfected into the kidney using the ultrasound-microbubble-mediated system. Results showed that overexpression of Smad7 blocked both renal fibrosis and inflammatory pathways in terms of Smad2/3 and NF-kappaB activation (P < 0.01), thereby inhibiting alpha-smooth muscle actin; collagen I, III, and IV accumulation; and expression of inflammatory cytokines (IL-1beta and IL-6), adhesion molecule/chemokine (intercellular adhesion molecule-1, monocyte chemoattractant protein-1), and inducible nitric oxide synthase (all P < 0.01). Leukocyte infiltration (CD4(+) cells and macrophages) was also suppressed (P < 0.005). Severe histologic damage (glomerular crescent formation and tubulointerstitial injury) and functional injury including proteinuria were significantly improved (all P < 0.05). This study provides important evidence that overexpression of Smad7 may have therapeutic potential for autoimmune kidney disease.     

Local delivery of angiotensin receptor blocker into the kidney ameliorates progression of experimental glomerulonephritis

Intrarenally synthesized angiotensin II (Ang II) may be involved in the progression of glomerulonephritis, leading to irreversible glomerulosclerosis. There is increasing evidence that systemic angiotensin receptor blocker (ARB) treatment has beneficial effect on the prognosis of progressive glomerulonephritis and diabetic nephropathy. However, the cellular and molecular mechanisms behind this therapeutic effect of ARB remain unclear. In this study, we used a novel strategy of local ARB delivery via type-1 collagen sponge, to treat progressive glomerulonephritis that would result in irreversible glomerulosclerosis in our previously established rat model. At days 9 and 14 after disease induction, mild proteinuria, 20.7+/-4.7 and 10+/-1.3 mg/day, was found. Local ARB treatment reduced proteinuria significantly to 3.19+/-3.2 and 5.25+/-0.95 mg/day (P < 0.01), respectively. Scoring of glomerular matrix expansion and sclerotic index revealed that local ARB treatment significantly ameliorated glomerular pathology. Ang II type 1 receptor mRNA expression was remarkably enhanced in the Ang II group and ARB treatment reversed this effect at 14 days. Local delivery of ARB significantly improved glomerular blood flow levels, compared to the untreated disease control group, from 710+/-18.25 to 859.44+/-22.86 microm/s, respectively. Local delivery of ARB into the kidney affected local RAS and thus improved the renal injury and function in the potentially progressive glomerulosclerosis of rat model.     

Pentoxifylline suppresses renal tumour necrosis factor-alpha and ameliorates experimental crescentic glomerulonephritis in rats.

BACKGROUND: Crescentic glomerulonephritis is a rapidly progressive form of glomerulonephritis, but treatment remains non-specific. The methylxanthine derivative pentoxifylline (PTX) is a clinically available phosphodiesterase inhibitor with anti-inflammatory and immunoregulatory activities. This study examined whether PTX has beneficial effects in a rat model of anti-glomerular basement membrane (GBM) crescentic glomerulonephritis. METHODS: Experimental crescentic glomerulonephritis was induced in Wistar rats by intravenous injection of rabbit anti-rat GBM serum and treated with either vehicle (phosphate-buffered saline) or PTX (0.1 g/kg/day) intravenously on a daily basis. Groups of six animals were euthanized at days 3, 7, 14 or 28 after induction of disease. Effects of PTX on renal function, histology and expression of cytokines, chemokines and adhesion molecules were determined. RESULTS: Compared with the vehicle-treated nephritic rats, PTX treatment beginning at the start of the nephritis significantly suppressed mRNA expression of tumour necrosis factor (TNF)-alpha, but not interleukin-1 beta, throughout the course of nephritis. Moreover, PTX decreased renal mRNAs for intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), regulated on activation, normal T-cell expressed and secreted (RANTES) and osteopontin (OPN) at all time points examined. These effects were associated with a significant inhibition of macrophage and T-cell infiltration, a reduction of 24-h urinary protein excretion (50-75%, P<0.05), an improvement of histological damage including glomerular crescent formation (60-70%, P<0.01) and a decrease of cortical mRNAs for type I (alpha 1) collagen and fibronectin. The efficacy of PTX could also be seen, though to a lesser extent, in rats with established nephritis. CONCLUSIONS: PTX is an effective anti-inflammatory and immunomodulatory agent capable of suppressing rat crescentic glomerulonephritis. Inhibition of renal TNF-alpha, ICAM-1, RANTES, MCP-1 and OPN expression may be a mechanism whereby PTX suppresses progressive renal injury in rat crescentic glomerulonephritis.     

The green tea polyphenol (-)-epigallocatechin-3-gallate ameliorates experimental immune-mediated glomerulonephritis

The unchecked overproduction of reactive oxygen and nitrogen species by inflammatory cells can cause tissue damage, intensify inflammation, promote apoptosis, and accelerate the progression of immune-mediated glomerulonephritis (GN). Here we tested whether the anti-inflammatory and antioxidant properties of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) favorably affect the development of immune-mediated GN. Pretreatment of 129/svJ mice with EGCG from 2 days before to 2 weeks after the induction of GN led to reduced proteinuria and serum creatinine, and marked improvement in renal histology when compared with vehicle-pretreated diseased mice. This pretreatment reduced oxidative stress, and normalized osteopontin, p65/nuclear factor-κB, inducible nitric oxide synthase, nitric oxide metabolites, p-Akt, phosphorylated extracellular signal-regulated kinases 1 and 2, p47phox, and myeloperoxidase, all of which were elevated in vehicle-pretreated diseased mice. Levels of glutathione peroxidase and peroxisome proliferator-activated receptor-γ (PPARγ), both reduced in the vehicle-pretreated diseased mice, were normalized. This renoprotective effect was reversed by concomitant administration of the PPARγ antagonist GW9662 throughout the EGCG pretreatment period. Importantly, mortality and renal dysfunction were significantly attenuated even when the polyphenol treatment was initiated 1 week after the onset of GN. Thus, EGCG reversed the progression of immune-mediated GN in mice by targeting redox and inflammatory pathways.     

Electroporation-mediated muscarinic M3 receptor gene transfer into rat urinary bladder

BACKGROUND: Muscarinic M3 (M3) receptor has been recognized as a major muscarinic receptor for smooth muscle contractions of the urinary bladder. Under the hypothesis that overexpression of M3 receptor in the urinary bladder would enhance urinary bladder contractions, we have transferred the M3 receptor gene into rat bladders using electroporation (EP) and evaluated the functional expression of the transferred gene. METHODS: Plasmids expressing luciferase, a green fluorescence protein and M3 receptor were injected into the rat bladder and square-wave electric pulses were immediately applied. Two days after gene transfer, we analyzed gene expression. Immunohistochemical staining was performed and the contractile responses from isolated bladder strips, which were induced KCl, carbachol and electrical field stimulation (EFS), were evaluated. RESULTS: The optimal conditions of electroporation were 8 pulses, 45 voltages, 50 milliseconds/pulses and 1 Hz. Under these conditions, luciferase gene expression was enhanced approximately 300-fold, compared to an injection of DNA only. Regarding immunohistochemistry with an anti-M3 receptor, an increase in immunoactivity was observed in the M3 receptor gene transferred rat bladder, compared to the bladder of the control rat. In rats with the transferred M3 receptor gene, carbachol- and EFS-induced maximum contractile responses of bladder smooth muscle strips significantly increased. CONCLUSIONS: These findings suggest that an in vivo EP procedure is an useful method for gene transfer into the bladder and that an overexpression of M3 receptor in the rat bladder enhances bladder contractility. This technique may become a new treatment modality for detrusor underactivity.     

Local delivery of a direct renin inhibitor into the kidney ameliorates progression of experimental glomerulonephritis

Background

Increasing evidence indicates that locally blocking renin–angiotensin system activity exerts a beneficial effect on glomerulonephritis (GN) progression leading to irreversible glomerulosclerosis. This is the first study on the pharmacological effect of the renal delivery of aliskiren, a direct renin inhibitor, in a progressive model of anti-Thy-1 GN.

Methods

Local blockade of renin activity was accomplished by subrenal capsular implantation of a collagen sponge with aliskiren. The pharmacological effect was evaluated by semiquantitative and quantitative analysis of immunohistological findings and by analysis of glomerular microcirculation using an intravital microscope system.

Results

Quantitative mesangial matrix analysis showed that local treatment with aliskiren significantly suppressed mesangial matrix expansion and ameliorated the glomerular sclerotic index in the progressive model of ATS GN. Immunofluorescent studies revealed that renin expression at the juxtaglomerular region was enhanced in the ATS?+?aliskiren group, and pathological expressions of α-smooth muscle cell actin and type I collagen in ATS GN were remarkably decreased by local treatment with aliskiren. Furthermore, local delivery of aliskiren significantly improved glomerular blood flow levels.

Conclusion

This study revealed that renally delivered aliskiren has a renoprotective effect on potentially progressive glomerulosclerosis.     

Pharmacologic control of angiotensin II ameliorates renal disease while reducing renal TGF-beta in experimental mesangioproliferative glomerulonephritis

We evaluated the effect of blocking angiotensin II (AngII) on the development of proteinuria and glomerular injury in antithymocyte serum (ATS) glomerulonephritis. Disease was induced in Sprague-Dawley rats by a single intravenous injection of rabbit ATS. Three groups of rats were considered: group 1 (n = 13), ATS rats with no therapy; group 2 (n = 13), ATS rats treated with angiotensin-converting enzyme inhibitor (40 mg/L lisinopril in the drinking water); and group 3 (n = 13), ATS rats treated with AngII receptor antagonist (50 mg/L L-158,809 in the drinking water). Treatment started 3 hours after ATS injection and lasted 4 days. An additional group of control rats (group 4, n = 13) received preimmune serum. At day 4, ATS rats developed proteinuria (46+/-5 mg/d v control 12+/-1 mg/d; P < 0.01), which was prevented by both lisinopril and L-158,809 (14+/-0.2 mg/d and 15+/-1.6 mg/d, respectively, P < 0.01 v ATS). Systolic blood pressure was comparable in ATS rats and in controls (119+/-4 mm Hg v 120+/-2 mm Hg). Systolic blood pressure values were significantly decreased after either lisinopril or L-158,809 (104+/-3 mm Hg and 101+/-5 mm Hg, respectively; P < 0.01 v ATS). Serum creatinine levels were similar in all groups. Quantitation of proliferating cells and macrophages by analysis of proliferating cell nuclear antigen-positive and ED1-positive cells/glomerular cross-section showed a marked increase in proliferating cell nuclear antigen-positive cells in glomeruli of ATS rats over controls (12.6+/-0.5 cells/glomerular cross-section v 1.9+/-0.2 cells/glomerular cross-section; P < 0.01), which was significantly (P < 0.01) prevented by both treatments (lisinopril, 5.7+/-1.0 cells/glomerular cross-section; L-158,809, 4.8+/-1.5 cells/glomerular cross-section). The increase in ED1-positive cells (10+/-0.7 cells/glomerular cross-section v controls, 1.8+/-0.2 cells/glomerular cross-section; P < 0.01) was also significantly (P < 0.01) reduced by lisinopril and L-158,809 (4.1+/-0.7 cells/glomerular cross-sections and 2.6+/-0.6 cells/glomerular cross-section, respectively). Blocking of AngII activity prevented almost completely the formation of microaneurysms in ATS rats (percent of glomeruli with microaneurysms: ATS, 11.5%+/-3.5%; ATS + lisinopril, 0.4%+/-0.2%; ATS + L-158,809, 0.8%+/-0.8%; controls, 0%). Because AngII is a potent inducer of renal transforming growth factor-beta (TGF-beta), a cytokine involved in the regulation of cell proliferation, matrix deposition, and monocyte migration (which is overexpressed in the kidney of ATS rats), we then evaluated the effect of AngII inhibitors on renal gene expression of TGF-beta1 and on urinary TGF-beta1. TGF-beta1 mRNA levels in kidneys of ATS rats were 3.6-fold higher than those of controls and were reduced by 46% and 32% after treatment with lisinopril and L-158,809, respectively. Urinary TGF-beta1 excretion increased in ATS (37.3+/-6.0 ng/d v controls, 13.8+/-3.4 ng/d; P< 0.01) but was normalized by lisinopril and L-158,809 (7.6+/-1.9 ng/d and 6.4+/-0.4 ng/d, respectively; P < 0.01). Thus, in ATS, blocking AngII synthesis prevents proteinuria and reduces glomerular cell proliferation and inflammatory cell infiltration, possibly by reducing excessive renal TGF-beta synthesis. These findings may be relevant for future strategies in the treatment of human mesangioproliferative glomerulonephritis.     

Preconditioning with endoplasmic reticulum stress ameliorates mesangioproliferative glomerulonephritis

Accumulating evidence suggests a pathophysiologic role of endoplasmic reticulum (ER) stress in kidney disease. This study investigated the potential of therapeutic approaches targeting ER stress in the anti-Thy1 model of mesangioproliferative glomerulonephritis in rats. Immunohistochemistry and Western blotting showed a time-dependent increase in the expression of the ER stress-inducible chaperones glucose-regulated protein 78 (GRP78) and oxygen-related protein 150 in isolated glomeruli, especially in the glomerular epithelial cells and mesangial cells, after induction of anti-Thy1 nephritis. For evaluation of whether preconditioning with ER stress ameliorates the severity of disease, rats were pretreated with a subnephritogenic dose of the ER stress inducer tunicamycin or thapsigargin for 4 d before disease was induced. Although preconditioning with ER stress had no effect on the degree of disease induction, it strongly ameliorated the manifestations of disease, evidenced by marked reductions in microaneurysm formation, mesangial proliferation, and adhesion of Bowman's capsule to the glomerular tuft. This improvement in histologic damage was associated with reduced proteinuria (39.4 +/- 10.5 versus 126.1 +/- 18.1 mg/d; P < 0.01) and with attenuated increases in glucose-regulated protein 78 and oxygen-related protein 150 expression. Of note, pretreatment with tunicamycin or thapsigargin decreased the excessive ER stress-induced intracellular signaling observed in anti-Thy1 nephritis. In conclusion, preconditioning with ER stress ameliorates the severity of disease in rats with anti-Thy1 nephritis. These findings suggest the possibility of therapeutic approaches targeting ER stress in mesangioproliferative glomerulonephritis.     

Tumor necrosis factor inhibitor gene transfer ameliorates lung graft ischemia-reperfusion injury

OBJECTIVE: Tumor necrosis factor is an important mediator of lung transplant ischemia-reperfusion injury, and soluble type I tumor necrosis factor receptor binds to tumor necrosis factor and works as a tumor necrosis factor inhibitor. The objectives of this study were to demonstrate that gene transfer of type I tumor necrosis factor receptor-IgG fusion protein reduces lung isograft ischemia-reperfusion injury and to compare donor endobronchial versus recipient intramuscular transfection strategies. METHODS: Three donor groups of Fischer rats (n = 6/group) underwent endobronchial transfection with either saline, 2 x 10(7) plaque-forming units of control adenovirus encoding beta-galactosidase, or 2 x 10(7) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein. Left lungs were harvested 24 hours later. Two recipient groups (n = 6/group) underwent intramuscular transfection with 2 x 10(7) plaque-forming units or 1 x 10(10) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein 24 hours before transplantation. All donor lung grafts were stored for 18 hours before orthotopic lung transplantation. Graft function was assessed 24 hours after reperfusion. Transgene expression was evaluated by means of enzyme-linked immunosorbent assay and immunohistochemistry of type I tumor necrosis factor receptor. RESULTS: Endobronchial transfection of donor lung grafts with 2 x 10(7) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein significantly improved arterial oxygenation compared with the saline and beta-galactosidase donor groups (366.6 +/- 137.9 vs 138.8 +/- 159.9 and 140.6 +/- 131.4 mm Hg, P =.009 and.010, respectively). Recipient intramuscular transfection with 1 x 10(10) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein improved lung graft oxygenation compared with that seen in the low-dose intramuscular group (2 x 10(7); 320.3 +/- 188.6 vs 143.6 +/- 20.2 mm Hg, P =.038). Type I tumor necrosis factor receptor-IgG fusion protein was expressed in endobronchial transfected grafts. In addition, intramuscular type I tumor necrosis factor receptor-IgG fusion protein expression was dose dependent. CONCLUSIONS: Donor endobronchial and recipient intramuscular adenovirus-mediated gene transfer of type I tumor necrosis factor receptor-IgG fusion protein improved experimental lung graft oxygenation after prolonged ischemia. However, donor endobronchial transfection required 500-fold less vector. Furthermore, at low vector doses, it does not create significant graft inflammation.     

Transforming growth factor-beta1 gene transfer ameliorates acute lung allograft rejection

BACKGROUND: The aim of the current work was to study the feasibility of functional gene transfer using the gene encoding for transforming growth factor-beta1, a known immunosuppressive cytokine, on rat lung allograft function in the setting of acute rejection. METHODS: The rat left lung transplant technique was used in all experiments, with Brown Norway donor rats and Fischer recipient rats. After harvest, left lungs were transfected ex vivo with either sense or antisense transforming growth factor-beta1 constructs complexed to cationic lipids, then implanted into recipients. On postoperative days 2, 5, and 7, animals were put to death, arterial oxygenation measured, and acute rejection graded histologically. RESULTS: On postoperative day 2, there were no differences in acute rejection or lung function between animals treated with transforming growth factor-beta1 and control animals. On postoperative day 5, oxygenation was significantly improved in grafts transfected with the transforming growth factor-beta1 sense construct compared with antisense controls (arterial oxygen tension = 411 +/- 198 vs 103 +/- 85 mm Hg, respectively; P =.002). Acute rejection scores from lung allografts were also significantly improved, corresponding to decreases in both vascular and airway rejection (vascular rejection scores: 2.0 +/- 0. 5 vs 2.8 +/- 0.6; P =.04; airway rejection scores: 1.3 +/- 0.7 vs 2. 3 +/- 0.8, respectively; P =.02). The amelioration of acute rejection was temporary and decreased by postoperative day 7. CONCLUSIONS: The feasibility of using gene transfer techniques to introduce novel functional genes in the setting of lung transplantation is demonstrated. In this model of rat lung allograft rejection, gene transfer of transforming growth factor-beta1 resulted in temporary but significant improvements in lung allograft function and acute rejection pathology.