Shuk-Man Ka

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.

Kidney-targeting Smad7 gene transfer inhibits renal TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways, and improves diabetic nephropathy in mice

Aims/hypothesisThe TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways have been shown to play a critical role in the development of renal fibrosis and inflammation in diabetic nephropathy. We therefore examined whether targeting these pathways by a kidney-targeting Smad7 gene transfer has therapeutic effects on renal lesions in the db/db mouse model of type 2 diabetes.MethodsWe delivered Smad7 plasmids into the kidney of db/db mice using kidney-targeting, ultrasound-mediated, microbubble-inducible gene transfer. The histopathology, ultrastructural pathology and pathways of TGF-β/SMAD2/3-mediated fibrosis and NF-κB-dependent inflammation were evaluated.ResultsIn this mouse model of type 2 diabetes, Smad7 gene therapy significantly inhibited diabetic kidney injury, compared with mice treated with empty vectors. Symptoms inhibited included: (1) proteinuria and renal function impairment; (2) renal fibrosis such as glomerular sclerosis, tubulo-interstitial collagen matrix abundance and renal inflammation, including Inos (also known as Nos2), Il1b and Mcp1 (also known as Ccl2) upregulation, as well as macrophage infiltration; and (3) podocyte and endothelial cell injury as demonstrated by immunohistochemistry and/or electron microscopy. Further study demonstrated that the improvement of type 2 diabetic kidney injury by overexpression of Smad7 was associated with significantly inhibited local activation of the TGF-β/SMAD and NF-κB signalling pathways in the kidney.Conclusions/interpretationOur results clearly demonstrate that kidney-targeting Smad7 gene transfer may be an effective therapy for type 2 diabetic nephropathy, acting via simultaneous modulation of the TGF-β/SMAD and NF-κB signalling pathways.

Decoy Receptor 3 Ameliorates an Autoimmune Crescentic Glomerulonephritis Model in Mice

Autoimmune crescentic glomerulonephritis (ACGN) is a variant of crescentic glomerulonephritis. The outcome of treatment of crescentic glomerulonephritis is poor. Binding of decoy receptor 3 (DCR3) to its ligand is capable of downregulating the alloresponsiveness of T cells. DCR3 has also been shown to benefit an experimental autoimmune model of diabetes. This study tested the hypothesis that a potential immune regulator, DCR3, could prevent the evolution of ACGN. With the use of an established ACGN model in mice, mice were treated with 100 microg/10 g body wt human DCR3 by hydrodynamics-based gene delivery at 14-d intervals. The results showed that the gene therapy resulted in (1) suppression of T and B cell activation and T cell proliferation; (2) a reduction in serum levels of proinflammatory cytokines; (3) improvement of proteinuria and renal dysfunction; (4) prevention of glomerular crescent formation, renal interstitial inflammation, and glomerulosclerosis; (5) a reduction in serum levels of autoantibodies and glomerular immune deposits; (6) inhibition of apoptosis in the spleen and kidney; (7) prevention of T cell and macrophage infiltration of the kidney; and (8) suppression of fibrosis-related gene expression in the kidney compared with empty vector-treated (disease control) ACGN mice. On the basis of these findings, it is proposed that human DCR3 exerts its preventive and protective effects on ACGN through modulation of T cell activation/proliferation, B cell activation, protection against apoptosis, and suppression of mononuclear leukocyte infiltration in the kidney.

Antroquinonol differentially modulates T cell activity and reduces interleukin-18 production, but enhances Nrf2 activation, in murine accelerated severe lupus nephritis.

OBJECTIVE Accelerated severe lupus nephritis (ASLN), with an acute onset of severe clinical manifestations and histopathologic renal lesions, may represent transformation of mild LN to a severe form of glomerulonephritis. Abnormal activation of T and B cells and/or oxidative stress may play a major role in the pathogenesis of ASLN. This study tested the hypothesis that antroquinonol, a purified compound and major effective component of Antrodia camphorata with antiinflammatory and antioxidant activities, might prevent the transformation of mild LN into higher-grade (severe) nephritis in a murine lupus model. METHODS Experimental ASLN was induced in (NZB×NZW)F1 mice by twice weekly intraperitoneal injections of Salmonella-type lipopolysaccharide (LPS). Starting 2 days after the first dose of LPS, mice were treated daily with antroquinonol, administered by gavage, for different durations up to 5 weeks. RESULTS Antroquinonol administration significantly ameliorated the proteinuria, hematuria, impairment of renal function, and development of severe renal lesions, especially cellular crescent formation, neutrophil infiltration, fibrinoid necrosis, and T cell proliferation in the glomerulus, as well as periglomerular interstitial inflammation. Mechanistic analyses revealed that antroquinonol 1) inhibited T cell activation/proliferation, but enhanced Treg cell suppression and reduced renal production of interleukin-18 (IL-18); 2) inhibited production of reactive oxygen species and nitric oxide, but increased activation of Nrf2 in the kidney; and 3) suppressed renal inflammation via blocking of NF-κB activation. CONCLUSION Antroquinonol may have therapeutic potential for the early treatment of ASLN via its differential regulation of T cell function and lowering of IL-18 production, but also via the promotion of Nrf2 activation.

Resveratrol inhibits NLRP3 inflammasome activation by preserving mitochondrial integrity and augmenting autophagy

The NLRP3 inflammasome is a caspase‐1‐containing multi‐protein complex that controls the release of IL‐1β and plays important roles in the development of inflammatory disease. Here, we report that resveratrol, a polyphenolic compound naturally produced by plants, inhibits NLRP3 inflammasome‐derived IL‐1β secretion and pyroptosis in macrophages. Resveratrol inhibits the activation step of the NLRP3 inflammasome by suppressing mitochondrial damage. Resveratrol also induces autophagy by activating p38, and macrophages treated with an autophagy inhibitor are resistant to the suppressive effects of resveratrol. In addition, resveratrol administration mitigates glomerular proliferation, glomerular sclerosis, and glomerular inflammation in a mouse model of progressive IgA nephropathy. These findings were associated with decreased renal mononuclear leukocyte infiltration, reduced renal superoxide anion levels, and inhibited renal NLRP3 inflammasome activation. Our data indicate that resveratrol suppresses NLRP3 inflammasome activation by preserving mitochondrial integrity and by augmenting autophagy. J. Cell. Physiol. 230: 1567–1579, 2015.

Osthole Mitigates Progressive IgA Nephropathy by Inhibiting Reactive Oxygen Species Generation and NF-κB/NLRP3 Pathway

Renal reactive oxygen species (ROS) and mononuclear leukocyte infiltration are involved in the progressive stage (exacerbation) of IgA nephropathy (IgAN), which is characterized by glomerular proliferation and renal inflammation. The identification of the mechanism responsible for this critical stage of IgAN and the development of a therapeutic strategy remain a challenge. Osthole is a pure compound isolated from Cnidiummonnieri (L.) Cusson seeds, which are used as a traditional Chinese medicine, and is anti-inflammatory, anti-apoptotic, and anti-fibrotic both in vitro and in vivo. Recently, we showed that osthole acts as an anti-inflammatory agent by reducing nuclear factor-kappa B (NF-κB) activation in and ROS release by activated macrophages. In this study, we examined whether osthole could prevent the progression of IgAN using a progressive IgAN (Prg-IgAN) model in mice. Our results showed that osthole administration resulted in prevention of albuminuria, improved renal function, and blocking of renal progressive lesions, including glomerular proliferation, glomerular sclerosis, and periglomerular mononuclear leukocyte infiltration. These findings were associated with (1) reduced renal superoxide anion levels and increased Nrf2 nuclear translocation, (2) inhibited renal activation of NF-κB and the NLRP3 inflammasome, (3) decreased renal MCP-1 expression and mononuclear leukocyte infiltration, (4) inhibited ROS production and NLRP3 inflammasome activation in cultured, activated macrophages, and (5) inhibited ROS production and MCP-1 protein levels in cultured, activated mesangial cells. The results suggest that osthole exerts its reno-protective effects on the progression of IgAN by inhibiting ROS production and activation of NF-κB and the NLRP3 inflammasome in the kidney. Our data also confirm that ROS generation and activation of NF-κB and the NLRP3 inflammasome are crucial mechanistic events involved in the progression of the renal disorder.

Osthole improves an accelerated focal segmental glomerulosclerosis model in the early stage by activating the Nrf2 antioxidant pathway and subsequently inhibiting NF-κB-mediated COX-2 expression and apoptosis

Inflammatory reactions and oxidative stress are implicated in the pathogenesis of focal segmental glomerulosclerosis (FSGS), a common chronic kidney disease with relatively poor prognosis and unsatisfactory treatment regimens. Previously, we showed that osthole, a coumarin compound isolated from the seeds of Cnidium monnieri, can inhibit reactive oxygen species generation, NF-κB activation, and cyclooxygenase-2 expression in lipopolysaccharide-activated macrophages. In this study, we further evaluated its renoprotective effect in a mouse model of accelerated FSGS (acFSGS), featuring early development of proteinuria, followed by impaired renal function, glomerular epithelial cell hyperplasia lesions (a sensitive sign that precedes the development of glomerular sclerosis), periglomerular inflammation, and glomerular hyalinosis/sclerosis. The results show that osthole significantly prevented the development of the acFSGS model in the treated group of mice. The mechanisms involved in the renoprotective effects of osthole on the acFSGS model were mainly a result of an activated Nrf2-mediated antioxidant pathway in the early stage (proteinuria and ischemic collapse of the glomeruli) of acFSGS, followed by a decrease in: (1) NF-κB activation and COX-2 expression as well as PGE2 production, (2) podocyte injury, and (3) apoptosis. Our data support that targeting the Nrf2 antioxidant pathway may justify osthole being established as a candidate renoprotective compound for FSGS.

Prolonging the expression duration of ultrasound-mediated gene transfection using PEI nanoparticles

Ultrasound (US) irradiation has been found to facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, its transfection efficiency is generally low, and the expression duration of transfected gene is short. Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. The purpose of this study is to determine whether PEI can also prolong the expression duration after US-mediated transfection. A mixture of pCMViLUC and 22-kDa linear PEI was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulsed US. The duration of expression was assessed periodically following US treatment. As expected, strong expression of luciferase could be found 30days after the treatment of DNA-PEI complex with US exposure, both in vitro and in vivo. However, without US, only very low transfection level could be obtained in vivo. The DNA/PEI complex showed protective effect against digestion of DNase I enzymes as compared with groups without PEI or to which PEI was added following the mixing of plasmid DNA with DNase I. PEI enhanced the US transfection efficiency by increasing both the intracellular uptake of plasmid DNA and the percentage of transfected cells. Most of the DNA uptake occurred at 3h after US exposure, suggesting that endocytosis took place. Moreover, the PEI-facilitated US gene transfection depended on the ratio of PEI and DNA (N/P ratio), which was different for in-vitro and in-vivo conditions. This system could be applied in future human gene therapies.

Citral alleviates an accelerated and severe lupus nephritis model by inhibiting the activation signal of NLRP3 inflammasome and enhancing Nrf2 activation

IntroductionLupus nephritis (LN) is a major complication of systemic lupus erythematosus. NLRP3 inflammasome activation, reactive oxygen species (ROS) and mononuclear leukocyte infiltration in the kidney have been shown to provoke the acceleration and deterioration of LN, such as accelerated and severe LN (ASLN). Development of a novel therapeutic remedy based on these molecular events to prevent the progression of the disease is clinically warranted.MethodsCitral (3,7-dimethyl-2,6-octadienal), a major active compound in a Chinese herbal medicine Litsea cubeba, was used to test its renoprotective effects in a lipopolysaccharide (LPS)-induced mouse ASLN model by examining NLRP3 inflammasome activation, ROS and COX-2 production as well as Nrf2 activation. The analysis of mechanisms of action of Citral also involved its effects on IL-1β secretion and signaling pathways of NLRP3 inflammasome in LPS-primed peritoneal macrophages or J774A macrophages.ResultsAttenuated proteinuria, renal function impairment, and renal histopathology, the latter including intrinsic cell proliferation, cellular crescents, neutrophil influx, fibrinoid necrosis in the glomerulus, and peri-glomerular infiltration of mononuclear leukocytes as well as glomerulonephritis activity score were observed in Citral-treated ASLN mice. In addition, Citral inhibited NLRP3 inflammasome activation and levels of ROS, NAD(P)H oxidase subunit p47phox, or COX-2, and it enhanced the activation of nuclear factor E2-related factor 2 (Nrf2). In LPS-primed macrophages, Citral reduced ATP-induced IL-1β secretion and caspase-1 activation, but did not affect LPS-induced NLRP3 protein expression.ConclusionOur data show that Citral alleviates the mouse ASLN model by inhibition of the activation signal, but not the priming signal, of NLRP3 inflammasome and enhanced activation of Nrf2 antioxidant signaling.

Citral is renoprotective for focal segmental glomerulosclerosis by inhibiting oxidative stress and apoptosis and activating Nrf2 pathway in mice.

The pathogenesis of focal segmental glomerulosclerosis (FSGS) is considered to be associated with oxidative stress, mononuclear leukocyte recruitment and infiltration, and matrix production and/or matrix degradation, although the exact etiology and pathogenic pathways remain to be determined. Establishment of a pathogenesis-based therapeutic strategy for the disease is clinically warranted. Citral (3,7-dimethyl-2,6-octadienal), a major active compound in Litsea cubeba , a traditional Chinese herbal medicine, can inhibit oxidant activity, macrophage and NF-κB activation. In the present study, first, we used a mouse model of FSGS with the features of glomerular epithelial hyperplasia lesions (EPHLs), a key histopathology index of progression of FSGS, peri-glomerular inflammation, and progressive glomerular hyalinosis/sclerosis. When treated with citral for 28 consecutive days at a daily dose of 200 mg/kg of body weight by gavage, the FSGS mice showed greatly reduced EPHLs, glomerular hyalinosis/sclerosis and peri-glomerular mononuclear leukocyte infiltration, suggesting that citral may be renoprotective for FSGS and act by inhibiting oxidative stress and apoptosis and early activating the Nrf2 pathway. Meanwhile, a macrophage model involved in anti-oxidative and anti-inflammatory activities was employed and confirmed the beneficial effects of citral on the FSGS model.