Yong Du

Reagent- and separation-free measurements of urine creatinine concentration using stamping surface enhanced Raman scattering (S-SERS).

We report a novel reagent- and separation-free method for urine creatinine concentration measurement using stamping surface enhanced Raman scattering (S-SERS) technique with nanoporous gold disk (NPGD) plasmonic substrates, a label-free, multiplexed molecular sensing and imaging technique recently developed by us. The performance of this new technology is evaluated by the detection and quantification of creatinine spiked in three different liquids: creatinine in water, mixture of creatinine and urea in water, and creatinine in artificial urine within physiologically relevant concentration ranges. Moreover, the potential application of our method is demonstrated by creatinine concentration measurements in urine samples collected from a mouse model of nephritis. The limit of detection of creatinine was 13.2 nM (0.15 µg/dl) and 0.68 mg/dl in water and urine, respectively. Our method would provide an alternative tool for rapid, cost-effective, and reliable urine analysis for non-invasive diagnosis and monitoring of renal function.

Green tea polyphenol (-)-epigallocatechin-3-gallate restores Nrf2 activity and ameliorates crescentic glomerulonephritis

Crescentic glomerulonephritis (GN) is the most severe form of GN and is associated with significant morbidity and mortality despite aggressive immunotherapy with steroids, cytotoxic drugs, and plasmapheresis. We examined the therapeutic efficacy of the green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG, 50 mg/kg BW/day x3weeks), a potent anti-inflammatory and anti-oxidant agent, on experimental crescentic GN induced in 129/svJ mice by administration of rabbit anti-mouse glomerular basement membrane sera. Routine histology and key molecules involved in inflammatory and redox signaling were studied. EGCG treatment significantly reduced mortality, decreased proteinuria and serum creatinine, and markedly improved renal histology when compared with vehicle-treated mice. The improvements in renal function and histology were accompanied by the restoration of Nrf2 signaling (which was impaired in vehicle-treated mice) as shown by increased nuclear translocation of Nrf2 and cytoplasmic glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione peroxidase. EGCG-treated mice also showed reduction in p-Akt, p-JNK, p-ERK1/2 and p-P38 as well as restoration of PPARγ and SIRT1 levels. Lower dose of EGCG (25 mg/kg BW/day x2 weeks) treatment also significantly decreased proteinuria and serum creatinine, and markedly improved renal histology when compared with vehicle-treated mice. Thus, our data illustrate the efficacy of EGCG in reversing the progression of crescentic GN in mice by targeting multiple signaling and inflammatory pathways as well as countering oxidative stress.

The association between reduced folate carrier-1 gene 80G/A polymorphism and methotrexate efficacy or methotrexate related-toxicity in rheumatoid arthritis: A meta-analysis.

Methotrexate (MTX), the most commonly used anti-rheumatic drug against RA, enters the cell via the action of the reduced folate carrier 1(RFC1). A major polymorphism of the RFC1 gene, 80G/A, has been reported to influence the activity of RFC1, resulting in variable intracellular MTX-polyglutamate (MTX-PG) levels. However, the association studies addressing the RFC1 80G/A polymorphism and MTX efficacy or toxicity in Rheumatoid arthritis (RA) has yielded conflicting results. In the present meta-analysis, we aimed to evaluate the association between the RFC1 80G/A polymorphism and MTX efficacy or toxicity in RA patients. A total 17 studies met our inclusion criteria. Among them, 12 studies with 2049 subjects reported the association between the RFC1 80G/A and MTX response, and 12 studies involving 2627 subjects were on MTX-related toxicity. Meta-analysis revealed significant association between RFC1 80G/A polymorphism and MTX efficacy (odds ratio (OR) for the A allele=1.29, 95% confidence interval (CI) 1.05-1.67, P=0.02; for AA genotype: OR=1.49, 95%CI 1.17-1.907, P=0.001). However, no association could be detected in the analysis of MTX-related toxicity. Stratification by ethnic population also indicated an association between this polymorphism and MTX efficacy in Asian group (P=0.002 for A allele; P=0.003 for AA genotype), but not in the Caucasian group (P=0.15 for A allele; P=0.05 for AA genotype). In both Asian and Caucasian sub-groups, no influence of the RFC1 80G/A polymorphism on MTX toxicity can be detected. In conclusion, the RFC1 G80A polymorphism is associated with responsiveness to MTX therapy, but may not be associated with MTX toxicity in RA patients.

Heightened cleavage of Axl receptor tyrosine kinase by ADAM metalloproteases may contribute to disease pathogenesis in SLE.

Systemic lupus erythematosus (SLE) is characterized by antibody-mediated chronic inflammation in the kidney, lung, skin, and other organs to cause inflammation and damage. Several inflammatory pathways are dysregulated in SLE, and understanding these pathways may improve diagnosis and treatment. In one such pathway, Axl tyrosine kinase receptor responds to Gas6 ligand to block inflammation in leukocytes. A soluble form of the Axl receptor ectodomain (sAxl) is elevated in serum from patients with SLE and lupus-prone mice. We hypothesized that sAxl in SLE serum originates from the surface of leukocytes and that the loss of leukocyte Axl contributes to the disease. We determined that macrophages and B cells are a source of sAxl in SLE and in lupus-prone mice. Shedding of the Axl ectodomain from the leukocytes of lupus-prone mice is mediated by the matrix metalloproteases ADAM10 and TACE (ADAM17). Loss of Axl from lupus-prone macrophages renders them unresponsive to Gas6-induced anti-inflammatory signaling in vitro. This phenotype is rescued by combined ADAM10/TACE inhibition. Mice with Axl-deficient macrophages develop worse disease than controls when challenged with anti-glomerular basement membrane (anti-GBM) sera in an induced model of nephritis. ADAM10 and TACE also mediate human SLE PBMC Axl cleavage. Collectively, these studies indicate that increased metalloprotease-mediated cleavage of leukocyte Axl may contribute to end organ disease in lupus. They further suggest dual ADAM10/TACE inhibition as a potential therapeutic modality in SLE.

Glutathione S-transferase Mu 2-transduced mesenchymal stem cells ameliorated anti-glomerular basement membrane antibody-induced glomerulonephritis by inhibiting oxidation and inflammation.

IntroductionOxidative stress is implicated in tissue inflammation, and plays an important role in the pathogenesis of immune-mediated nephritis. Using the anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN) mouse model, we found that increased expression of glutathione S-transferase Mu 2 (GSTM2) was related to reduced renal damage caused by anti-GBM antibodies. Furthermore, mesenchymal stem cell (MSC)-based therapy has shed light on the treatment of immune-mediated kidney diseases. The aim of this study was to investigate if MSCs could be utilized as vehicles to deliver the GSTM2 gene product into the kidney and to evaluate its potential therapeutic effect on anti-GBM-GN.MethodsThe human GSTM2 gene (hGSTM2) was transduced into mouse bone marrow-derived MSCs via a lentivirus vector to create a stable cell line (hGSTM2-MSC). The cultured hGSTM2-MSCs were treated with 0.5mM H2O2, and apoptotic cells were measured by terminal dUTP nick-end labeling (TUNEL) assay. The 129/svj mice, which were challenged with anti-GBM antibodies, were injected with 106 hGSTM2-MSCs via the tail vein. Expression of hGSTM2 and inflammatory cytokines in the kidney was assayed by quantitative PCR and western blotting. Renal function of mice was evaluated by monitoring proteinuria and levels of blood urea nitrogen (BUN), and renal pathological changes were analyzed by histochemistry. Immunohistochemical analysis was performed to measure inflammatory cell infiltration and renal cell apoptosis.ResultsMSCs transduced with hGSTM2 exhibited similar growth and differentiation properties to MSCs. hGSTM2-MSCs persistently expressed hGSTM2 and resisted H2O2-induced apoptosis. Upon injection into 129/svj mice, hGSTM2-MSCs migrated to the kidney and expressed hGSTM2. The anti-GBM-GN mice treated with hGSTM2-MSCs exhibited reduced proteinuria and BUN (58% and 59% reduction, respectively) and ameliorated renal pathological damage, compared with control mice. Mice injected with hGSTM2-MSCs showed alleviated renal inflammatory cell infiltration and reduced expression of chemokine (C-C motif) ligand 2 (CCL2), interleukin (IL)-1β and IL-6 (53%, 46% and 52% reduction, respectively), compared with controls. Moreover, hGSTM2-MSCs increased expression of renal superoxide dismutase and catalase, which may associate with detoxifying reactive oxygen species to prevent oxidative renal damage.ConclusionsOur data suggest that the enhanced protective effect of GSTM2-transduced MSCs against anti-GBM-GN might be associated with inhibition of oxidative stress-induced renal cell apoptosis and inflammation, through over-expression of hGSTM2 in mouse kidneys.

Classifying murine glomerulonephritis using optical coherence tomography and optical coherence elastography

Acute glomerulonephritis caused by antiglomerular basement membrane marked by high mortality. The primary reason for this is delayed diagnosis via blood examination, urine analysis, tissue biopsy, or ultrasound and X-ray computed tomography imaging. Blood, urine, and tissue-based diagnoses can be time consuming, while ultrasound and CT imaging have relatively low spatial resolution, with reduced sensitivity. Optical coherence tomography is a noninvasive and high-resolution imaging technique that provides superior spatial resolution (micrometer scale) as compared to ultrasound and CT. Changes in tissue properties can be detected based on the optical metrics analyzed from the OCT signals, such as optical attenuation and speckle variance. Furthermore, OCT does not rely on ionizing radiation as with CT imaging. In addition to structural changes, the elasticity of the kidney can significantly change due to nephritis. In this work, OCT has been utilized to quantify the difference in tissue properties between healthy and nephritic murine kidneys. Although OCT imaging could identify the diseased tissue, its classification accuracy is clinically inadequate. By combining optical metrics with elasticity, the classification accuracy improves from 76% to 95%. These results show that OCT combined with OCE can be a powerful tool for identifying and classifying nephritis. Therefore, the OCT/OCE method could potentially be used as a minimally invasive tool for longitudinal studies during the progression and therapy of glomerulonephritis as well as complement and, perhaps, substitute highly invasive tissue biopsies. Elastic-wave propagation in mouse healthy and nephritic kidneys.

Rapid, noninvasive quantitation of skin disease in systemic sclerosis using optical coherence elastography.

Abstract. Systemic sclerosis (SSc) is a connective tissue disease that results in excessive accumulation of collagen in the skin and internal organs. Overall, SSc has a rare morbidity (276 cases per million adults in the United States), but has a 10-year survival rate of 55%. Currently, the modified Rodnan skin score (mRSS) is assessed by palpation on 17 sites on the body. However, the mRSS assessed score is subjective and may be influenced by the experience of the rheumatologists. In addition, the inherent elasticity of skin may bias the mRSS assessment in the early stage of SSc, such as oedematous. Optical coherence elastography (OCE) is a rapidly emerging technique, which can assess mechanical contrast in tissues with micrometer spatial resolution. In this work, the OCE technique is applied to assess the mechanical properties of skin in both control and bleomycin (BLM) induced SSc-like disease noninvasively. Young’s modulus of the BLM-SSc skin was found be significantly higher than that of normal skin, in both the in vivo and in vitro studies (p<0.05). Thus, OCE is able to differentiate healthy and fibrotic skin using mechanical contrast. It is a promising new technology for quantifying skin involvement in SSc in a rapid, unbiased, and noninvasive manner.

Loss of diacylglycerol kinase epsilon in mice causes endothelial distress and impairs glomerular Cox-2 and PGE2 production

Thrombotic microangiopathy (TMA) is a disorder characterized by microvascular occlusion that can lead to thrombocytopenia, hemolytic anemia, and glomerular damage. Complement activation is the central event in most cases of TMA. Primary forms of TMA are caused by mutations in genes encoding components of the complement or regulators of the complement cascade. Recently, we and others have described a genetic form of TMA caused by mutations in the gene diacylglycerol kinase-ε (DGKE) that encodes the lipid kinase DGKε (Lemaire M, Fremeaux-Bacchi V, Schaefer F, Choi MR, Tang WH, Le Quintrec M, Fakhouri F, Taque S, Nobili F, Martinez F, Ji WZ, Overton JD, Mane SM, Nurnberg G, Altmuller J, Thiele H, Morin D, Deschenes G, Baudouin V, Llanas B, Collard L, Majid MA, Simkova E, Nurnberg P, Rioux-Leclerc N, Moeckel GW, Gubler MC, Hwa J, Loirat C, Lifton RP. Nat Genet 45: 531-536, 2013; Ozaltin F, Li BH, Rauhauser A, An SW, Soylemezoglu O, Gonul II, Taskiran EZ, Ibsirlioglu T, Korkmaz E, Bilginer Y, Duzova A, Ozen S, Topaloglu R, Besbas N, Ashraf S, Du Y, Liang CY, Chen P, Lu DM, Vadnagara K, Arbuckle S, Lewis D, Wakeland B, Quigg RJ, Ransom RF, Wakeland EK, Topham MK, Bazan NG, Mohan C, Hildebrandt F, Bakkaloglu A, Huang CL, Attanasio M. J Am Soc Nephrol 24: 377-384, 2013). DGKε is unrelated to the complement pathway, which suggests that unidentified pathogenic mechanisms independent of complement dysregulation may result in TMA. Studying Dgke knockout mice may help to understand the pathogenesis of this disease, but no glomerular phenotype has been described in these animals so far. Here we report that Dgke null mice present subclinical microscopic anomalies of the glomerular endothelium and basal membrane that worsen with age and develop glomerular capillary occlusion when exposed to nephrotoxic serum. We found that induction of cyclooxygenase-2 and of the proangiogenic prostaglandin E2 are impaired in Dgke null kidneys and are associated with reduced expression of the antithrombotic cell adhesion molecule platelet endothelial cell adhesion molecule-1/CD31 in the glomerular endothelium. Notably, prostaglandin E2 supplementation was able to rescue motility defects of Dgke knockdown cells in vitro and to restore angiogenesis in a test in vivo. Our results unveil an unexpected role of Dgke in the induction of cyclooxygenase-2 and in the regulation of glomerular prostanoids synthesis under stress.

Leukocyte beta-catenin expression is disturbed in systemic lupus erythematosus

Wnt/β-catenin signaling is relatively understudied in immunity and autoimmunity. β-catenin blocks inflammatory mediators and favors tolerogenic dendritic cell (DC) phenotypes. We show here that leukocytes from lupus-prone mice and SLE patients express diminished β-catenin transcriptional activity, particularly in myeloid cells, although other leukocytes revealed similar trends. Serum levels of DKK-1, an inhibitor under transcriptional control of Wnt/β-catenin, were also decreased in lupus-prone mice. Surprisingly, however, preemptive deletion of β-catenin from macrophages appears to have no effect on lupus development, even in mice with varying genetic loads for lupus. Although myeloid-specific loss of β-catenin does not seem to be important for lupus development, the potential role of this transcription factor in other leukocytes and renal cells remain to be elucidated.

What Do Mouse Models Teach Us about Human Systemic Lupus Erythematosus

Abstract Systemic lupus erythematosus (SLE) is a complex and heterogeneous autoimmune disease characterized by the presence of autoantibodies against several autoantigens, with the involvement of multiple organ systems. From 1965 to 2015, research using various murine lupus models has allowed researchers to make significant progress toward understanding the pathogenic mechanisms and treatment of human SLE. It is clear that multiple factors, including genetic susceptibility loci, various immune cells, molecular mediators, and environmental factors, contribute to the genesis and progression of lupus. In addition to a long list of mouse-to-human translational studies, an increasing number of human-to-mouse translational studies are also being performed in the field of lupus. The laboratory mouse continues to be a versatile tool for dissecting out the complexities enshrouding lupus pathogenesis.