Identification of candidate genes for Sjögren's syndrome using MRL/lpr mouse model of Sjögren's syndrome and cDNA microarray analysis

Sj?gren's syndrome is a chronic autoimmune disease characterized by focal lymphocytic infiltration of lacrimal and salivary glands, but the precise mechanism of this syndrome is poorly understood. To clarify the mechanism of onset and progression of Sj?gren's syndrome, it is necessary to identify Sj?gren's syndrome-related genes. For this purpose, we used MLR/MpJ-lpr/lpr (MRL/lpr) mouse as a model of human secondary Sj?gren's syndrome and analyzed specific mRNA expression pattern in MRL/lpr mouse salivary glands by in-house cDNA microarray. Among arrayed 2304 genes, 13 genes were isolated as highly expressed genes in MRL/lpr mouse salivary gland in comparison with MRL/MpJ-+/+ (MRL/+) mouse tissue. Subsequently, we performed RT-PCR analysis and confirmed the high expression level of nine genes; caspase3, Ly-6C.2, vimentin, Mel-14 antigen, cathepsin B, mpt1, Laptm5, Gnai2 and UCP2. Five of the nine genes have already been identified in patients with Sj?gren's syndrome or mice models of the syndrome, but the remaining four genes; mpt1, Laptm5, Gnai2, and UCP2 have not been reported previously as Sj?gren's syndrome-related genes. Although, further experiments are necessary to examine the relationship between these four genes and Sj?gren's syndrome, our system of mouse model of Sj?gren's syndrome combined with in-house cDNA microarray is suitable for the isolation of Sj?gren's syndrome-related genes.     

Production of nitric oxide,but not prostacyclin,is reduced in klotho mice

A novel murine model of aging (kl/kl mice) has been developed by in vivo mutagenesis. We analyzed endothelial function in this strain. Ring preparations of the thoracic aorta were obtained from 6- to 9-week old wild-type (+/+) and heterozygous (kl/+) klotho mice. The aortas of kl/+ mice showed an exaggerated contractile response to norepinephrine and attenuated vasodilator responses to acetylcholine and lecithinized superoxide dismutase (SOD) compared to +/+ mice. The response to sodium nitroprusside was unaltered in kl/+ mice. The contraction in response to norepinephrine was augmented by treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-5) M) to a greater extent in +/+ mice than in kl/+ mice. Treatment with L-NAME abolished the vasodilator responses to both acetylcholine and lecithinized SOD. NO metabolites (NO2- and NO3-) and cGMP concentrations in the urine were significantly reduced in kl/+ mice compared to +/+ mice. However, the urinary excretion of 6-keto-prostaglandin F1alpha was unaltered. There was little immunostaining for NO synthase and vascular endothelial growth factor (VEGF) in the aorta of kl/+ mice. No immunostaining for NO synthase was noted in the aorta of kl/kl mice. The expression of the klotho gene product may have a role in the regulation of VEGF expression and is tightly linked to endothelial release of NO.     

Troglitazone improves endothelial function and augments renal klotho mRNA expression in Otsuka Long-Evans Tokushima Fatty (OLETF) rats with multiple atherogenic risk factors.

Targeted disruption of the klotho gene induces multiple phenotypes characteristic of human aging, including arteriosclerosis, pulmonary emphysema and osteoporosis. Moreover, we previously observed that insufficient klotho expression in mice leads to endothelial dysfunction. In the present study, we used Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which exhibit hypertension, obesity, severe hyperglycemia and hypertriglyceridemia, and are thus considered an animal model of atherogenic disease, to test the effects of oral administration of troglitazone (200 mg/kg) on renal klotho mRNA expression and endothelial function. Systolic blood pressure, body weight, plasma glucose and triglyceride levels were all significantly higher in 30-week-old OLETF rats than in controls (LETO; Long-Evans Tokushima Otsuka) (p<0.05, n=7). In addition, endothelium-dependent relaxation of the aorta in response to 10(-5) M acetylcholine was significantly attenuated in OLETF rats (p<0.05, n=7), as was renal expression of klotho mRNA. Administration of troglitazone for 10 weeks significantly reduced systolic blood pressure, plasma glucose and triglyceride levels in OLETF rats, while augmenting endothelium-dependent aortic relaxation and renal klotho mRNA expression. These findings suggest that troglitazone protects the vascular endothelium against damage caused by the presence of multiple atherogenic factors.