All-trans retinoic acid suppresses bone morphogenetic protein 4 in mouse diabetic nephropathy through a unique retinoic acid response element.

Diabetic nephropathy (DN) causes mesangial matrix expansion, which results in glomerulosclerosis and renal failure. Collagen IV (COL4) is a major component of the mesangial matrix that is positively regulated by bone morphogenetic protein 4 (BMP4)/suppressor of mothers against decapentaplegic (Smad1) signaling. Because previous studies showed that retinoids treatment had a beneficial effect on kidney disease, we investigated the therapeutic potential of retinoids in DN, focusing especially on the regulatory mechanism of BMP4. Diabetes was induced with streptozotocin in 12-wk-old male Crl:CD1(ICR) mice, and, 1 mo later, we initiated intraperitoneal injection of all-trans retinoic acid (ATRA) three times weekly. Glomerular matrix expansion, which was associated with increased BMP4, phosphorylated Smad1, and COL4 expression, worsened in diabetic mice at 24 wk of age. ATRA administration alleviated DN and downregulated BMP4, phosopho-Smad1, and COL4. In cultured mouse mesangial cells, treatment with ATRA or a retinoic acid receptor-α (RARα) agonist significantly decreased BMP4 and COL4 expression. Genomic analysis suggested two putative retinoic acid response elements (RAREs) for the mouse Bmp4 gene. Chromatin immunoprecipitation analysis and reporter assays indicated a putative RARE of the Bmp4 gene, located 11,488-11,501 bp upstream of exon 1A and bound to RARα and retinoid X receptor (RXR), which suppressed BMP4 expression after ATRA addition. ATRA suppressed BMP4 via binding of a RARα/RXR heterodimer to a unique RARE, alleviating glomerular matrix expansion in diabetic mice. These findings provide a novel regulatory mechanism for treatment of DN.

Urinary type IV collagen excretion is involved in the decline in estimated glomerular filtration rate in the Japanese general population without diabetes: A 5-year observational study.

Urinary type IV collagen (U-Col4) and albumin excretion is evaluated to monitor the development of diabetic kidney disease. However, U-Col4 excretion in the general population without diabetes has not yet been fully elucidated. In this study, 1067 participants without diabetes and with urinary albumin-creatinine ratio <300 mg/gCr (normo- or microalbuminuria) who underwent an annual health examination in 2004 were enrolled and observed for 5 years. They were divided according to the amount of U-Col4 or urinary albumin excreted. The decline in estimated glomerular filtration rate (eGFR) was calculated. In participants with eGFR ≥80 mL/min, abnormal U-Col4 excretion was indicated as a significant independent risk factor for 10% eGFR change per year, which is one of the prognostic factors for the development of end-stage kidney disease. Moreover, in contrast to urinary albumin excretion, U-Col4 excretion was not related to age or kidney function, suggesting that some individuals with abnormal U-Col4 excretion can have an independent hidden risk for the development of kidney dysfunction. In conclusion, it is important to measure U-Col4 excretion in the general population without diabetes to determine changes in renal features in every individual and help detect future complications such as diabetic kidney disease. If U-Col4 excretion is abnormal, kidney manifestation should be carefully followed up, even if the kidney function and urinalysis findings are normal.

Urinary IgG4 and Smad1 Are Specific Biomarkers for Renal Structural and Functional Changes in Early Stages of Diabetic Nephropathy.

Diabetic nephropathy (DN) is the major cause of end-stage kidney disease, but early biomarkers of DN risk are limited. Herein we examine urinary IgG4 and Smad1 as additional early DN biomarkers. We recruited 815 patients with type 2 diabetes; 554 patients fulfilled the criteria of an estimated glomerular filtration rate (eGFR) >60 mL/min and no macroalbuminuria at baseline, with follow-up for 5 years. Patients without macroalbuminuria were also recruited for renal biopsies. Urinary IgG4 and Smad1 were determined by enzyme-linked immunoassays using specific antibodies. The specificity, sensitivity, and reproducibility were confirmed for each assay. Increased urinary IgG4 was significantly associated with lower eGFR. The level of urinary IgG4 also significantly correlated with surface density of peripheral glomerular basement membrane (Sv PGBM/Glom), whereas Smad1 was associated with the degree of mesangial expansion-both classic pathological findings in DN. Baseline eGFR did not differ between any groups; however, increases in both urinary IgG4 and Smad1 levels at baseline significantly predicted later development of eGFR decline in patients without macroalbuminuria. These data suggest that urinary IgG4 and Smad1 at relatively early stages of DN reflect underlying DN lesions and are relevant to later clinical outcomes.

NKT cells from normal and tumor-bearing human livers are phenotypically and functionally distinct from murine NKT cells.

A major group of murine NK T (NKT) cells express an invariant Valpha14Jalpha18 TCR alpha-chain specific for glycolipid Ags presented by CD1d. Murine Valpha14Jalpha18(+) account for 30-50% of hepatic T cells and have potent antitumor activities. We have enumerated and characterized their human counterparts, Valpha24Vbeta11(+) NKT cells, freshly isolated from histologically normal and tumor-bearing livers. In contrast to mice, human NKT cells are found in small numbers in healthy liver (0.5% of CD3(+) cells) and blood (0.02%). In contrast to those in blood, most hepatic Valpha24(+) NKT cells express the Vbeta11 chain. They include CD4(+), CD8(+), and CD4(-)CD8(-) cells, and many express the NK cell markers CD56, CD161, and/or CD69. Importantly, human hepatic Valpha24(+) T cells are potent producers of IFN-gamma and TNF-alpha, but not IL-2 or IL-4, when stimulated pharmacologically or with the NKT cell ligand, alpha-galactosylceramide. Valpha24(+)Vbeta11(+) cell numbers are reduced in tumor-bearing compared with healthy liver (0.1 vs 0.5%; p < 0.04). However, hepatic cells from cancer patients and healthy donors release similar amounts of IFN-gamma in response to alpha-galactosylceramide. These data indicate that hepatic NKT cell repertoires are phenotypically and functionally distinct in humans and mice. Depletions of hepatic NKT cell subpopulations may underlie the susceptibility to metastatic liver disease.

Delineation of a CD1d-restricted antigen presentation pathway associated with human and mouse intestinal epithelial cells.

BACKGROUND & AIMS: CD1d, a major histocompatibility complex (MHC) class I-related molecule that is responsible for the presentation of glycolipid antigens to subsets of natural killer T (NK-T) cells, is expressed by intestinal epithelial cells (IECs). However, CD1d-restricted antigen presentation has not yet been examined on IECs. METHODS: A mouse intestinal epithelial cell line (MODE-K), a human epithelial cell line (T84), T84 cells transfected with CD1d and/or MHC class II, and freshly isolated human IECs were examined for their ability to present model glycolipid antigens to NK-T cells as defined by interleukin (IL)-2 or IL-4 secretion. RESULTS: MODE-K and freshly isolated human IECs exhibited dose-dependent, CD1d-restricted presentation of the functional glycolipid antigen, alpha-galactosylceramide (alpha GalCer), to the mouse NK-T cell hybridoma, DN32.D3. The human IEC line, T84, mainly presented alpha GalCer when transfected with human CD1d. Presentation of alpha GalCer by CD1d-transfected T84 cells (T84d) to DN32.D3 cells was greater along the basal surface in comparison with the apical surface. Induction of the MHC class II antigen presentation machinery by cotransfecting T84d with the MHC class I transactivator (CIITA) did not alter this polarity of presentation. Neither MODE-K nor T84 cells transfected with CD1d, CD1d plus CIITA, or CD1d plus HLA-DR were able to present glycolipid antigens requiring intracellular processing. The MODE-K cell line could also present alpha GalCer to primary mouse NK-T cells. CONCLUSIONS: CD1d is expressed functionally on IECs with a polarity of presentation (basal > apical) predicting a role in presentation of mucosal glycolipid antigens to local CD1d-restricted T cells.

The CD1d natural killer T-cell antigen presentation pathway is highly conserved between humans and rhesus macaques.

Natural killer T (NKT) cells play an important role in controlling cancers, infectious diseases and autoimmune diseases. Although the rhesus macaque is a useful primate model for many human diseases such as infectious and autoimmune diseases, little is known about their NKT cells. We analyzed V alpha 24TCR+ T cells from rhesus macaque peripheral blood mononuclear cells stimulated with alpha-galactosylceramide (alpha-GalCer) and interleukin-2. We found that rhesus macaques possess V alpha 24TCR+ T cells, suggesting that recognition of alpha-GalCer is highly conserved between rhesus macaques and humans. The amino acid sequences of the V-J junction for the V alpha 24TCR of rhesus macaque and human NKT cells are highly conserved (93% similarity), and the CD1d alpha1-alpha2 domains of both species are highly homologous (95.6%). These findings indicate that the rhesus macaque is a useful primate model for understanding the contribution of NKT cells to the control of human diseases.

NK T cells contribute to expansion of CD8(+) T cells and amplification of antiviral immune responses to respiratory syncytial virus.

CD1d-deficient mice have normal numbers of T lymphocytes and natural killer cells but lack Valpha14(+) natural killer T cells. Respiratory syncytial virus (RSV) immunopathogenesis was evaluated in 129xC57BL/6, C57BL/6, and BALB/c CD1d(-/-) mice. CD8(+) T lymphocytes were reduced in CD1d(-/-) mice of all strains, as shown by cell surface staining and major histocompatibility complex class I tetramer analysis, and resulted in strain-specific alterations in illness, viral clearance, and gamma interferon (IFN-gamma) production. Transient activation of NK T cells in CD1d(+/+) mice by alpha-GalCer resulted in reduced illness and delayed viral clearance. These data suggest that early IFN-gamma production and efficient induction of CD8(+)-T-cell responses during primary RSV infection require CD1d-dependent events. We also tested the ability of alpha-GalCer as an adjuvant to modulate the type 2 immune responses induced by RSV glycoprotein G or formalin-inactivated RSV immunization. However, immunized CD1-deficient or alpha-GalCer-treated wild-type mice did not exhibit diminished disease following RSV challenge. Rather, some disease parameters, including cytokine production, eosinophilia, and viral clearance, were increased. These findings indicate that CD1d-dependent NK T cells play a role in expansion of CD8(+) T cells and amplification of antiviral responses to RSV.

Natural killer T cell ligand alpha-galactosylceramide enhances protective immunity induced by malaria vaccines.

The important role played by CD8(+) T lymphocytes in the control of parasitic and viral infections, as well as tumor development, has raised the need for the development of adjuvants capable of enhancing cell-mediated immunity. It is well established that protective immunity against liver stages of malaria parasites is primarily mediated by CD8(+) T cells in mice. Activation of natural killer T (NKT) cells by the glycolipid ligand, alpha-galactosylceramide (alpha-GalCer), causes bystander activation of NK, B, CD4(+), and CD8(+) T cells. Our study shows that coadministration of alpha-GalCer with suboptimal doses of irradiated sporozoites or recombinant viruses expressing a malaria antigen greatly enhances the level of protective anti-malaria immunity in mice. We also show that coadministration of alpha-GalCer with various different immunogens strongly enhances antigen-specific CD8(+) T cell responses, and to a lesser degree, Th1-type responses. The adjuvant effects of alpha-GalCer require CD1d molecules, Valpha14 NKT cells, and interferon gamma. As alpha-GalCer stimulates both human and murine NKT cells, these findings should contribute to the design of more effective vaccines against malaria and other intracellular pathogens, as well as tumors.