Effect of Adiponectin on Kidney Crystal Formation in Metabolic Syndrome Model Mice via Inhibition of Inflammation and Apoptosis

<div><p>The aims of the present study were to elucidate a possible mechanism of kidney crystal formation by using a metabolic syndrome (MetS) mouse model and to assess the effectiveness of adiponectin treatment for the prevention of kidney crystals. Further, we performed genome-wide expression analyses for investigating novel genetic environmental changes. Wild-type (+/+) mice showed no kidney crystal formation, whereas ob/ob mice showed crystal depositions in their renal tubules. However, this deposition was remarkably reduced by adiponectin. Expression analysis of genes associated with MetS-related kidney crystal formation identified 259 genes that were >2.0-fold up-regulated and 243 genes that were <0.5-fold down-regulated. Gene Ontology (GO) analyses revealed that the up-regulated genes belonged to the categories of immunoreaction, inflammation, and adhesion molecules and that the down-regulated genes belonged to the categories of oxidative stress and lipid metabolism. Expression analysis of adiponectin-induced genes related to crystal prevention revealed that the numbers of up- and down-regulated genes were 154 and 190, respectively. GO analyses indicated that the up-regulated genes belonged to the categories of cellular and mitochondrial repair, whereas the down-regulated genes belonged to the categories of immune and inflammatory reactions and apoptosis. The results of this study provide compelling evidence that the mechanism of kidney crystal formation in the MetS environment involves the progression of an inflammation and immunoresponse, including oxidative stress and adhesion reactions in renal tissues. This is the first report to prove the preventive effect of adiponectin treatment for kidney crystal formation by renoprotective activities and inhibition of inflammation and apoptosis.</p></div

Confirmation of selected genes detected by microarray analysis.

<p>A–F. Among the selected genes with significant expression changes in the microarray analysis, the expression of several characteristic genes belonging to 6 categories based on GO analysis was evaluated by immunohistochemical staining. Magnification is ×20 (in box: ×400). 6 categories are as follows. A. Inflammation and immune-related gene group: LYZ1, Lysozyme 1.CD44, CD44 antigen; MHC-class 2, major histocompatibility complex-class2. B. Apoptosis-related gene group: STAT3, signal transducer and activator of transcription 3; AURKA, aurora kinase A, Thymidine kinase 1, C. Cell repair and proliferation-related gene group: MCM5, minichromosome maintenance deficient 5. D. Adhesion and fibrosis-related gene group: Fn, Fibronectin. E. Oxidative stress-related gene group: 8OHdG, 8-Hydroxydeoxyguanosine. F: transporter-related gene group; SLC12A1, solute carrier family 12, member 1.</p

Expression analyses of <i>Spp1</i>, <i>Sod2</i>, <i>Ccl2</i>, and <i>Adipoq</i>.

<p>A. The results of quantitative PCR for the expression of <i>Spp1</i>, <i>Sod2</i>, <i>Ccl2</i>, and <i>Adipoq</i>. Expression levels are expressed relative to <i>Actb</i> (<i>actin-beta</i>) transcript levels. <i>Spp1</i>, secreted phosphoprotein 1; <i>Ccl2</i>, C-c chemokine ligands 2; <i>Sod2</i>, superoxide dismutase 2; <i>Adipoq</i>, adiponectin. Data are indicated as the mean ± SE. *, p<0.05 between groups at the same time point. †, p<0.01 compared with the same group at day 0. B. Immunohistochemical staining for OPN, MCP-1, SOD, and APN expression. OPN, osteopontin; MCP-1, monocyte chemotactic protein-1; SOD, superoxide dismutase; APN, adiponectin. Magnification is ×20 (in box: ×400). C. The quantification of immunohistochemical staining. Data are indicated as the mean ± SD. *, p<0.05 between groups at the same time point. †, p<0.05 compared with the same group at day 0.</p

Confirmation that selected genes detected by microarray analysis showed differential expression between different treatment groups.

<p>The expression of several characteristic genes belonging to 5 categories based on GO analysis was evaluated by quantitative PCR. Expression levels are expressed relative to <i>Actb</i>. Data are indicated as the mean ± SE. *, p<0.05 between the groups at the same time point. †, p<0.01 compared with the same group at day 0. 5 categories are as follows. A. Inflammation and immune-related gene group: <i>Lcn2</i>, lipocalin2; <i>Cd44</i>, CD44 antigen; <i>Lyz1</i>, lysozyme1. B. Apoptosis-related gene group: <i>Stat3</i>, signal transducer and activator of transcription 3; <i>Aurka</i>, aurora kinase A. C. Cell repair and proliferation-related gene group: <i>Mcm5</i>, minichromosome maintenance deficient 5. D. Adhesion and fibrosis-related gene group: <i>Vcam1</i>, vascular cell adhesion molecule 1; <i>Col3a1</i>, collagen, type III, alpha 1. E. Transporter-related gene group: <i>Slc12a1</i>, solute carrier family 12, member 1; <i>Slc7a13</i>, solute carrier family 7, member 13.</p

Detection and quantification of calcium oxalate kidney crystal formation.

<p>A. Kidney sections of wild type (+/+), obesity (ob/ob), and adiponectin-treated ob/ob (ob/ob+APN) mice at day 6. The upper images show calcium oxalate crystal deposits in Pizzolato-stained sections. The lower images show non-stained sections observed by polarized light optical microphotography (PLOM). Magnification is ×20 (in box: ×400). B. Quantification of kidney crystals in +/+, ob/ob, and ob/ob+APN mice. Data are indicated as the mean ± SD. a; p = 0.0004, b; p = 0.005.</p

TUNEL staining and the number of TUNEL-positive cells.

<p>Data are indicated as the mean ± SD. *, p<0.05 between groups at the same time. **, p<0.01 between groups at the same time point. †, p<0.01 compared with the same group at day 6.</p

The scheme of the microarray analysis design.

<p>A. Experimental design and gene expression changes between groups. <i>Δ</i> refers to a gene group with >2.0- or <0.5-fold expression change between 2 experimental groups. <i>ΔA</i> means gene expression involving GOx administration. <i>ΔB</i> means gene expression involving obesity. <i>ΔC</i> means gene expression involving GOx administration and kidney stone formation. <i>ΔD</i> means gene expression involving obesity and kidney stone formation. <i>ΔE</i> means gene expression involving obesity, GOx administration, and kidney stone formation. <i>ΔF</i> means gene expression involving APN administration and kidney stone prevention. +GOx, glyoxylate administration; +APN, adiponectin treatment. B. The selection scheme for kidney stone formation-related gene groups (Venn diagram). The gray-stained area is represented by (<i>ΔC</i>∩<i>ΔD</i>∩<i>ΔE</i>)\(<i>ΔA</i>∪<i>ΔB</i>) and is the kidney stone formation-specific gene group under a MetS environment.</p

Confirmation of selected genes detected by microarray analysis.

<p>Western blot analysis for OPN, MCP-1, SOD, APN, LYZ1, AURKA, TK1, MCM5, NGAL, CD44, STAT3, SLC12A1, SLC7A13, VCAM1, COL3A1 and FN protein expression. Each molecular weight demonstrated as the bands were as follows. OPN, 75 and 55 kDa; MCP-1,17 kDa; SOD, 37 kDa; APN, 64 kDa; LYZ1, 17 kDa; AURKA, 40 kDa; TK1, 25 kDa; MCM5, 90 kDa; NGAL, 25 kDa; CD44, 80–95 kDa; STAT3, 92 kDa; SLC12A1, 47 kDa; SLC7A13, 52 kDa; VCAM1, 100 kDa; COL3A1, 138 kDa; FN, 212 kDa and β-actin, 37 kDa.</p

Clinical and laboratory findings of cases 1–3.

<p>DSD, disorders of sex development; MP, micropenis; HS, hypospadias; CO, cryptorchidism.</p><p>The hormone values below the reference range are boldfaced, and those above the reference range are italicized.</p>a<p>Reference values of the age-matched control individuals are shown in the parenthesis.</p

Genes affected by the cryptic deletions.

<p>Genes affected by the cryptic deletions.</p