Optimal delipidation solvent to secure extracellular matrix from human perirenal adipose tissue.

The objective of this study was to select the optimal delipidation solvent for preparation of human perirenal adipose tissue-derived extracellular matrix (ECM). Human perirenal adipose tissue can be obtained in large amounts during surgery, and it can be an alternative source of human ECM. Delipidation is an essential procedure for the ECM preparation, because lipid strongly inhibits regeneration of target tissue. Isopropanol has been widely used as a delipidation solvent for adipose tissue. However, because adipose tissue is mostly composed of nonpolar lipid, a nonpolar solvent might be more effective for delipidation. We evaluated the delipidation efficiency of acetone, chloroform, methanol, ether, ethanol, isopropanol, water, chloroform/methanol, ethanol/heptane, ether/methanol, hexane/ethanol, and butanol/methanol solvents for ECM extraction from human perirenal adipose tissue. Among them, acetone-treated adipose tissue showed the greatest delipidation efficiency (93.05%), significantly lower residual DNA content, and the greatest residual collagen concentration (42.49 ± 0.05 µg/g). In addition, acetone-treated tissue also had well-preserved ultrastructure with high porosity and significantly low in vitro cytotoxicity. These results suggested that acetone may be an optimal delipidation solvent for extraction of ECM from human perirenal adipose tissue.

Renal Protective Effect of Beluga Lentil Pretreatment for Ischemia-Reperfusion Injury.

MATERIALS AND METHODS: Mice were divided into four groups: normal, untreated, low- (2 mg), and high-dose (8 mg) beluga lentil treatment groups. Beluga lentil was orally administered for 2 weeks, followed by bilateral renal ischemia for 20 min and reperfusion for 30 min. Blood samples and kidney tissues were collected and analyzed to investigate renal function, histopathology, epithelial and endothelial cell damage, apoptosis, oxidative stress, and inflammatory responses. RESULTS: The pretreated groups maintained renal function, with significantly lower blood urea nitrogen (BUN) and creatinine levels, compared with the other groups. The histopathological analysis showed reduced proximal tubule injury and decreased injury-related molecule (kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL)) secretion in the pretreated groups compared with the other groups. Terminal deoxynucleotidyl transferase dUTP nick-end labeling- (TUNEL-) positive cells and the secretion of apoptosis-related molecules (Fas and caspase 3) were significantly reduced in the pretreated groups compared with the other groups. The pretreated groups showed positive microvessel-associated gene (cluster of differentiation (CD31)) expression and negative adhesion molecule (intracellular adhesion molecule 1 (ICAM-1)) expression. An antioxidant effect was observed in the pretreatment groups, with reduced malonaldehyde (MDA) expression and increased antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GPx)) secretion. In the pretreated groups, F4/80+ macrophages and CD4+ T cell infiltration were inhibited and proinflammatory cytokine (interleukin- (IL-) 1ß, IL-6, and tumor necrosis factor- (TNF-) α) levels decreased; however, the levels of anti-inflammatory cytokines (transforming growth factor- (TGF-) ß, IL-10, and IL-22) increased. CONCLUSIONS: Beluga lentil pretreatment demonstrated protective effects against I/R-induced renal damage, via antiapoptotic, anti-inflammatory, and antioxidant activities.

IL-10 Deficiency Aggravates Renal Inflammation, Fibrosis and Functional Failure in High-Fat Dieted Obese Mice.

BACKGROUND: High-fat diet-induced obesity is one of the major cause of chronic renal failure. This obesity-related renal failure is mainly caused by inflammatory processes. However, the role of the major anti-inflammatory cytokine interleukin (IL)-10 has not been researched intensively. METHODS: To evaluate the effect of IL-10 deficiency on obesity-related renal failure, the in vivo study was carried with four animal groups; (1) Low-fat dieted C57BL/6 mice, (2) Low-fat dieted IL-10 knockout (KO) mice, (3) High-fat dieted C57BL/6 mice and (4) High-fat dieted IL-10 KO mice group. The analysis was carried with blood/urine chemistry, H&E, Oil-Red-O, periodic acid-Schiff and Masson's trichrome staining immunohistochemistry and real-time PCR methods. RESULTS: At week 12, high-fat dieted IL-10 KO mice showed 1) severe lipid accumulation in kidneys, cholesterol elevation (in total, serum kidney) and low-density lipoprotein increasion through the SCAP-SREBP2-LDLr pathway; (2) serious histopathologic alterations showing glomerulosclerosis, tubulointerstitial fibrosis and immune cell infiltration; (3) increased pro-inflammatory cytokines and chemokines expression; (4) enhanced renal fibrosis; and (5) serious functional failure with high serum creatinine and BUN and proteinuria excretion compared to other groups. CONCLUSION: IL-10 deficiency aggravates renal inflammation, fibrosis and functional failure in high-fat dieted obese mice, thus IL-10 therapy could be applied to obesity-related chronic renal failure.

Optimization of extracellular matrix extraction from human perirenal adipose tissue.

Human adipose tissue includes useful substrates for regenerative medicine such as the extracellular matrix (ECM), but most perirenal fat tissue is wasted after kidney surgery. Since a lot of adipose tissue can be procured after a kidney, we extracted ECM from human perirenal adipose tissue and optimized the extraction process. To verify the efficacy for ECM extraction, we compared the products in several steps. Perirenal adipose tissue was either finely homogenized or underwent crude manual dissection. The amount of extracted ECM was quantified with ELISA for verification of the initial tissue downsizing effect. To validate the drying effect for fast and complete delipidation, tissues were prepared in a dry or wet phase, and residual lipids were visualized with Oil-Red-O staining. The extracted lipid was assayed at each time point to quantify the appropriate delipidation time. To select the optimal decellularization method, tissues were treated with physical, chemical, or enzymatic method, and the residual cell debris were identified with histological staining. The biochemical properties of the ECM extracted by the above methods were analyzed. The ECM extracted by fine homogenization showed a significantly enhanced amount of collagen, laminin and fibronectin compared to the crude dissection method. The dried tissue showed fast and complete lipid elimination compared to the wet tissue. Complete delipidation was achieved at 45 min after acetone treatment. Additionally, 1% triton X-100 chemical treatment showed complete decellularization with well-preserved collagen fibers. Biochemical analysis revealed preserved ECM proteins, a high cell proliferation rate and normal cell morphology without cell debris or lipids. The established process of homogenization, drying, delipidation with acetone, and decellularization with Triton X-100 treatment can be an optimal method for ECM extraction from human perirenal adipose tissue. Using this technique, human perirenal adipose tissue may be a valuable source for tissue engineering and regenerative medicine.