Journal: Antioxidants

Article Title: Transcriptomic Redox Dysregulation in a Rat Model of Metabolic Syndrome-Associated Kidney Injury

doi: 10.3390/antiox14060746

Figure Lengend Snippet: Experimental protocol and sample allocation in metabolic syndrome (MetS) rat model. ( A ) The experimental protocol for MetS induction in Wistar rats is illustrated for 11 weeks. After one week of acclimation, rats were randomly assigned to either the control group (blue timeline) or the MetS group (orange timeline) starting at 8 weeks of age (week 0) fed with normal or high-fat diet (HFD), respectively. At week 3, the MetS group received a single intraperitoneal injection of streptozotocin (STZ) to induce insulin deficiency, while the control group received a vehicle. Fasting blood glucose (FBG) was measured weekly from week 3 to week 10. Metabolic and oxidative stress (OxS) assessments, including lipid profile testing, an oral glucose tolerance test (OGTT), and free oxygen radicals test (FORT)/free oxygen radicals defense (FORD) assays, were conducted at week 7. At week 10, final blood and urine samples were collected for CCL5 measurements, FBG evaluation, OxS markers, and renal oxidative injury analysis, followed by euthanasia. ( B ) The sample distribution for different analyses is summarized. All 20 control rats and 30 MetS rats underwent assessments of body weight (BW), FBG, lipid profiles (TG, triglyceride; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein), and serum C-C motif chemokine ligand 5 (CCL5) levels. Subgroups were allocated for specific analyses: blood and cardiovascular parameters, OxS and redox gene expression profiling, renal CCL5 expression analysis by RT-PCR and qPCR, urine parameters, and renal oxidative injury assessments for 4-hydroxy-2-nonenal(4-HNE) and 3-nitrotyrosine (3-NT).

Article Snippet: Slides were blocked with 10% fetal bovine serum (FBS) and incubated overnight at 4 °C with primary antibodies against CCL5 (sc-365826, dilution 1:50; Santa Cruz Biotechnology, Dallas, TX, USA), 4-hydroxynonenal (4-HNE) (MAB3249, dilution 1:250; R&D Systems, Minneapolis, MN, USA), and 3-nitrotyrosine (3-NT) (sc-32757, dilution 1:150; Santa Cruz Biotechnology).

Techniques: Control, Injection, Gene Expression, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Antioxidants

Article Title: Transcriptomic Redox Dysregulation in a Rat Model of Metabolic Syndrome-Associated Kidney Injury

doi: 10.3390/antiox14060746

Figure Lengend Snippet: Schematic workflow of the experimental design and analysis pipeline. Metabolic syndrome (MetS) was established in rats using a high-fat diet combined with streptozotocin (STZ) injection. Systemic oxidative stress (OxS) was assessed via the free oxygen radicals test and the free oxygen radicals defense assay performed on whole blood samples. Renal OxS was evaluated by immunohistochemical staining for 4-hydroxynonenal and 3-nitrotyrosine. Transcriptomic profiling of 84 redox-related genes was conducted using a PCR array to identify differentially expressed targets. CCL5 was subsequently validated by various PCR assays in kidney tissues, and protein expression was measured in both serum and renal tissues.

Article Snippet: Slides were blocked with 10% fetal bovine serum (FBS) and incubated overnight at 4 °C with primary antibodies against CCL5 (sc-365826, dilution 1:50; Santa Cruz Biotechnology, Dallas, TX, USA), 4-hydroxynonenal (4-HNE) (MAB3249, dilution 1:250; R&D Systems, Minneapolis, MN, USA), and 3-nitrotyrosine (3-NT) (sc-32757, dilution 1:150; Santa Cruz Biotechnology).

Techniques: Injection, Immunohistochemical staining, Staining, Expressing

Journal: Antioxidants

Article Title: Transcriptomic Redox Dysregulation in a Rat Model of Metabolic Syndrome-Associated Kidney Injury

doi: 10.3390/antiox14060746

Figure Lengend Snippet: Oxidative stress status in serum and renal tissues. ( A ) Systemic oxidative stress was evaluated using the free oxygen radicals test (FORT) and the free oxygen radicals defense (FORD) assays at week 7, i.e., 4 weeks after STZ injection in the metabolic syndrome (MetS) group and week 10 (prior to sacrifice). Note that FORT levels at week 7 were slightly but significantly lower in MetS rats compared to controls, while FORD levels remained unchanged. By week 10, MetS rats exhibited significantly elevated FORT levels and reduced FORD levels relative to controls. Control group: n = 6; MetS group: n = 12. ( B ) Representative immunohistochemical staining of renal tissues at 40× magnification for a marker of lipid peroxidation, 4-hydroxynonenal (4-HNE), showed weak staining in the cortex and outer medulla of control rats, but it was markedly increased in MetS kidneys. ( C ) Representative immunohistochemical staining of renal tissues at 40× magnification for a marker of protein tyrosine nitration after OxS, 3-nitrotyrosine (3-NT), showed low levels in controls and strong expression in MetS kidneys, particularly in the tubules and interstitial regions. Insets in ( B , C ) show staining patterns at higher magnification (200×). The white arrows indicate glomeruli, long white arrows indicate proximal tubules, black arrows indicate distal tubules, and asterisks (*) denote interstitial cells. ( D ) Quantitative analysis of 4-HNE and 3-NT expression in renal tissue confirmed significantly higher OxS marker levels in MetS rats compared to controls. (n = 12 per group). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control; ns = not significant.

Article Snippet: Slides were blocked with 10% fetal bovine serum (FBS) and incubated overnight at 4 °C with primary antibodies against CCL5 (sc-365826, dilution 1:50; Santa Cruz Biotechnology, Dallas, TX, USA), 4-hydroxynonenal (4-HNE) (MAB3249, dilution 1:250; R&D Systems, Minneapolis, MN, USA), and 3-nitrotyrosine (3-NT) (sc-32757, dilution 1:150; Santa Cruz Biotechnology).

Techniques: Injection, Control, Immunohistochemical staining, Staining, Marker, Nitration, Expressing