Bn 2

Serum CRP was obtained from overnight fasting blood and was measured using high-sensitivity assay by immunochemistry - nephelometry - (BN II; Siemens).

Total Ig in serum samples were assessed by a nephelometry method (BN II, Siemens Health Care). In order to normalize the IgG or IgA concentration, serums were diluted to 20 μg/ml for IgG measure and 35 μg/ml for IgA measure in PBS (Gibco). Twenty-five microliters of fixed samples containing 2.5 × 10⁵ fungal forms were distributed in a 1.5 ml tube (Eppendorf). Twenty-five microliters of normalized serum were then distributed in each tube and incubated for 20 min at 4 °C. Two hundred microliters of PBS were then added and samples were centrifuged at 21,000 g during 10 min. Supernatant was removed and fungi were incubated with a 25 μl mix of goat anti-human IgG Alexa Fluor 647 (Jackson ImmunoResearch) or goat anti-human IgA FITC (Jackson Immunoresearch) or the isotype control, goat IgG A647/FITC (Jackson Immunoresearch), at 4 °C during 20 min. After one wash with PBS, stained fungi were resuspended in 200 μl of PBS. Acquisition of fungi was performed on a FACS Canto II flow cytometer (BD) using the FACS Diva (BD) software. Unstained spores or budding forms were used as negative control to identify background fluorescence. Monoclonal antibodies with irrelevant specificity (anti-TNF-α human IgG1; MSD laboratory) revealed Fc binding. Median fluorescence intensity (MFI) was used to assess the Ig response.

Blood samples were obtained by tail venepuncture to examine the glucose levels. Blood samples taken from the heart of anaesthetised rats were immediately analysed to measure serum levels of urea, creatinine, triglycerides, total cholesterol and albumin using an automatic biochemistry analyser (AU5800, Beckman Coulter Inc., Brea CA, USA). Urine was collected for 1 day before sacrifice to detect proteinuria using the nephelometry method (Siemens BN II, Deerfield, IL, USA).

Twenty-four-hour urine samples were collected from individual rats housed in metabolic cages with free access to water but without access to food. Proteinuria was determined using the nephelometry method (Siemens BN II, Deerfield, IL, USA). On days 10, 14, 21 and 28 after ADR induction, 10 rats per group were euthanized under anesthesia. Kidney tissue and blood samples were obtained from the anesthetized rats. The blood samples were immediately analyzed with an automatic biochemistry analyzer (Roche, Cobasc 311, Mannheim, Germany) to measure the serum levels of albumin, total protein, triglycerides and cholesterol[24 (link)]. The renal tissues were processed for morphological studies, immunofluorescence microscopy and molecular biology experiments.
We conducted our human subject research with the approval of the Institutional Review Board of the Second Affiliated Hospital of Harbin Medical University in Harbin, China. All participants provided written informed consent according to the latest version of the Helsinki Declaration on human research ethics.
Human kidney tissues were collected during renal biopsy at the Second Affiliated Hospital of Harbin Medical University and were processed for immunofluorescence staining as subsequently described. Human urine was collected for 24 hours prior to treatment.

Every subject was required to complete a questionnaire including information on demographic characteristics and medical history. After an overnight fast, they were examined in the morning by trained nurses. The concentrations of SUA, serum albumin, fasting plasma glucose, hemoglobin A1c (HbA1c), hemoglobin, serum creatinine (Scr), blood urea nitrogen (BUN), total cholesterol (TC), triglycerides (TGs), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and C-reactive protein (CRP) were measured by using a 717A automatic biochemical analyzer (Hitachi H7600). Urine creatinine (Ucr) and urine albumin (UALB) were measured through an automatic urine analyzer (Sysmex AX4280) and an automatic protein analyzer (Siemens BNII). The urinary albumin-to-creatinine ratio (U-ACR, mg/g) was calculated as UALB/Ucr. Body mass index (BMI) was defined as body weight (kilogram, kg) divided by the square of height (meter, m²). Blood pressures were measured by using a standard sphygmomanometer after the subjects rested for 15 minutes.