Urea nitrogen direct kit

NTS serum was prepared as previously described (5 (link)). In brief, mice glomeruli were isolated by differential sieving and sonicated. Sheep were hyperimmunized with mouse glomeruli. The NTS was heat-inactivated and absorbed with murine blood cells. Age- and sex-matched mice (WT, Axl-KO, Mer-KO, and A/M-KO) were injected intravenously with 7.5ml/kg of nephrotoxic serum prepared from sheep (5 (link)). Mice were then followed with measurement of proteinuria (Uristix, Bayer Corporation, Elkhart, IN) and serum urea level using Urea Nitrogen Direct kit (Stanbio Laboratory, Boerne, TX). These animals were euthanized to obtain renal specimens at days 3-, 7-, and 14-post injection.

For measuring metabolic activity, PrestoBlue (Thermo Fisher Scientific) was added to culture media of rat hepatocytes on day 3 of culture at the concentration suggested by the manufacturer and incubated for 2 hours at 37°C, 10% CO₂. Following the incubation, media samples were used for the measurement.
For albumin and urea assays, the media collected from both rat and human hepatocytes at day 3 of culture was used. Level of albumin secreted in rat hepatocytes was determined following a direct, competitive enzyme-linked immunosorbent assay (ELISA) protocol as described before[25 (link)]. Albumin secreted by human hepatocytes was determined using Human Albumin ELISA kit (Abcam) following manufacturer’s instructions. The amount of urea secreted was determined using urea nitrogen direct kit (Stanbio) following the manufacturer’s instructions. The results were corrected to compensate for well-well differences in cell number using the cell counts.

Mice were divided into three groups (WT, WT-R428, and Axl-KO). R428 (125mg/kg/day) or vehicle was orally administered at day -2. All three groups were then iv injected with 7.5 ml/kg of anti-GBM sera prepared from sheep. The severity of nephritis was followed by measuring BUN with a Urea Nitrogen Direct kit (Stanbio Laboratory, Boerne, TX). Animals were euthanized at days 7 and 14 post-injection. Kidney samples were collected and embedded in OCT medium and snap frozen in liquid nitrogen. Sections (4 μm) were processed for hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining. Sections were examined in a blinded fashion. The glomeruli were screened and the severity of GN was graded on a 0–4 scale as previously described [21 (link)]. Briefly, 0, normal; 1, mild increase in mesangial cellularity and matrix; 2, moderate increase in mesangial cellularity and matrix, with thickening of the GBM; 3, substantial increase in the thickness and irregularity of the GBM; and 4, segmental necrosis, crescents, and hyalinized end-stage glomeruli.

Female C57BL/6 mice were purchased from The Taconic Laboratory. All experiments were performed in compliance with federal laws and institutional guidelines. The animal protocol was approved by the Augusta University Institutional Animal Care and Use Committee (no. A3307-01). 12-week-old mice (18–20 g) were used for all experiments. Established cloned glomerular endothelial cell clones were employed as described previously ^{28 (link)}. Urea nitrogen direct kit (Stanbio Laboratory, Boerne, TX), PKC-α inhibitor Ro-320432, (which displays 10-fold greator selectivity for PKC-α, 4-fold greater selectivity for PKC-β over other isoforms; EMD Millipore, Billerica, MA), PKC activator, Phorbol 12,13-dibutyrate (PDBu) (Sigma-Aldrich); EDC (Thermo Scientific, Rockford, IL), Mito-ID Membrane Potential Detection Kit (Enzo Life Sciences, Farmingdale NY), Pierce LDH Cytotoxicity Kit (Thermo Scientific), Nephrotoxicity PCR array (Qiagen, Maryland), MitoTracker Green FM (Thermo Scientific, Rockford, IL) wereused.