Ab136933

To quantify aldosterone and C-reactive protein (CRP) in plasma, an aldosterone ELISA kit (ab136933) and rat CRP ELISA kit (PTX1, ab108827), respectively, were obtained from Abcam (Cambridge, UK). Appropriately diluted plasma samples were reacted using the kit in according to the manufacturer’s suggestion.
The composition of apolipoprotein/lipoprotein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an exact amount of protein loaded in the gel (5 µg of total protein per lane) from individual HDL₃, and the levels of apolipoprotein expression were measured by immunodetection. Anti-human apoA-I antibody (ab7613) and anti-apo-B antibody (ab20737) were ordered from Abcam (Cambridge, UK). Relative band intensity (BI) was compared through band scanning with Chemi-Doc^® XRS+ (Bio-Rad, Hercules, CA, USA) using Image Lab software (Version 5.2, Bio-Rad, Hercules, CA, USA). Blot images are imported into the Quantity One software and then contrast was adjusted in a way so that the bands were fully noticeable on the blot image. The area around the band was chosen; additionally, the background intensity was deducted from the blot image. The appropriate bands were used to calculate band intensities and exported to excel for analysis.

One month after surgery, 100 µL retro-orbital blood samples were collected in anesthetized animals (isoflurane: 1.5%) to quantify plasma creatinine by enzymatic method. At sacrifice, blood samples were collected allowing to measure plasma creatinine, soluble vascular cell adhesion molecule 1 (sVCAM-1) and fibroblast growth factor-23 (FGF-23) were assessed by enzyme-linked immunosorbent assays (ab100750, Abcam and EZMFGF23–43K, EMD Millipore respectively). In addition, the plasma levels of 14,15-EET, the preferential substrate of sEH, its metabolite 14,15-DHET, and the pro-inflammatory hydroxyeicosatetraenoic acids (5-HETE, 12-HETE and 15-HETE), derived from the lipoxygenase (LOX) metabolites of arachidonic acid, were quantified by LC-MS/MS using a previously published method (Duflot et al., 2017 (link); Duflot et al., 2019 (link)). The ratio of 14,15-DHET-to-14,15-EET was used as an index of sEH activity.
24-h urine was collected 1 and 3 months after surgical procedure using metabolic cages. Urine albumin and aldosterone were measured at 3 months using enzyme-linked immunosorbent assays (ab108792 and ab136933, respectively; Abcam, Paris, France). Na⁺ excretion was quantified using standard procedure.

Aldosterone plasma levels were quantified by ELISA using a commercially available Aldosterone kit (ab136933, Abcam, Cambridge, UK), following the manufacturer’s instructions. To increase accuracy, the assay was preceded by an extraction step performed similarly to that for corticosterone and 11-deoxycorticosterone, with the sole difference that extracts were reconstituted in 250 µL of the assay buffer of the aldosterone ELISA kit.

Serum samples were collected on day 29 after the T2DM rats were sacrificed. Insulin was quantified through the measurement of the optical density at 450 nm by using a Mercodia Ultrasensitive rat insulin ELISA kit (Mercodia AB, Uppsala, Sweden) and aldosterone was quantified through the measurement of the optical density at 405 nm corrected by the measurement at 590 nm by using an aldosterone ELISA kit (ab136933; Abcam, Cambridge, UK).