The Institutional Animal Care and Use Committee at University of Houston approved these protocols.
Ethical approval
All methods in this study are reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org/) that maximize the quality and reliability of published research, and enabling others to better scrutinize, evaluate and reproduce it. Humans are not involved in this study. All the methods were carried out in accordance with relevant guidelines and regulations.
Animals
Male OZR, 11-14 weeks old were purchased from Envigo, Indianapolis. The animals were acclimatized for a week at the University of Houston animal care facility upon arrival. In vivo experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee at the University of Houston. The animals were fed with NSD (0.4%; TekLad TD.99215, Harlan laboratories) or HSD (4%, TekLad TD.92034, Harlan laboratories) and treated with AT2R agonist C21 (0.3 mg/kg/day i.p.) for 2 days. The specificity of C21 in vivo as well as in vitro studies in our laboratory has been tested by blocking its effects with the AT2R antagonist PD123319. [31-33] Rats were placed in metabolic cages during the study for urine collection. Body weight, food intake, water consumption and urine output were measured at 24 and 48 hrs. At the end of the study, blood was collected through cardiac puncture under isoflurane (2-3%) anesthesia, processed for plasma, and stored at -80°C. Kidney cortices were collected and a part of it was embedded in OCT and stored at -80°C. Detailed methods on measurement of proteinuria and albuminuria, immunoblotting, Separation of phospho-megalin, phospho-Akt and phospho-GSK3b, immunofluorescence and colocalization, creatinine and GFR measurements, atomic absorption spectroscopy, quantitative RT-PCR analysis and list of chemicals is provided in the supplemental material.
Proteinuria and albuminuria
Urinary protein was measured by pyrogallol red (PR)-molybdate method. Briefly, to 5 μL of centrifuged urine sample, 200 μL PR-molybdate reagent was added and allowed to react for 10 minutes at 37°C. Absorbance was read at 600 nm to measure total protein (mg/mL). Urinary albumin was determined by Nephrat II competitive ELISA kit (catalog# NR002 Ethos biosciences) according to manufacturer’s protocol. Urinary protein and albumin were normalized with urine volume (mL/hr) and reported as excretion rate in mg/hr.
Immunoblotting
The expression of podocin, nephrin, pAkt and pGSK3b in the kidney cortices was determined by western blot analysis. Equal amount of protein (20 μg for podocin and nephrin, and 100mg for pAkt and pGSK3b) was loaded at 4-20% SDS-PAGE, transferred to activated PVDF membrane, and immunoblotted with anti-podocin, anti-nephrin, anti-phospho-Akt (Ser-473) and anti-phospho-GSK3b (Ser9) respectively. β-Actin was used as loading control for podocin and nephrin and total Akt and total GSK3b were used to normalize pAKT and pGSK3b. For dot blot analysis, an equal amount of protein (10 μg) was directly spotted onto the activated PVDF membrane. The membrane was then incubated with specific anti-megalin or anti-cubilin antibody in 5% BSA-PBST (phosphate buffered saline containing 0.05% tween-20) overnight at 4°C. The membrane was washed with PBST (5 mL, 10 min x 3), immunoprobed with relevant secondary HRP-conjugated antibodies, namely goat-anti-mouse IgG secondary antibody, goat-anti-rabbit IgG secondary antibody, for 1 hour at room temperature, washed with PBST and the electrochemiluminescence signal was recorded and the bands density was analyzed (BioRad ChemicDoc Imaging System).
Separation of phospho-megalin, phospho-Akt and phospho-GSK3b
To determine the phosphorylated proteins, SuperSep Phos-tag (50μmol/l), 7.5%, 17well, 83×100×3.9 mm (FUJIFILM Wako Pure Chemical Corporation catalog# 198-17981) was used. Phos-tag gel is a novel method which separates phospho-proteins based on migration and band shift which relies on complex formation ability. Phospho-proteins separate at a higher level compared with the non-phosphorylated form. Phos-tag allows to study phospho-proteins independent of phospho-specific antibody. Moreover, the stripping procedure is not required, hence, this is the method of choice to study phospho-proteins independent of loading control (e.g., GAPDH, beta-actin, etc.). The samples were prepared using RIPA lysis buffer without EDTA (150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, Tris-HCl and 1% SDS with protein phosphatase inhibitor) and samples were separated for 9 hrs. at 10 mA. Before transferring the gel on the PVDF membrane, the gel was washed with transfer buffer containing 10mmol/L EDTA and 3 times for 10 mins each. The gel was then immersed in transfer buffer without EDTA for 10 min. Transfer buffer without EDTA was used to transfer the proteins on the PVDF membrane. Immunoblotting with megalin antibody, Akt or GSK3b antibody was performed traditionally as explained earlier. The chemiluminescence signal was recorded and analyzed densitometrically by BioRad ChemicDoc Imaging System. The data is represented as the density ratio of the phospho- to non-phospho bands.
Immunofluorescence and colocalization
Approximately 20 μm thick sections were used for immunofluorescence experiment. The sections were incubated and permeabilized/blocked with 0.4% BSA, 0.2% saponin and 1% of the animal serum (donkey) in which the secondary antibody is raised in 1X PBS for 1 hr. at room temperature. This blocking buffer was discarded and 1X PBS containing the anti-megalin, anti-cubilin antibody and/or anti-LAMP1 in 0.2% BSA and 0.1% saponin was added to the sections and incubated overnight at 4°C. The sections were washed with 1X PBS (10 min x 4) and secondary antibodies for megalin, cubilin, and/or LAMP1 in 1X PBS containing 0.2% BSA and 0.1% saponin was added and incubated for 2 hr. at room temperature. The sections were washed (10 min x 4) with 1X PBS and incubated with DAPI (D1306, Thermo Fisher Scientific, 1:3000, 5 mg/mL stock) for 10 minutes followed by washing 3-times with PBS. The sections and coverslip were mounted on slides with glycerol and were imaged using Leica confocal microscope (DMi8).
Creatinine and GFR measurements
Urinary creatinine was measured using BioAssay systems kit (catalog# DICT500) according to the manufacturer’s protocol. The data was reported as mg/day. Plasma creatinine was measured by Arbor Assays kit (catalog# KB02-H1) according to the manufacturer’s protocol. The values were reported as mg/dL. The GFR was calculated creatinine clearance method.
Atomic absorption spectroscopy
This method was used to measure urinary and plasma sodium. The standards and samples were prepared according to the company’s protocol and the data was calculated using Beer’s law.
Quantitative RT-PCR Analysis for mRNA Expression
Total RNA from frozen kidneys was extracted using the RNAEasy kit (Qiagen) according to the manufacturer’s protocol. A total of 500 ng of RNA were reversed transcribed into cDNA using ReverTra Ace qPCR RT Master Mix with gDNA remover (Diagnocine). This cDNA was used to semi-quantitate cytokines (TNF-a, IL-10, TGF-b, megalin and cubilin) using Thunderbird SYBR qPCR master mix (Diagnocine) in CFX Connect RT-PCR (Bio-Rad). Specific quantitative PCR primers for TNF-a (catalog# RP300044), TGF-b (catalog# RP300111) and IL-6 (catalog# RP300072) were purchased from Sino Biological and megalin (catalog# 316614765 F [Sequence: TGG AAT CTC CCT TGA TCC TG], catalog# 316614766 R [Sequence: TGT TGC TGC CAT CAG TCT TC]) and cubilin (catalog# 316614763 F [Sequence: GCA CTG GCA ATG AAC TAG CA ], catalog# 316614764 R [Sequence: TGA TCC AGG AGC ACT CTG TG]) from Integrated DNA Technologies. Expression of each gene was normalized to b-actin (catalog# VRPS-97, Real Time Primers, LLC), and relative fold expression values were calculated using a DD threshold cycle method.
Chemicals
Anti-podocin (Santa Cruz, catalog# sc-518088), anti-nephrin (Santa Cruz, catalog# sc-377246), anti-phospho-Akt (Ser-473) (Cell Signaling catalog# 9271), anti-phospho-GSK3b (Ser9) (Cell Signaling catalog# 9336), β-actin (catalog# sc-47778), total Akt (Cell Signaling catalog# 9272) and total GSK3b (Cell Signaling catalog# 9315), anti-megalin (Santa Cruz, catalog# sc-515750), anti-cubilin antibody (Santa Cruz, catalog# sc-518059), anti-LAMP1 (Development Studies Hybridoma Bank catalog# 1D4B), Alexa fluor 488 anti-mouse for megalin, Alexa fluor 488 anti-rabbit for cubilin, and/or Alexa fluor 568 anti-rat for LAMP1.
Statistical analysis
The data were analyzed using GraphPad Prism Version 9.1.2 (225). Data are represented as mean ± s.e.m. Statistical analysis was performed using one-way or two-way ANOVA with Fisher’s LSD for multiple comparisons and *p<0.05 vs NSD and #p <0.05 vs HSD considered statistically significant.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.