Animal models

Eight-week-old male db/db mice and their non-diabetic db/m littermates were obtained from Jicui Bioscience Co. Ltd. (Jiangsu, China), and were used as the type 2 diabetes models. All mice were housed individually, subjected to a 12-hour light/dark cycle, given free access to water, and were nourished with a standard laboratory diet. Twenty of the db/db mice were given injections of either adeno-associated virus (AAV)-shNedd4l-EGFP or the control virus (Yuanjing Biosciences, Guangdong, China). The Animal Care Committee of Tianjin Medical University strictly regulated all animal experiments according to its established guidelines and regulations.

Cell culture

Proximal tubular epithelial cells of human origin, HK-2, were procured from the Cell Bank of the Chinese Academy of Sciences, based in Shanghai, China, and subsequently cultured in DMEM medium, which was fortified with a 10% concentration of fetal bovine serum. The HK-2 cells were subjected to culture conditions that consisted of either a normal glucose concentration of 5.5 mM or a high glucose (HG) concentration of 25.5 mM over a period of 48 h. Additionally, 20.0 mM mannitol was used as an osmolality control. The cell transfection process was executed using the plasmids pcDNA3.1-CaMKKβ (FulenGen, Guangdong, China), Nedd4L-siRNA (Ribobio, Guangdong, China) and CaMKKβ-siRNA (Ribobio, Guangdong, China), along with the Lipofectamine 2000 reagent (Invitrogen, California, USA) in compliance with the guidelines provided by the manufacturer.

Renal histological analysis

As previously described [22], renal histological examination was conducted. Briefly, protocols provided by the manufacturer were followed to stain kidney sections with Hematoxylin and Eosin (HE), Masson’s trichrome, and Periodic acid-Schiff (PAS). For immunohistochemical (IHC) analysis, kidney sections were blocked and incubated with primary antibodies. Subsequent incubation with secondary antibodies was performed before visualization using diaminobenzidine. The final step of counterstaining was performed with hematoxylin.

Western blotting analysis

Total proteins or mitochondrial proteins were isolated, separated and transferred onto polyvinylidene fluoride membranes (Millipore, Boston, USA). The membranes were incubated overnight with primary antibodies. The primary antibodies and their respective dilutions were NEDD4L, p-AMPK, AMPK, DRP-1, and HSP60 (all with a dilution ratio of 1:1000, Cell Signaling Technology, Danvers, USA), CaMKKβ and MFN-2 (1:1000 dilution, Abcam, Cambridge, UK), and phospho-DRP1 (Ser616) (1:1000 dilution, Biorbyt, Cambridge, UK). Following this procedure, the sections were incubated with secondary anti-mouse/rabbit antibodies (Sungene Biotech, Tianjin, China). The protein bands were subsequently visualized using ECL Blotting Detection Reagents (Invigentech, California, USA). The final quantification of these blots was achieved using densitometry via the application of ImageJ software.

Isolation of mitochondrial protein

The mitochondrial fractions were extracted with a Mitochondria/Cytosol Fractionation Kit (KeyGEN BioTECH, Jiangsu, China) according to the manufacturer’s instructions.

Immunofluorescence (IF) staining

For IF staining of cells and tissues, the protocols used were identical to those used for IHC analysis. Following incubation with the primary antibody, the slides were subjected to serial incubation with goat anti-rabbit IgG/FITC at a concentration of 1:100 (Proteintech, Chicago, USA). Subsequently, the slides were imaged using an automated Leica DMI 4000 B inverted microscope equipped with a Leica DFC300 FX camera.

Assessment of mitochondrial morphology

After several treatments, the viable cells were incubated with MitoTracker (Cell Signaling Technology, Danvers, USA) following the manufacturer’s guidelines. Subsequently, these cells were inspected via confocal microscopy.

Albumin uptake analyses

The endocytosis assay was conducted according to a previously established procedure. In brief, HK2 cells were washed with warm Hanks’ balanced salt solution (without phenol red) and subsequently incubated with 0.5 mg/ml TRITC-bovine serum albumin (BSA) (Sigma-Aldrich, USA) for one-quarter of an hour at 37 °C. After incubation, six washes were performed on the cells using cold Hanks’ balanced salt solution (at 4 ℃), followed by lysis in PBS buffer. A confocal fluorescence microscope (Leica Microsystems, Germany) was used to detect the fluorescence within the lysate.

Detection of ROS

To assess intracellular and mitochondrial ROS production, cells were incubated with H2DCFDA (Keygenbio, China) or Mitosox (Keygenbio, China), respectively, and examined by microscopy. Intracellular ROS production in kidney tissues was evaluated utilizing 4-mm-thick cryostat sections, which were then stained with dihydroethidium (DHE) acquired from EVERBRIGHT, USA.

Co-immunoprecipitation (Co-IP)

The immunoprecipitation (IP) experiments were conducted in accordance with the methods previously reported [23]. Briefly, cells were harvested and lysed in IP lysis buffer, which consisted of 50 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 0.5% NP-40, and 1× protease inhibitor cocktail. Following an incubation period of one hour at 4 °C, the cell lysates were centrifuged at 14,000 × g for 10 min at 4 °C. Both NEDD4L and CaMKKβ were immunoprecipitated from 500 mg of the digitonin-solubilized proteins using Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, CA, USA) according to the manufacturer’s guidelines. The immunocaptured proteins were then subjected to SDS‒PAGE/immunoblotting to analyze protein‒protein interactions.

Statistical analyses

SPSS 19.0 software was used for statistical analysis. All the values are expressed as the mean ± SEMs. The data were analyzed using one-way analysis of variance (ANOVA) or the least significant difference (LSD) test. A value of P < 0.05 was considered to indicate statistical significance.