Animal experiments and procedures

Proximal tubule-specific Pacs-2 knockout (PT-Pacs-2−/−) mice were generated by the Cre-LoxP recombination strategy as previously described (Li et al. 2022). Cre-negative littermate wild-type mice (Pacs-2fl/fl) were used as controls. PT-Pacs-2−/− mice and their control group Pacs-2fl/fl mice were fed with a high-fat diet (HFD) (45% fat, ResearchDiets, USA) for 1 month at 4 weeks of age, followed by intraperitoneal injection of 100 mg/kg streptozotocin (STZ) (Sigma-Aldrich, USA). After the injection, mice with random blood glucose levels > 12 mmol/l were included for the experiment. They were named diabetic PT-Pacs-2−/− mice and diabetic Pacs-2fl/fl mice, respectively. Then the mice were maintained for another 20 weeks of HFD feeding. Mice fed with a standard fat diet (SFD) were used as non-diabetic controls, called control Pacs-2fl/fl mice and control PT-Pacs-2−/− mice, respectively. The mice were randomly assigned to four groups. At the end of the last week, mice were sacrificed and anesthetized with sodium pentobarbital. Urine and kidney tissues were collected for various experiments. The sample size was estimated from the effect of the study. All the experiments were approved by the Medical Ethics Committee of Central South University and followed the NIH guidelines for the care and use of laboratory animals.

Assessment of physiological features and renal function

Body weight and blood glucose were measured every 2 weeks. Urine albumin was measured by a Mouse MAU (Microalbuminuria) ELISA Kit (Sangon Biotech, China) according to the specification of this kit. Urine creatinine levels were measured in the same samples using CicaLiquid-N CRE (Kanto Chemical Co Inc, Japan). Urine N-acetyl-β-d-glucosaminidase (NAG) levels were measured with a NAG assay kit (Jiancheng Bioengineering, China) following the manufacturer’s protocol.

Morphological analysis

Kidney tissues from mice were fixed with 4% paraformaldehyde (PFA) for 14–16 h, embedded in paraffin, sectioned, and stained with hematoxylin–eosin (HE), periodic acid Schiff (PAS), Masson staining, and observed under light microscopy. Tubular damage and glomerular damage were assessed using a semiquantitative scoring system, as described previously (Sun et al. 2011).

Transcriptome analysis

The transcriptome analysis was conducted by Seqhealth Technology Co., LTD (Wuhan, China). Total RNA from the renal cortex of control Pacs-2fl/fl mice and control PT-Pacs-2−/− mice was extracted using RNAiso Plus (TaKaRa, Japan) according to the manufacturer’s instructions. The RNA concentration was determined by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and the integrity was tested by 1.0% agarose gel electrophoresis. 5 μg total RNAs were used for mRNA sequencing library preparation. After the library quality inspection is qualified, ~ 300 bp products were sequenced in HiSeq X10 system (Illumina, San Diego, CA, USA). For the RNA-seq data analysis, the data was aligned to the mouse reference genome to obtain comprehensive transcript information and differentially expressed genes (DEGs) as described previously (Ma et al. 2021).

Immunohistochemistry (IHC)

Paraffin-embedded renal sections were deparaffined in xylene, rehydrated in ethanol and antigen repaired in citrate buffer. After blocking endogenous peroxidase activity with peroxidase blocking solution, the sections were exposed to 5% bovine serum albumin (BSA) and sequentially incubated with primary antibody, anti-Adipophilin (1:200, proteintech, 15294-1-AP, China) overnight at 4 ℃. After washing, the sections were incubated with Goat Anti-Rabbit secondary antibody (1:200, Servicebio, G1213, China), treated with diaminobenzidine, and counterstained with hematoxylin. Finally the sections were dehydrated, soaked in xylene, air-dried, and sealed. Ten images under brightfield were randomly taken for per section in a blinded fashion. Images were obtained using a Nikon microscope and analyzed with Image-Pro plus 6.0.

Immunofluorescence (IF)

Paraffin-embedded renal sections were blocked in 5% BSA followed by primary antibody, Anti-SOAT1 (1:200, abclonal, A6311, China), incubation overnight at 4 ℃. After washing with Phosphate-buffered saline (PBS), sections were incubated with Alexa Fluor-488-conjugated goat anti-rabbit antibody (1:1000, abcam, ab150077, USA) at 37 °C for 1 h. The nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI) (SouthernBiotech, China). Five images were randomly taken for each group in a blind fashion. Images were obtained using a Nikon microscope and analyzed with ImageJ software as described previously (Gao et al. 2020).

Oil Red O staining

Oil Red O staining was performed in a common manner as previously described (Pei et al. 2016; Yuan et al. 2017). Briefly, the mouse kidney tissues were fixed in 4% PFA for 24–48 h and was dehydrated in 30% sucrose solution overnight. Then the tissues were embedded in O.C.T. compound and cryocut cross-sections (5 mm) were prepared. The frozen sections were air-dried at room temperature and then stained with Oil Red O dye (Servicebio, China) according to the manufacturer’s instructions. HK-2 cells were fixed in 4% PFA for 30 min. After washing with PBS, HK-2 cells were stained with Oil Red O dye for 30 min and then dipped into 60% isopropanol three times. The deposition of lipid droplets in tissue or HK-2 cells was observed under a Nikon microscope. Images were obtained in a blinded manner and analyzed with ImageJ software.

Cell culture and treatments

The human proximal tubular cell line (HK-2 cells) was obtained from ATCC and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 supplemented with 10% FBS and 100 U/mL penicillin plus 0.1 mg/mL streptomycin. One day before transfection, cells were grown to ~ 70% confluence. Then HK-2 cells were transfected with siRNAs or plasmids by Lipofectamine 3000 (Invitrogen, USA) in accordance with the manufacturer’s instructions. pcDNA3 hPACS-2 Flag plasmid was a gift from Professor Gary Thomas (Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA). PACS-2 siRNA was purchased from RiboBio Co., Ltd (China). SOAT1 siRNA were purchased from Tsingke Biotechnology Co., Ltd (China). Scrambled siRNAs (Tsingke Biotechnology Co., Ltd, China) and empty vector pcDNA 3.1 vector were applied to parallel cultures as negative controls. Subsequently, cells were stimulated with high glucose plus palmitic acid (HGPA; final concentration 30 mmol/L glucose and 300 μmol/L of the saturated free fatty acid palmitate [16:0]) for 24 h then harvested (Wu et al. 2021a; Sun et al. 2021; Lin et al. 2022; Wu et al. 2021b). The control group used mannitol and fatty acid free BSA (Solarbio, China) as the isotonic solvent control group.

Bodipy staining

HK-2 cells were washed using PBS and stained with BODIPY493/503 (Invitrogen, USA) 1000-fold diluted for 30 min according to the manufacturer’s instructions. Cell nuclei were counterstained hoechst and images acquired by laser confocal microscopy.

Cholesterol and free fatty acid content determination

Total cholesterol (TC), free cholesterol (FC) and free fatty acid (FFA) content in HK-2 cells was determined using the Micro Total Cholestenone (TC) Content Assay Kit (Solarbio, China), Micro Free Cholestenone (FC) Content Assay Kit (Solarbio, China) and Free fatty acid detection kit (Solarbio, China) respectively, following the manufacturer’s protocol. Amount of cholesteryl ester (CE) were determined by subtracting the amount of free cholesterol from total cholesterol as described previously (Huang et al. 2018).

Real time qRT-PCR

The total RNA extracted from the renal cortex and cells using RNAiso Plus was reverse transcribed into cDNA using PrimeScriptTM Reagent Kit (TaKaRa, Japan) with Bio-Rad iCycler system (Bio-Rad, Hercules, CA). Real time qRT-PCR was performed using TB GreenTM Premix Ex Taq II reagent (TaKaRa, Japan) with 7300 Real-Time PCR System (Applied Biosystems). The sequences of the primers are listed in Additional file 2: Table S1. The data are presented as fold changes (2−ΔΔCt) normalized to β-actin.

Western blot analysis

Western blot analysis was performed as described previously (Xu et al. 2016). In brief, protein from mice renal cortex as well as cell pellets was extracted using RIPA buffer (CWBIO, China) containing protease and phosphatase inhibitor cocktail (CWBIO, China). BCA protein assay (Thermo Fisher Scientific, USA) was used for the quantification of protein concentrations. Equal amounts of proteins were used in the Western blot analysis. Primary antibodies specific for PACS2 (1:1000, proteintech, 19508-1-AP, China), FN (1:1000, abcam, ab2413, USA), α-SMA (1:3000, abcam, ab32575, USA), SOAT1 (1:1000, abclonal, A6311, China), SREBF1 (1:500, santa cruz, sc-13551, USA), SREBF2 (1:500, santa cruz, sc-13552, USA), Beta Actin (1:5000, proteintech, 60008-1-Ig, China) were used for western blot.

Statistical analysis

Data are expressed as mean ± SD. Statistical analyses were performed with GraphPad Prism (version 8.0). The values were analyzed by one-way ANOVA followed by post hoc Tukey’s test or two-way ANOVA followed by post hoc Tukey’s test. Significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001.