Ethics statement of patient urine samples
The Ethics Committee of Southwest Hospital, Third Military Medical University (Army Medical University) approved the present study (approval number: KY2020085). Prior to be included in the study, all participants signed relevant informed consent. Participants were identified using a number, not their name. It was conducted in adherence with the Declaration of Helsinki. All methods were carried out in accordance with the institutional guidelines of the ethics committee at Southwest Hospital, Third Military Medical University (Army Medical University) (Chongqing, China).
Urine sample collection
Eight urine samples of patients with clinically diagnosed diabetes were collected from the inpatients who signed a consent form. These inpatients received formal treatment at Endocrinology Department, Southwest Hospital, Third Military Medical University (Army Medical University). The samples used were the urine left over from routine urine collections, which were midstream samples. All samples were assigned a study number that connected them to clinical information from the chart to deidentify them. The samples were used for ELISA analysis.
Ethics statement of animal study
All animal experiments were implemented in accordance with the Guide for the Care and Use of Laboratory Animals announced by the US National Institutes of Health (NIH Publication, 8th Edition, 2011) and were granted permission by the Animal Experiment Ethics Committee of the Army Medical University (the Third Military Medical University).
The knock-in mice that mimicked MAP4 hyperphosphorylation in specific-sites (S737 and S760, MAP4 KI mice) were performed as previously described (Li et al., 2018). Wild-type (WT) littermates were used as controls for experiments with corresponded MAP4 KI male or female mice. The age of mice ranged from 10-74 weeks.The C57BL/6J or MAP4 KI mice (10 to 74 weeks) applied for STZ (Cat# S0130, Sigma, Darmstadt, Germany)-induced diabetes were starved for 8-12h and then injected with a single intraperitoneal dose of STZ in sodium citrate buffer at 150 mg/kg body weight. Mice with a glucose concentration exceeding 16.7 mmol/L were considered diabetic. Mice were anesthetized with pentobarbital sodium (1%, 50 mg/kg) by intraperitoneal injection and sacrificed 5 weeks after STZ injection. Experiments involving animals were performed in accordance with United Kingdom Home Office and European Union guidelines and were approved by the Animal Care Centre of the Third Military Medical University.
Biochemical tests of urine and blood samples
Mouse random urine was collected and centrifuged, and blood samples were collected by retro-orbital puncture from anesthetized animals after fasting 8-12h. Urine albumin, urine creatinine (CR), serum levels of urea nitrogen (UN), CR and cystatin c (Cys-c) were determined using commercial kits.
Histology and ultrastructural pathology
Renal tissue was cut and fixed with 10% formalin, embedded in paraffin or made in frozen, and sectioned at 5-μm thickness for routine histopathology or Masson’s trichrome staining. Six serial sections from each mouse were used to measure the glomerular volume: the volume of the glomerular was calculated as previously described (Najafian et al., 2002, Chen et al., 2017). For transmission electron microscope (TEM) observation, tissues of renal cortical were fixed in 2.5% glutaraldehyde followed by dehydration, vibratome sliced and recut on a microtome and stained with uranyl acetate and lead citrate overnight. The degree of foot process fusion was determined by measuring the number of junctions per 1 micron length of glomerular basement membrane (GBM) as described previously (Asanuma et al., 2005).
Podocyte culture and treatment
Conditioned immortalized murine podocytes were obtained and cultured as our study described previously (Shankland et al., 2007, Chen et al., 2017). Cells were used for experiments as stated. After transfection of adenovirus overexpressing MAP4(Ala), MAP4(Glu), MKK6(Glu), or CMV-null for 36h, the cells were treated with 5.6 mM D-glucose, 5.6 mM D-glucose + 25 mM mannitol or 30 mM D-glucose for 48h. During the stimulation, SB203580 (Calbiochem Cat# 559389, 10 μM, Darmstadt, Germany) was applied to pretreat cells for 1h prior to glucose treatment.
Site-directed mutagenesis of MAP4 and MKK6, construction and transduction of recombinant adenoviruses overexpression MAP4(Glu), MAP4(Ala) and MKK6(Glu) were performed as described previous (Hu et al., 2014, Li et al., 2015, Li et al., 2018). MAP4(Ala) mimicked MAP4 (S737 and S760) dephosphorylation, MAP4(Glu) mimicked MAP4 (S737 and S760) phosphorylation and MKK6(Glu) was an upstream kinase of p38/MAPK, which was consistently activated p38/MAPK.
Podocytes or frozen sections were used as described previously in our reports (Li et al., 2018). Antibodies: α-tubulin (Proteintech Group Cat# 11224-1-AP, RRID:AB_2210206, Rosemont, IL, USA), Phalloidin (Thermo Fisher Scientific Cat# A12379, RRID:AB_2315147, Waltham, MA, USA), Wilms Tumor protein (WT-1) (Abcam Cat# ab89901, RRID:AB_2043201, Cambridge, Cambridgeshire, United Kingdom), fibroblast-specific protein 1 (FSP1) (Abcam Cat# ab197896, RRID:AB_2728774), Nephrin (R and D Systems Cat# AF3159, RRID:AB_2155023, Minneapolis, MN, USA), Desmin (R and D Systems Cat# AF3844, RRID:AB_2092419).
In Situ Cell Death Detection Kit, fluorescein (Cat# 11684795910, Roche, Basel, Switzerland) were used for apoptosis assay of frozen kidney sections (WT-1 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) co-staining) or podocytes.
Cell proliferation assay
Cell proliferation determined by 5-ethynyl-2'-deoxyuridine (EdU) assay was carried out using the Click-iT® EdU imaging detecting kit according to the manufacturer's instructions (Cat# BCK488-IV-IM-S, Sigma).
Western blot (WB) analysis
Renal tissue samples and podocytes were used for WB experiments following our previously described procedures (Li et al., 2018). Antibodies: WT-1 (Abcam Cat# ab89901, RRID:AB_2043201), FSP1 (Abcam Cat# ab197896, RRID:AB_2728774), Nephrin (R and D Systems Cat# AF3159, RRID:AB_2155023), Desmin (R and D Systems Cat# AF3844, RRID:AB_2092419), GAPDH (Proteintech Group Cat# 60004-1-Ig, RRID:AB_2107436), MAP4 (Bethyl Cat# A301-489A, RRID:AB_999616, Montgomery, Texas, USA), p38 MAPK (P38) (Cell Signaling Technology Cat# 9212, RRID:AB_330713, Danvers, MA, USA), Phospho-p38 MAPK (p-P38) (Cell Signaling Technology Cat# 9211, RRID:AB_331641). Rabbit polyclonal antibodies against p-MAP4 (S787) and p-MAP4 (S737) were made in-house and validated as reported previously (Li et al., 2018).
Enzyme-Linked Immunosorbent Assay (ELISA)
Specific ELISA kits were purchased from the Mlbio Group to detect the concentrations of MAP4 and p-MAP4 in urine samples. Detection procedures were conducted according to the manufacturer’s instructions.
Measurement of transepithelial electric resistance (TER)
Cellular barrier properties were assessed using assessment of TER across confluent podocytes with Millicell-ERS (MERS00002, Millipore, Darmstadt, Germany) (Li et al., 2015). A 24-well transwell system (Greiner Bio-One, 0.4-mm pore size, 6.5-mm diameter, transparent, Costar) was inserted into the 24-well plate. Podocytes were plated in transwell chambers. The volumes of culture medium were 100 and 600 μl in the upper and lower compartments, respectively, in which cells were allowed to grow for 2 days (considered day 0). The resistance (R sample) was measured, the value for the blank well represented the blank resistance (R blank). The following calculation was performed: epithelial monolayer resistance = (R sample-R blank) * effective membrane area (the area of a 24-well transwell chamber is 0.336 cm2).
Measurement of the permeability to fluorescein isothiocyanate (FITC)-dextran Cells were grown on transwell compartments as above described. The permeability was measured by the addition of FITC-dextran (40 kDa, Sigma-Aldrich) for 1h (Li et al., 2015).
Retinal angiography imaging
Angiography imaging was obtained with spectralis HRA + OCT (Heidelberg Engineering, Heidelberg, Germany). In brief, pupils were dilated using topical tropicamide phenylephrine eye drop (Mydrin-P, Santen. Oy, Japan). Mice were anesthetized as described above. Fluorescein sodium salt (5ml:0.5g, Alcon Laboratories, Freiburg, Germany) was injected intraperitoneally as fluorescent tracer. Imaging was obtained at 488-nm absorption and 495-nm emission using a 55° lens. Images of the central retina were taken, with the optic nerve located in the center of the image.
All values are means ± SEM (for bar graphs) or SD (for univariate scatter plots). Statistical differences between groups were assessed using two-tailed Student's t test or one-way analysis of variance (ANOVA) post hoc tests, as appropriate. For all studies, values of P < 0.05 were considered statistically significant.