Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice (original) (raw)

Animal work. Diabetic db/db, mice and their control littermates db/m were obtained from The Jackson Laboratory (strain: BKS.Cg-Dock7m+/+Leprdb/J) and bred in-house. All animals were maintained on a normal chow diet with free access to water and housed in a room with a 12-hour light/12-hour dark cycle and an ambient temperature of 22°C. Type 1 diabetes was induced in mice as previously described (13)

Drp1S600A-knockin mice. We designed a Drp1S600A-knockin construct using a replacement targeting strategy that involved 3 cloning steps. First, a 3.5-kb genomic DNA encompassing exons 15–17 and the proximal intronic regions of the Drp1 gene was PCR amplified and subcloned directly into a pLNTK vector. This DNA fragment was sequencing verified and used as the 5′ homologous recombination arm. Second, a neomycin-resistant minigene cassette flanked by flippase recognition target (FRT) sequences was added to the 3′ end of the 5′ arm in the pLNTK backbone. Third, a 5.1-kb genomic fragment encompassing exons 18–19 and the proximal intronic regions was PCR amplified and cloned into a pGEM-T-Easy vector and then sequence verified. A T-to-G transversion in exon 18 (resulting in a corresponding S600A amino acid mutation) was introduced by site-directed mutagenesis (Takara Bio USA). The resulting DNA fragment was used as the 3′ arm and was cloned into the 3′ end of the Neo cassette to complete the knockin construct. The targeting construct was restriction digested, purified, and electroporated into C57BL/6N-Tac ES cells at the BCM Mouse Embryonic Stem Cell Core Facility (https://bcm.corefacilities.org/service_center/show_external/3152/mouse-embryonic-stem-cell-core). Successful gene targeting was determined by Southern blot analysis. Briefly, genomic DNA was digested with the restriction enzymes ScaI and XhoI (New England BioLabs). The WT allele generated a 12.7-kb ScaI DNA fragment, whereas the targeting construct introduced an Xhol I site. The introduced restriction site produced a 5.8-kb fragment after restriction enzyme digestion. A 3′ probe was generated by PCR for Southern blot analysis. Six targeted clones were then further verified by PCR and sequencing for the Drp1S600A mutation. Two of these clones were injected into blastocysts to achieve germline transmission. We generated 16 chimera pups from which targeted founders were established through Southern blot analysis using tail DNA.

Tissue culture. Conditionally immortalized mouse podocytes were a gift from Jochen Reiser (Rush University, Chicago, Illinois, USA). Briefly, cells were cultured at 33°C in RPMI (Corning, 10-041-CV) containing 10% FBS (FBS Opti-gold, GenDepot, F0900-050) and 20 U/ml mouse recombinant IFN-γ (MilliporeSigma, I4777) to enhance expression of a thermosensitive T antigen. The cells were differentiated at 37oC in DMEM (Corning, 10-014-CV) supplemented with 5% FBS without IFN-γ on 10-cm dishes coated with collagen type I (Gibco, Thermo Fisher Scientific, A10483-01) for 7 to 12 days. For imaging, the differentiated cells were trypsinized, dissociated, and plated onto collagen type I–coated coverslips. Podocytes prepared for experiments involving HG (25 mM) conditions were serum deprived for 24 hours prior to addition of HG. Control cells were cultured with 5 mM NG.

Southern blot analysis and PCR genotyping. Approximately 10 μg genomic DNA was subjected to restriction enzyme digestion by XhoI and ScaI (New England BioLabs). Digested DNA was separated by gel electrophoresis (0.9% agarose in 1× TAE buffer, 35 V, 8 h). Gels were depurinated, denatured, and neutralized before transfer onto nitrocellulose membranes. Membranes were UV cross-linked and prehybridized in hybridization buffer (PerfectHyb Plus, MilliporeSigma, H7033). PCR was performed to generate a biotinylated 3′ probe. The Southern probe was hybridized and then the membrane washed and subsequently incubated with HRP-labeled streptavidin (Vector Laboratories, SA-5004). Bands were revealed using chemiluminescence detection kits (Thermo Fisher Scientific). Mouse tail DNA was isolated using the REDExtract-N-Amp Tissue PCR Kit (MilliporeSigma Aldrich, XNAT) according to the manufacturer’s instructions. The primers used for PCR genotyping were Drp1 MF and Drp1 MR (CCATCTGCAGGTGGTGGGATTGGAG and GACATCACAAACTCATCCAACA GT). PCR was performed as described by the REDExtract-N-Amp protocol, and the products were resolved on a 2% agarose gel using the TAE buffer system.

Cartesian allelic discrimination plot. TaqMan genotyping was performed with preoptimized PCR primer pairs (SNP forward/reverse, CCCTCTTTTAGCCAGTTCCAGTTG/CAATAACCTCACAATCTCGCTGTTC) and 2 probes for allelic discrimination (FAM/VIC, AAAACTGGCCGCCCGA/CAAGAAAACTGTCTGCCCGA; Thermo Fisher Scientific, AHZAGZD). Each assay contained a pair of unlabeled primers and 3 TaqMan probes (1 with a FAM dye label and 1 with a VIC dye label). The relative levels of fluorescence from the labeled probes detecting either the mutant or WT sequence for each sample were plotted on the x and y axes, respectively.

Metabolic and physiological parameters. Mice were individually housed in metabolic cages and fasted overnight but were provided water ad libitum. Blood glucose levels were determined the next morning with a glucose meter (Walgreens True Metrix). Urine was collected and urinary albumin concentration measured with a mouse albumin ELISA kit (Exocell, 1011). Urinary creatinine was measured using the QuantiChrom Creatinine Assay Kit (Bioassay Systems, DICT500).

Western blot analysis. Total protein lysates were extracted using RIPA buffer (50 mM Tris-HCl, pH 8.0, with 150 mM NaCl, 1.0% igepal CA-630 [NP-40], 0.5% sodium deoxycholate, and 0.1% SDS). Protein concentration was determined by BCA Protein Assay (Pierce, Thermo Fisher Scientific, 23228). Total protein lysate (20 μg) was diluted in 5× Laemmli buffer, loaded on a 4%–20% gradient SDS-PAGE (Bio-Rad,) and transferred onto PVDF membranes (Roche, 03010-040-001). Membranes were probed with the indicated primary antibody and a fluorescent secondary antibody followed by visualization with a Odyssey Infrared Imaging System (LI-COR Biosciences). The primary antibodies used for Western blotting were: anti-Drp1 (BD, 611113; RRID: AB_398424); anti–FLAG tag (MilliporeSigma, F7425; RRID: AB_439687); anti-MFF1 (Cell Signaling Technology, 86668; RRID: AB_2734126); and anti-GFP (MilliporeSigma, AB3080; RRID: AB_2630379). The secondary antibodies used for Western blotting were: goat anti–mouse Dylight 680 (Thermo Fisher Scientific, 35519; RRID: AB_1965956); goat anti–mouse Dylight 800 (Thermo Fisher Scientific, SA510172; RRID: AB_2556752); goat anti–rabbit Dylight 680 (Thermo Fisher Scientific, 35519; RRID: AB_1965956); and goat anti–rabbit Dylight 800 (Thermo Fisher Scientific, SA535571; RRID: AB_2556775).

Cell and tissue staining. For cell culture, podocytes were washed with cold PBS, fixed in 4% formaldehyde, and permeabilized with 0.1% Triton X-100 (Acros, 21568-0010). Cells were blocked in 1% BSA (Jackson ImmunoResearch, 001-000-162), 50 mM Tris, pH 7.6, 155 mM sodium chloride (TBS), and 0.1% Triton X-100. The cells were incubated overnight at 4°C with the appropriate primary antibodies in blocking buffer. Coverslips were washed 3 times in TBS and incubated with the appropriate secondary antibodies in blocking buffer for 1 to 2 hours at room temperature (RT). When indicated, the secondary antibody solution had 1:100 rhodamine/phalloidin (Life Technologies, Thermo Fisher Scientific, R415; RRID: AB_2572408) added at the same time to label actin. Coverslips were washed 3 times in TBS and mounted onto slides. Images were obtained using a DeltaVision Elite deconvolution inverted microscope (GE Healthcare, see below). Quantification was performed using ImageJ software (NIH). For tissue sections, formalin-fixed, paraffin-embedded tissue sections were cut at approximately 5-μm thickness and mounted onto slides. The slides were deparaffinized and rehydrated through an ethanol series with a final wash in distilled water. Antigen retrieval was performed by incubating the slides in 20 mM Tris and 1 mM EDTA at 95°C for 1 hour. Sections were then washed twice with 1× TBS, and nonspecific binding was blocked in blocking buffer (see above) for 1 hour at an ambient temperature. After blocking, the sections were incubated overnight at 4°C with 1:100 dilutions of primary antibodies in blocking buffer. The sections were washed 3 times in TBS and incubated with secondary antibodies for 1 to 2 hours at RT in blocking buffer. The sections were washed 3 times with TBS, and nuclei were counterstained with DAPI (Thermo Fisher Scientific, 62248). Finished slides were mounted with ProLong Gold Antifade Mounting Reagent (Molecular Probes, P36934). The antibodies used for immunohistochemistry were: anti-Drp1 (BD, CA, 611113; RRID: AB_398424); anti–p–Drp1 (S637) (Biorbyt, Orb127984; RRID: AB_2734124); anti-Tomm20 (Genetex, GTX32928; RRID: AB_2734125); anti–cofilin 1 (Abcam, ab131519; RRID: AB_11155720); anti-Arp3 (C-term) (ECM Biosciences, AP4581; RRID: AB_2734127); anti-SDHA (Abcam, ab14715; RRID:AB_301433); donkey anti–rabbit Alexa Fluor 488 (Thermo Fisher Scientific, A21206; RRID: AB_2535792); donkey anti–rabbit Alexa Fluor 594 (Thermo Fisher Scientific, A21207; RRID: AB_141637); donkey anti–rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A21244; RRID: AB_141663); donkey anti–mouse Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, A21202; RRID: AB_141607); and donkey anti–mouse Alexa Fluor 594 (Thermo Fisher Scientific, A21203; RRID: AB_141633). The sections were examined using a Nikon A1Si inverted confocal microscope. The images were quantified using ImageJ software, version 2.00. Postanalyses of colocalization were performed using ImageJ coloc2.

Mesangial expansion and WT1 staining. Formalin-fixed, paraffin-embedded tissue sections were rehydrated as described above. The sections were subjected to PAS staining to evaluate the general histologic changes in glomerular and tubular structures. Mesangial fraction/volumes from PAS-stained images were estimated using ImageJ. The glomerular tuft was outlined in the images, simultaneously providing the cross-sectional area of the tuft. ImageJ thresholding was used to highlight the regions of similar intensity of red staining within the region of interest (glomerulus). The fractional area of the glomerulus with positive staining was then calculated. WT1 quantification was achieved using fluorescence immunohistochemistry. Tissue sections were subjected to WT1 staining (Abcam, ab89901, RRID: AB_1965956) and DAPI counterstaining. The acquired images were analyzed using ImageJ software. The images were viewed and the glomerular region of interest outlined to obtain a cross-sectional area. The number of WT1-positive nuclei and the total number of nuclei within the region of interest were determined. The results are presented as either WT1-positive nuclei per mm2 or the percentage of total nuclei observed. Five mice were analyzed per group, and fifty glomeruli were measured per mouse.

Confocal and deconvolution microscopy. Confocal image collection was performed on a Nikon A1Si inverted confocal microscope equipped with 4 solid-state lasers under acousto-optic tunable filter (AOTF) control. The objective lenses used were either a Nikon CFI60 Plan Fluor ×40 oil (NA 1.3) or a Nikon CFI Apo TIRF ×60 oil. Multicolor channel fluorescence images were captured sequentially using 405-, 488-, 561-, and 640-nm excitation wavelengths and the following emission filters (range limits): 425–475 nm, 500–550 nm, 570–620 nm, and 663–738 nm, respectively. Wide-field and deconvolution images were captured using a DeltaVision Elite deconvolution inverted microscope (GE Healthcare), equipped with a PCO sCMOS camera. The lenses used were a U Plan FLN ×40 (1.3 NA oil) and a Plan Apo N ×60 (1.42 NA oil). Fluorescence images were captured using quadruple polychroic mirrors (DAPI/FITC/TRITC/Cy5 or DAPI/FITC/Alexa 594/Cy5) and the corresponding sets of excitation and emission filter sets mounted in filter wheels. The specifications for each filter set were: DAPI 390/18 nm and 435/48 nm; FITC 475/28 nm and 525/48 nm; TRITC 542/27 nm and 597/45 nm; mCherry 575/25 nm and 625/45 nm; and Cy5 632/22 nm and 679/34 nm (excitation and emission respectively, expressed as peak/bandwidth).

IP for MS. Cultured podocytes were cross-linked (XL) by adding 37.5% formaldehyde to culture medium with a final concentration of 0.5%, followed by incubation at 37°C for 8 minutes. The cross-linking was quenched by adding glycine to a final concentration of 0.2 M. The cells were collected and washed twice with ice-cold PBS. The cells were lysed in 3 packed cell volumes of NETN buffer (25 mM Tris/pH7.5, 170 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1× phosphatase inhibitor, and 1× protease inhibitor cocktail) by sonication (SONICS Ultrasonic Processors VCX; 30 s on, 1 min rest, 25% amp, 6 cycles). After centrifugation for 20 minutes at 100,000 g, the supernatant was collected as whole-cell lysate (WCL) for IP experiments. For each IP experiment, 12 mg WCL was incubated for 30 minutes at 4°C with 30 μl Protein A–Dynabeads slurry (MilliporeSigma, catalog GE17-0780-01) for preclearance. The resulting supernatant was removed and cleared by ultracentrifugation (44,000 g for 15 min). The supernatants were then incubated with 30 μl M2 antibody–conjugated beads (MilliporeSigma, catalog F1804; RRID: AB_262044) for 1 hour at 4°C. The bead-bound complexes were washed 3 times with NETN buffer (100 mM NaCl, 20 mM Tris, pH 8.0, 0.5 mM EDTA, 0.5% NP-40). The washed beads were boiled with 1× NuPAGE LDS sample buffer with 50 mM 2-mercaptoenthanol and subjected to 4% to 20% Tris-glycine SDS-PAGE (Novex Gel, Invitrogen, Thermo Fisher Scientific). The gels were stained with Coomassie brilliant blue, and visualized proteins bands were excised into 4 gel pieces according to molecular size.

IP validations. Stable podocytes were generated by subcloning FLAG-tagged Drp1S600A or Drp1S600D into the TRE-responsive vector. The podocytes were then transiently transfected with native MFF1, CFL1-EGFP (gift from James Bamburg, Addgene plasmid no. 50859 [ref. 50]), or Arp3-EGFP (gift from Matthew Welch, Addgene plasmid no. 8462 [ref. 51]) mammalian expression vectors. Drp1 and MFF expression was induced with doxycycline (200 nM) for 72 hours. To study the interaction of Drp1 with MFF1 and CFL1, each group of cells (vector control, S600A, and S600D) were seeded onto 10 collagen I–coated tissue culture dishes (100-mm) at 1 × 105 cells/dish in NC DMEM (5% FBS, 5 mM glucose-DMEM) and differentiated for 7 days at 37°C. Cells were then serum-starved overnight before treatment with HG DMEM (5% FBS, 25 mM glucose) for 72 hours. Cells were washed, scraped into PBS, and cross-linked with 0.5% formaldehyde and PBS for 10 minutes at RT. Glycine was added to 0.2 M to quench the cross-link. Cells were washed 3 times with PBS and lysed by sonication in a 3× packed cell volume of lysis buffer NETN (50 mM Tris, pH 7.3, 170 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with protease inhibitor and phosphatase inhibitor cocktails. Lysates were precleared with Protein A/G PLUS Beads (Santa Cruz Biotechnology) at 4°C for 30 minutes before IP with FLAG-M2 beads (anti-FLAG M2 Affinity Gel, MilliporeSigma, catalog A2220; RRID: AB_10063035) or anti-MFF1 antibody (Cell Signaling Technology, catalog 86668; RRID: AB_2734126) and protein A/G PLUS beads (Santa Cruz Biotechnology). Beads were washed with lysis buffer 5 times and eluted with 0.1 mg/ml 3× FLAG peptide (for FLAG-M2 beads, MilliporeSigma F4799). Samples had cross-links undone by subjection to 65°C for 1 hour in Laemmli buffer (for protein A/G beads). Proteins were subjected to 4% to 20% SDS-PAGE (Bio-Rad) and immunoblotted with antibodies against FLAG (anti–FLAG tag, MilliporeSigma, catalog F7425; RRID: AB_439687); GFP (anti-GFP, MilliporeSigma, catalog AB3080; RRID: AB_2630379); MFF1; and cofilin 1 (anti–cofilin 1, Cytoskeleton, catalog ACFL02-A; RRID: AB_10708808). To study the interaction of Drp1 with Arp3, cells were lysed in M-PER buffer (Thermo Fisher Scientific, catalog 78501), and IP and Western blotting were carried out as above.

GST-pulldown assay. A GST-pulldown assay was carried out as previously described (52). Briefly, GST-tagged Drp1 isoforms (WT, S600A, and S600D) or GST vector control (pGEX-2T, GE Healthcare) were transformed into BL21 (DE3) (New England BioLabs) and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (MilliporeSigma, catalog I6758) at 25°C overnight (18 h). Immobilized GST proteins were purified according to the manufacturer’s protocol. GST proteins (2 μg) were incubated with 5 μg purified Arp2/3 complex (Cytoskeleton Inc., catalog RP01P-A) in 1 ml GST-binding buffer (20 mM Tris-HCl at pH 7.5, 25 mM KCl, 1 mM MgCl2, 50 mM NaCl) at 4°C for 2 hours. Beads were washed 5 times with binding buffer, boiled in SDS sample buffer, and subjected to 4% to 15% PAGE (Bio-Rad, 4561086). Proteins were transferred to a solid support and blotted with Arp3 mAb (1:200, MilliporeSigma; RRID: AB_476749) and Arp2 polyclonal Ab (1:100, ECM Biosciences, RRID: AB_1944438), respectively.

Vectors and recombinant DNA. Genomic integration of tetracycline-inducible transgenic genes was accomplished using the piggybac transposon system. Cells were transfected with the nonintegratable PiggyBac transposase pCMV-PB to mediate translocation of the transposable vectors into the genomic DNA without extended expression of the transposase. Integration involved 2 vectors in each case. The transposase was transfected along with a transposable Tet-on and transposable transgenic vectors. The transposable vectors had independent selection antibiotics. Vector backbone has been described previously (26). This vector had the TRE-tight promoter. For selection purposes, the vectors contained either puromycin or G418 behind the SV40 promoter. Cells were then selected with G418 and puromycin.

Mitochondrial measurements. Images were acquired by deconvolution microscopy. ImageJ was used to analyze the resulting images. AR measurements were performed by assigning each mitochondria into an ellipse. The mitochondrial AR was determined as the major and minor axes of the ellipse expressed as a fraction. Length was the major axis. The form factor was defined as (Pm2)/(4pAm), where Pm is the length of the mitochondrial outline, and Am is the area of the mitochondrion. Mitochondrial area was measured as the 2D area (nm2) projected to the focal plane of the image. Circularity was 4π(Am/Pm2). These measurements were made using ImageJ and previously published methods. The morphology of at least 120 mitochondria was determined for each condition.

SEM. Fixed samples containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, were washed with 0.1 M cacodylate buffer, pH 7.3, post-fixed with 1% cacodylate-buffered osmium tetroxide, and washed with 0.1 M cacodylate buffer and then in distilled water. Afterwards, the samples were sequentially treated with Millipore-filtered 1% aqueous tannic acid, washed in distilled water, treated with Millipore-filtered 1% aqueous uranyl acetate, and then rinsed thoroughly with distilled water. The samples were dehydrated with a graded series of increasing concentrations of ethanol and then transferred to a graded series of increasing concentrations of hexamethyldisilazane (HMDS) and air-dried overnight. The samples were mounted onto double-stick carbon tabs (Ted Pella) that had been previously mounted onto glass microscope slides. Next, the samples were coated under vacuum using a Balzer MED 010 evaporator (Technotrade International) with platinum alloy for a thickness of 25 nm and then immediately flash carbon-coated under vacuum. The samples were transferred to a desiccator for examination at a later date. The samples were examined and imaged using a JEOL JSM-5910 scanning electron microscope at an accelerating voltage of 5 kV.

TEM. The samples were fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, and then washed in 0.1 M sodium cacodylate buffer and treated with 0.1% Millipore-filtered cacodylate-buffered tannic acid, post-fixed with 1% buffered osmium, and stained en bloc with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in LX-112 medium. The samples were polymerized in a 60°C oven for approximately 3 days. Ultrathin sections were cut in a Leica Ultracut microtome (Leica), stained with uranyl acetate and lead citrate in a Leica EM Stainer, and examined in a JEOL JEM 1010 transmission electron microscope at an accelerating voltage of 80 kV. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques).

MS. The gel pieces were subject to in-gel digestion using trypsin (GenDepot, T9600). The tryptic peptide was dried under vacuum and then resuspended in 10 μl loading solution (5% methanol containing 0.1% formic acid). One-half of the suspended peptide was injected using the Nano-HPLC 1000 system (Thermo Fisher Scientific) coupled to the Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). The peptides (2 μm) were loaded onto an in-house Reprosil-Pur Basic C18 trap column (Dr. Maisch GmbH), which was 2 cm × 100 μm in size. Then, the trap column was washed with loading solution and switched in-line with an in-house 5-cm × 150-μm column packed with 2 μm Reprosil-Pur Basic C18 beads equilibrated in 0.1% formic acid in water. The peptides were separated with a 75-minute discontinuous gradient of 2% to 24 % acetonitrile and 0.1% formic acid at a flow rate of 800 nl/min. Separated peptides were directly electrosprayed into the mass spectrometer. The instrument was operated in data-dependent mode, with acquisition of fragmentation spectra of the top 35 strongest ions and under the direct control of Xcalibur software (Thermo Fisher Scientific). The parent MS spectrum was acquired in the Orbitrap with the full MS range of 375 to 1300 m/z at a resolution of 140,000. The CID fragmented tandem MS (MS/MS) spectrum was acquired in the ion trap in rapid-scan mode. The MS/MS spectra obtained were searched against the target-decoy Human RefSeq database (released June 2015, containing 73,637 entries; https://www.ncbi.nlm.nih.gov/refseq/) in the Proteome Discoverer 2.1 interface (Thermo Fisher Scientific) with the Mascot algorithm (Mascot 2.4, Matrix Science). Variable modification of oxidation of methionine and protein N-terminal acetylation was allowed. The precursor mass tolerance was confined within 20 ppm, allowing a fragment mass tolerance of 0.02 Dalton and a maximum of 2 missed cleavages. The assigned peptides were filtered with a 1% FDR. The iBAQ algorithm was used to calculate protein abundance to compare relative amounts between different proteins in the sample using an in-house data-processing algorithm (53). Simply, iBAQ was calculated on the basis of normalization of the summed peptide intensity divided by the number of the theoretically observable tryptic peptide of certain proteins.

Latrunculin A and shRNA experiments. Podocytes were differentiated on collagen-I–coated coverslips. Cells were treated with 50 nM latrunculin A for the duration of the 48-hour HG (25 mM) treatment in 5% FBS and DMEM in normal cells or for 48 hours in 5% FBS and DMEM with NG (5 mM) and 50 nM doxycycline in the case of Drp1S600D-expressing cells. The cells were transduced with lentiviral shRNAs 1 day prior to the start of the glucose treatments. GIPZ mouse CFL1 shRNA (Dharmacon, V3LMM_521443 and V3LMM_449309), GIPZ mouse ACTR3 shRNA (Dharmacon, V3LMM_34878 and V3LMM_444971), and GIPZ nonsilencing control (Dharmacon, RHS4346) were used to prepare lentiviruses at the Baylor College of Medicine Gene Vector Core.

In silico analyses. Functional protein interaction analysis was performed using STRING (https://string-db.org/). For KEGG pathway analysis, the KEGG Automatic Annotation Server–KEGG (KAAS-KEGG) (http://www.genome.jp/tools /kaas/) was used, and for gene enrichment analysis, GSEA (http://software.broadinstitute.org/gsea/index.jsp) was used.

IVM for live animal imaging. We monitored the presence of mitochondrial ROS in the kidney in real time in vivo by IVM imaging using diabetic mice that expressed a redox-sensitive GFP biosensor. IVM imaging of the kidney was performed by the Intravital Microscopy Core at the Houston Methodist Research Institute. Kidney imaging was performed by exposing the kidney with a lateral incision. Image acquisition was performed over selected fields of view, with a resolution of 512 × 512 pixels and an optical slice thickness of 7.1 μm. The IVM system uses an upright Nikon A1R laser scanning confocal microscope with a resonance scanner, motorized and heated stage, and Nikon long-working distance 4× and 20× dry plan apochromat objectives. Images were obtained with a 2-channel setup in which fluorescence was collected at 525 nm, exciting sequentially with a 405-nm and 488-nm laser at a rate of 5 fps. Images were analyzed and exported using Nikon Elements. To measure the mitochondrial ROS, the intensity ratio of the areas covered by the biosensor was quantified with Nikon Elements.

Human archival kidney biopsy. Human kidney biopsy samples were obtained from archival biopsy specimens collected at the Houston Methodist Hospital. Histological sections of formalin-fixed, paraffin-embedded biopsy samples were subjected to immunofluorescence staining with antibodies against synaptopodin (GeneTex, GTX39067, RRID: AB_11161967); p-Drp1 (S637) (Biorbyt, orb127984); Alexa Fluor 647 goat anti-rabbit (Invitrogen, Thermo Fisher Scientific, A21244); and Alexa Fluor 488 donkey anti-mouse (Invitrogen, Thermo Fisher Scientific, A21202).

Statistics. All group data are expressed as the mean ± standard error of the mean. Comparisons of multiple groups were performed using 1-way ANOVA followed by Tukey’s multiple comparisons test. Comparisons between 2 groups were performed using a 2-tailed Student’s t test, with a P value of less than 0.05 considered statistically significant. Statistical analyses were performed using GraphPad Prism 6.0b (GraphPad Software).

Study approval. All animal studies were approved and conducted according to the Principles of Laboratory Animal Care (NIH publication no. 85023, revised 1985) and the guidelines of the IACUC at the University of Texas MD Anderson Cancer Center. Animal IVM studies were performed in accordance with the guidelines of the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011) and based on approved protocols by the IACUCs of Baylor College of Medicine and Houston Methodist Research Institute. Human kidney biopsy samples were obtained from archival biopsy specimens collected at the Houston Methodist Hospital under a protocol approved by the hospital’s IRB. Written informed consent was obtained from all patients prior to their inclusion in the study.