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. 2025 Nov 25;122(47):e2504565122.
doi: 10.1073/pnas.2504565122. Epub 2025 Nov 17.

Rac1 promotes proximal tubule kidney repair by coupling the actin cytoskeleton to mitochondrial function

Olga M Viquez #  1 , Meiling Melzer #  1 , Shensen Li #  1 , Matthew Tantengco #  1 , Xinyu Dong  1 , Eric Sha  1 , Jeffery Huang  1   2 , Evan S Krystofiak  3   4 , Rachel C Hart  3   4 , Wentian Luo  1 , Christian Warren  5 , Al-Borhan Bayazid  1 , Richard Zhang  1 , Ryoichi Bessho  1 , Volker H Haase  1   5   6 , Cord Brakebusch  7 , Takanari Inoue  8 , Craig Brooks  1   3 , Matthew H Wilson  1   5   9 , Andrew S Terker  1 , Juan P Arroyo  1   5 , Ambra Pozzi  1   5   6 , Roy Zent #  1   3   5 , Fabian Bock #  1   3   5
Affiliations

Rac1 promotes proximal tubule kidney repair by coupling the actin cytoskeleton to mitochondrial function

Olga M Viquez et al. Proc Natl Acad Sci U S A. .

Abstract

The kidney proximal tubule (PT) is a specialized polarized epithelium that functions as a high capacity resorptive machine. PT cells are exquisitely sensitive to ischemia due to their high metabolic rate. The small GTPase Rac1 regulates epithelial function by promoting polarity through its effects on the actin cytoskeleton. We show that Rac1, in the setting of the recovery of the PT from ischemic injury, plays a critical role in reconstituting cellular bioenergetics by promoting actin cytoskeleton formation around damaged mitochondria. This mechanism removes damaged mitochondria through mitophagy and preserves PT metabolic capacity and reabsorption function. Loss of Rac1 causes intracellular lipid accumulation, energy depletion, and PT cell atrophy. Thus, Rac1 promotes the repair of PT cells by enhancing mitochondrial bioenergetics, rather than by regulating cell polarity via a mechanism that links the actin cytoskeleton to metabolic demands and cell morphology.

Keywords: actin cytoskeleton; kidney repair; mitochondria.

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Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

Figure 1.
Figure 1.. Loss of Rac1 results in tubular atrophy late after AKI.
(A) Representative macroscopic appearance of whole kidneys of mice 3 months after AKI. Scale bar, 0.635 cm. (B) Quantification of kidney size (as longitudinal length in cm) with n=4 mice/group. (C) Periodic Acid-Schiff (PAS)-stained paraffin kidney sections (top two rows, Scale bars: 20 μm) followed by megalin-stained kidney cortex sections (middle rows, Scale bars: 100 μm) and sirius red staining (bottom two rows, Scale bars: 200 μm) of control (Rac1f/f) and mutant (γGT:Rac1f/f) mice at baseline without injury and 3 months after AKI. Areas for insets are indicated by a black dashed box. (D) Quantification of atrophic tubules as total number per cortical high-power field (HPF). (E) Quantification of megalin positive (+) tubules as total number per cortical HPF. (F) Quantification of fibrosis (as percentage of sirius red–positive area per HPF). Data is representative of n=5 mice/group, all at baseline and 3 months after AKI. (G) Electron micrograph (TEM) of mice 3 months after AKI showing cellular structure of cortical tubular cells. BB: brush border, BM: basement membrane, vac: vacuole, M: mitochondria, N: nucleus. Scale bars: 2 μm. n=3 mice/group. Means are ± SD. * p< 0.05. ns, not significant.
Figure 2.
Figure 2.. Rac1 promotes proximal tubular cell structure after AKI.
(A) Representative Periodic Acid-Schiff (PAS)-stained paraffin kidney sections (top two panels, Scale bars: 20 μm) followed by megalin-stained kidney cortex sections (middle panels, Scale bars: 100 μm) and sirius red staining (bottom two panels, Scale bars: 200 μm) of control (Rac1f/f) and mutant (γGT:Rac1f/f) mice at baseline without injury and 1 month after AKI. (B) Quantification of atrophic tubules as total number per cortical high-power field (HPF). (C) Quantification of megalin positive (+) tubules as total number per cortical HPF. (D) Quantification of fibrosis (as percentage of Sirius Red–positive area per HPF). n=5 mice/group in B-D. (E) Representative DIC images overlaid with LTL (magenta) and DAPI (blue) of cortical tubules of control and mutant mice at baseline and 1 month after AKI. Areas for insets are indicated by a yellow dashed box. The bottom row shows schematics of control and mutant cell morphologies after AKI depicting the disruption of the basolateral surface pattern in the mutants. Scale bars: 10 μm. n=3–4 mice/group. (F) Quantification of basolateral tubular folds as % tubular area. (G) Representative electron micrographs (TEM) of cortical tubules showing basolateral cell structure. The brush border (BB) and basement membrane (BM) are annotated in red. Areas for insets are indicated by a blue dashed box. (H) Scale bars: 2 μm. n=3–4 mice/group. The last column shows schematics of the morphological changes occurring in mutant PTs with the loss of basolateral folds. Means are ± SD. * p< 0.05; ns, not significant.
Figure 3.
Figure 3.. Rac1 promotes the recovery of PT function and mitochondrial bioenergetics.
(A) Graphs showing urine glucose, phosphate, amino acids, and retinol-binding protein 4 (RBP4), all normalized to urine creatinine. Urine was collected at baseline (bl) without injury, early after AKI (which is day 1 after delayed nephrectomy), and 1 month after AKI in control and mutant mice. *control vs mutant (AKI 1mo). (B) RBP4 immunostaining (brown) of cortical proximal tubules in control and mutant kidneys at baseline and 1 month after AKI. Scale bars: 10 μm. (C) Quantification of PTs with apical RBP4 as number per HPF. (D) Measurement of Na-K ATPase activity (expressed as ouabain-sensitive release of phosphate) of kidney biopsy samples from control and mutant mice 1 month after AKI with ex vivo treatments as indicated (+CCCP or ATP). (E) ATP levels in fresh cortex tissue preparations of control and mutant mice 1 month after AKI. (F) Oil-Red-O (ORO, black) fluorescence overlaid with LTL (magenta, to mark PTs) and DAPI (blue), Scale bars: 10 μm. (G) Electron micrographs (TEM) with lipid droplets indicated by red arrows. Scale bars: 2 μm. BM, basement membrane; BB, brush border. Both panels (F and G) display control and mutant mice 1 month after AKI. (H) Quantification of ORO positive cells per tubule with dots representing averages per mouse. (I) Flow cytometry density dot plots of mitotracker-stained (Deep Red for mass against CM-H2TMRos-Orange for function) kidneys 1 month after AKI with the dysfunctional mitochondria gate shown. (J) Quantification of the gate shown in I as parent percentage. (K) Ex vivo tissue seahorse measurement of oxygen consumption rates (OCR) over time of cortical kidney biopsies of control and mutant mice at baseline and 1 month after AKI with averages quantified in the right panel. (L) Seahorse energy profile with OCR plotted against ECAR (extracellular acidification rate) of control and mutant kidneys 1 month after AKI. (M) Lactate levels in cortex tissue preparations of control and mutant mice 1 month after AKI. n=3–5 mice/group for all panel. Means are ± SD. * p< 0.05; ns, not significant.
Figure 4.
Figure 4.. Rac1 promotes mitochondrial organization, structure and F-actin formation after injury.
(A) Super-resolution confocal images (Airy Scan) of Cox4 (cyan) labeled mitochondria in cortical proximal tubules (LTL, magenta). Scale bars: 10 μm. Areas for insets (middle row) are indicated by a white dashed box. The bottom row is a binarized and skeletonized version of the mitochondria in the middle panel. (B) Quantification of mitochondrial aspect ratio, network branches, and branch length with each dot representing one mouse sample with n= 3/group. (C) Electron micrographs (TEM) of proximal tubular mitochondria with the area for insets (top panels) indicated by a black dashed box in the bottom panels. BB, brush border. BM, basement membrane. Scale bars: 1 μm. (D) Super-resolution confocal images of Phalloidin (F-actin, green) and Cox4-labeled mitochondria (magenta) in the proximal tubule. Area for insets (top panel) indicated by a white dashed box in the bottom panels. Scale bars: 5 μm. (E) Quantification of peri-mitochondrial F-actin intensity (AU, arbitrary units). Images and quantifications are representative of n=3–5 mice/group. A-E: all panels show control (Rac1f/f) and mutant (γGT:Rac1f/f) mice at baseline without injury and 1 month after AKI. Means are ± SD. * p< 0.05; ns, not significant.
Figure 5.
Figure 5.. Rac1KO PT cells in vitro are unable to undergo mitochondrial repair after injury.
(A) Experimental schematic of the in vitro mitochondrial repair model. Cells were treated with CCCP to depolarize and damage mitochondria. CCCP was then washed out to stimulate repair. (B) PKmito-labeled (in black) control and Rac1KO TKPTs cells are shown at baseline before CCCP treatment (bl), upon CCCP induction (injured), and 1.5h after washout of CCCP (washout). Area for insets (top panel) indicated by a red dashed box in the bottom panels. Scale bars: 10 μm. (C) Violin plots of mitochondrial aspect ratios (length over width) of 43–87 mitochondria. Representative of three experiments. (D) Confocal time lapse imaging of PKmito labeled mitochondria of control and Rac1KO cells after CCCP washout. Fusion and fission are highlighted by green and blue arrowheads, respectively. Scale bars: 5μm. (E) Fusion and fission odds ratios of mitochondrial live imaging tracks based on Mitometer in Rac1KO vs controls cells (logarithmic scale) after CCCP washout. (F) Frequency distributions of mitochondrial solidity (ratio of mitochondrial area to its enclosing convex hull) of 220–294 mitochondrial tracks of control and Rac1KO cells. P value for control vs Rac1KO. (G) Representative flow cytometry density dot plots (side scatter vs TMRM fluorescence) of TMRM-labeled control and mutant cells. The gates for TMRM low and TMRM high cell populations are shown. (H) Stacked bar graphs of the gated percentages as indicated in G quantifying TMRM low (in red) and high (in gray) cell populations. (I) Oxygen consumption rates (OCR) in the Seahorse Real-Time ATP rate assay of control and mutant cells of the indicated experimental groups. The time points for the additions of mitochondrial inhibitors (Oligo, oligomycin; Rot/AA, rotenone/antimycin A) are indicated by black arrows. (J) Quantification of mitochondrial ATP Production rates summarizing three experiments normalized to 1000 cells. Means are ± SD. * p< 0.05; ns, not significant.
Figure 6.
Figure 6.. Rac1 regulates mitochondrial repair by promoting actin-driven mitophagy.
(A) Schematic of the working principle of the Fis1-Lifeact-GFP mitochondrial actin reporter. (B) Airy Scan confocal images of PK-mito labeled mitochondria (magenta) and Fis-1-Lifeact (green) of control and Rac1KO TKPTS cells before (baseline) and after CCCP treatment. Scale bar: 5 μm. (C) Quantification of peri-mitochondrial F-actin intensity (in normalized arbitrary units, AU) of the indicated groups of at least 20 mitochondria/group. (D) Schematic of the working principle of the ActuAtor System. Rapamycin induces dimerization of Tom-20-FRB and ActA-FKBP recruiting F-actin (via ActA) to mitochondria. (E) Left panels: Airy Scan confocal images of the Tom20-ECFP reporter (mitochondria, white) and ActA-mCherry (blue), scale bars: 5 μm. Right panels: Confocal images of F-actin (Phalloidin, cyan), DAPI (blue), and mitochondria (Tom20, magenta). Scale bars: 5 μm. Both left and right panels show CCCP treated and Actuator-expressing Rac1KO cells before (top row) and after (bottom row) the addition of Rapamycin (Rapa, 100nM). (F) Quantification (box and whiskers plot) of peri-mitochondrial F-actin (Phalloidin) intensity of Actuator-expressing and CCCP-treated Rac1KO cells without (Rapa−) and with rapamycin (Rapa+). At least 20 mitochondria per group. A-F representative of three experiments. (G) Oxygen consumption rates (OCR) in the Seahorse Mito Stress test of the indicated experimental groups after washout of CCCP in ActuAtor-expressing TKPTS cells. (H) Quantification of basal and maximal mitochondrial respiration and OCR-linked ATP Production rates based on the assay and groups shown in G summarizing at least three experiments. All data are normalized to 1000 cells. (I) Experimental schematic: control TKPTS cells were treated with CCCP with or without the Arp2/3 inhibitor CK666. CCCP/CK666 were washed out and cells were then treated with the Rac1 Activator 3744270. (J) Super-resolution confocal imaging of peri-mitochondrial F-actin (Fis1-LifeAct, green) and mitochondria (PKmito, magenta) of untreated, CCCP-, CK666-, CCCP- and CK666-treated PT cells. CK666 and CCCP were washed out and cells were treated with the Rac1 activator 3744270 during recovery. Scale bar 5μm. (K) Quantification of peri-mitochondrial F-actin intensity pooled from two experiments. The group numbers correspond to the numbers below the images in J. (L) Oxygen consumption rates (OCR) in the Seahorse Mito Stress test of the indicated experimental groups, all after washout of either CCCP, CCCP and CK666, or CCCP and CK666 followed by Rac1 activator treatments. (M) Quantification of maximal mitochondrial respiration and OCR-linked ATP Production rates based on the assay and groups shown in L. All data are normalized to 1000 cells. Areas for insets are outlined by solid white boxes. Shown are means ± SD. *p<0.05. ns, not significant.
Figure 7.
Figure 7.. Rac1 repairs mitochondria by promoting actin-driven mitophagy.
(A) Schematic of the working principle of the Cox8-EGFP-mCherry mitophagy reporter. (B) Airy Scan confocal images of the Cox8-EGFP-mCherry reporter of the CCCP-treated control (+/− CK666) and mutant cells showing the EGFP channel in cyan and mCherry in red. Mitophagy appears as red-only puncta. Scale bars: 10 μm. (C) Quantification of red-only puncta per cell of at least 10 cells per condition. (D) Confocal immunofluorescence of LC3 (green) puncta in outer mitochondrial membranes (Tom20, magenta) of ActuAtor-expressing untreated and CCCP-treated Rac1KO TKPTS cells with or without the induction of peri-mitochondrial F-actin (+Rapa). The bottom row depicts single color channel images of LC3. Scale bars 10μm. (E) Quantification (box and whiskers plot) of LC3+ puncta per cell of at least 10 cells per group. (F) Flow cytometry histograms of Mtphagy dye-labeled ActuAator-expressing Rac1KO TKPTS cells without (−Rapa) or with (+Rapa) the induction of peri-mitochondrial F-actin. (G) Quantification of Mean Fluorescence Intensity (MFI) of the experiment in F. (H) Schematic of the experiment testing the role of mitophagy in Rac1-dependent mitochondrial repair. Control and KO cells were treated with CCCP and UMI-77, both were washed out, and mitochondrial organization and dynamics were live imaged after the indicated time interval. (I) PKmito labeled mitochondria (black) are shown in the top row. Scale bars: 5 μm. Skeletonized images of the same mitochondria (in white) are shown in the middle row. The last row shows representative fusion tracks as calculated by Mitometer. (J) Quantification (violin plots) of mitochondrial branch length of n = 60–176 mitochondria per group. (K) Fusion Odds Ratios (logarithmic x-scale) calculated using Mitometer comparing KO vs controls or KO vs KO + UMI-77. (L) Oxygen consumption rates (OCR) in the Seahorse Mito Stress test. (M) Quantification of maximal mitochondrial respiration and OCR-linked ATP Production normalized to 1000 cells. I-M all show control, Rac1KO, and UMI-77 Rac1KO cells after washout of CCCP. All Data are representative of at least two to three experiments. Areas for insets are outlined by solid white boxes. Means are ± SD. * p< 0.05; ns, not significant.
Figure 8.
Figure 8.. Rac1 regulates mitophagy after AKI in vivo.
(A) Schematic of the in vivo experiment. UMI-77 (+U) was injected intraperitoneally (i.p.) after AKI (AKI-IRI) every other day (qod) for 1 month. Mice were assessed at 1 month and 3 months after AKI. (B) Electron micrographs showing mitochondrial structure and morphology. Scale bars: 1 μm. Bottom two rows: DIC images overlaid with LTL (magenta) and DAPI (blue) with cartoons illustrating the changes in PT surface morphology. Scale bars: 10 μm. Areas for insets are outlined by a solid yellow box. (C) Quantification of mitochondrial length by TEM. (D) Quantification of mitochondria with damaged-appearing cristae per high-power field. (E) Quantification proximal tubular area (as %) appearing with of longitudinal basolateral folds. (F) Ex vivo tissue seahorse measurement of oxygen consumption (OCR) over time of cortical kidney biopsies. (G) Quantification of average oxygen consumption rates (OCR). (H) Seahorse energy profile with OCR plotted against ECAR (extracellular acidification rate) with the energy states indicated in blue. B-H all show control (Rac1f/f), Rac1KO (γGT:Rac1f/f), and UMI-77 (U) treated Rac1KO mice 1 month after AKI with data averaged from at least three mice per group. (I) Periodic Acid-Schiff (PAS)-stained paraffin kidney sections (left panels) followed by megalin-stained kidney cortex sections (right panels). Areas for insets are indicated by a black dashed box. Scale bars: 50 μm. (J) Quantification of atrophic tubules as average total number per cortical high-power field (HPF). (K) Quantification of megalin positive (+) tubules as average total number per cortical HPF. I-K: n=3–4 mice/group. I-K all show control (Rac1f/f), Rac1KO (γGT:Rac1f/f), and UMI-77 (U) treated Rac1KO mice 3 months after AKI. (L) Schematic showing the proposed mechanism whereby Rac1 repairs proximal tubular function and bioenergetics by promoting actin-driven removal of damaged mitochondria with a low metabolic capacity (orange). Means are ± SD. * p< 0.05; ns, not significant.
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