Journal: bioRxiv

Article Title: Tubular Mitochondrial Pyruvate Carrier Disruption Elicits Redox Adaptations that Protect from Acute Kidney Injury

doi: 10.1101/2023.01.31.526492

Figure Lengend Snippet: (A) Bar graph comparing kidney Mpc1 mRNA levels after vehicle treatment or cisplatin-, ischemia reperfusion (IR)-, and rhabdomyolysis (Rhabdo)-induced AKIs. Samples were collected 72 hours after cisplatin injury and 24 hours after IR and rhabdomyolysis injuries. (n = 6/group; *** p < 0.001 by unpaired t test with Welch’s correction). (B) Representative Western blot of kidney MPC1 protein abundance after AKI. Samples were collected 72 hours after cisplatin injury and 24 hours after IR and rhabdomyolysis injuries. β-ACTIN was blotted as a loading control. (C) Representative immunostaining images of MPC1 (red), lotus tetragonolobus lectin (LTL, green, proximal tubule marker), or peanut agglutinin (PNA, green, distal tubule marker), and DAPI (blue) in whole kidney, outer cortex (OC) and cortico-medullary junction (CM) kidney sections 30 hours following vehicle treatment or rhabdomyolysis-induced AKI. (Images captured at 15x magnification; whole kidney scale bar = 1,000 μm; OC and CM scale bar = 50 μm). ( D ) Representative fluorescence image of kidney sections of mT/mG/Ggt1-Cre mice confirming GFP+ renal tubular epithelial cells (green, #, GFP) and tdTomato+ non-RTEC cells (red, *, tdT) stained with Dapi (blue). (Scale bar = 100 μm). (E) Bar graph comparing Mpc1 mRNA levels in flow-sorted Non-RTEC (tdTomato+) and RTEC (GFP+) cells 24 hour after vehicle treatment or rhadbomyolysis-induced AKI. (n = 5/group, ***p < 0.001 by unpaired t test with Welch’s correction). (F) Representative Western blot of MPC1 and VDAC protein abundance in flow-sorted Non-RTEC (tdTomato+) and RTEC (GFP+) cells 24 hour after AKI. β-ACTIN was blotted as a loading control. Data are presented as means ± SEM.

Article Snippet: The MPC1 f/f mice were bred with mice that express Cre recombinase under the control of the tubule specific Pax8 Cre promoter (Jackson Laboratory, 028196) ( , ).

Techniques: Western Blot, Control, Immunostaining, Marker, Fluorescence, Staining

Journal: bioRxiv

Article Title: Tubular Mitochondrial Pyruvate Carrier Disruption Elicits Redox Adaptations that Protect from Acute Kidney Injury

doi: 10.1101/2023.01.31.526492

Figure Lengend Snippet: (A) Schematic illustrating the generation of tubular Mpc1 null allele, MPC TubKO mice (TubKO). (B-C) Bar graphs showing body weights ( B ) and serum cystatin C concentration ( C ) in WT and MPC TubKO mice. (n = 5/group, 8-week-old mice). (D) Bar graph comparing mouse kidney Mpc1 mRNA levels in WT and MPC TubKO mice. (n = 4/group, 7 – 12-week-old mice, ** p < 0.01 by unpaired t test with Welch’s correction). (E-G) Representative Western blot of kidney MPC1 and MPC2 protein abundance ( E ) and quantification of normalized MPC1 ( F ) and MPC2 ( G ) levels in WT and MPC TubKO mice. Tubulin was blotted as loading control and used as the protein quantification normalizer. (n = 4 – 6/group, 7 – 12-week-old mice, *** p < 0.001 and ** p < 0.01 by by unpaired t test with Welch’s correction). (H) Representative immunostaining images of kidney MPC1 (green) and lotus tetragonolobus lectin (LTL, green, proximal tubule marker) or peanut agglutinin (PNA, green, distal tubule marker) in whole kidney (WK), outer-cortex (OC), and cortico-medullary junction (CM) in WT and MPC TubKO mice. (Images taken at 4x (WK) or 20x (OC and CM) magnification, scale bar = 500 μm). Data are presented as means ± SEM.

Article Snippet: The MPC1 f/f mice were bred with mice that express Cre recombinase under the control of the tubule specific Pax8 Cre promoter (Jackson Laboratory, 028196) ( , ).

Techniques: Concentration Assay, Western Blot, Control, Immunostaining, Marker

Journal: bioRxiv

Article Title: Tubular Mitochondrial Pyruvate Carrier Disruption Elicits Redox Adaptations that Protect from Acute Kidney Injury

doi: 10.1101/2023.01.31.526492

Figure Lengend Snippet: ( A ) Line graph showing the survival curve of WT and MPC TubKO mice following rhabdomyolysis (Rhabdo)-induced AKI. (n = 10 – 11/group, 8 – 12-week-old mice, * p < 0.05 by Mantel-Cox log-rank test). ( B-C ) Bar graphs showing serum cystatin C ( C ), and blood urea nitrogen ( D , BUN) levels prior to (D0) and on day 1 (D1, 24-hours) and day 2 (D2, 48 hours) after vehicle treatment or rhabdomyolysis-induced AKI in WT and MPC TubKO mice. (n = 10 – 11/group, 8 – 12-week-old mice, * p< 0.05, ** p < 0.01, *** p < 0.001 by two-way ANOVA followed by Turkey’s multiple comparison tests). ( E-F ) Bar graphs showing kidney Ngal ( D ) and Kim1 ( E ) mRNA levels one day (24 hours) after vehicle treatment or rhabdomyolysis-induced AKI in WT and MPC TubKO mice. (n = 4/group for vehicle treatment, n = 12 – 13/group for Rhabdo, 8 – 12-week-old mice, * p < 0.05 by two-way ANOVA with Tukey’s multiple comparison test). ( G-H ) Bar graphs showing quantification of histologically assessed tubular injury score ( F ) and tunel positive tubular cells ( G ) one day (24 hours) after vehicle treatment or rhabdomyolysis-induced AKI in WT and MPC TubKO mice. (n = 4/group for vehicle treatment, n = 12 – 13/group for Rhabdo, 8 – 12-week-old mice, ** p < 0.01 and *** p < 0.001 by two-way ANOVA with Tukey’s multiple comparison test). ( H-J ) Heatmaps showing Spearman correlation between variables analyzed following vehicle treatment or rhabdomyolysis-induced AKI. Correlation calculated in WT mice comparing Mpc1 mRNA levels, tubular injury, Ngal and Kim1 mRNA levels, tunel score, and tubular GSH with AKI ( H ). Spearman correlation performed in WT ( I ) and MPC TubKO ( J ) mice comparing GSH and antioxidant defense system markers following rhabdomyolysis-induced AKI. Data are presented as means ± SEM.

Article Snippet: The MPC1 f/f mice were bred with mice that express Cre recombinase under the control of the tubule specific Pax8 Cre promoter (Jackson Laboratory, 028196) ( , ).

Techniques: Comparison, TUNEL Assay