Research ArticleKidney Disease

Circulating uromodulin inhibits systemic oxidative stress by inactivating the TRPM2 channel

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Science Translational Medicine  02 Oct 2019:
Vol. 11, Issue 512, eaaw3639
DOI: 10.1126/scitranslmed.aaw3639
  • Fig. 1 THP inhibits activation of the RAC1/p-JNK signaling pathway.

    (A) Transcriptomic analysis of laser microdissected S3 proximal tubules from THP−/− and THP+/+ mouse kidney. KO, knockout; WT, wild type. (B) Canonical pathway analysis (Ingenuity) of differentially expressed transcripts and pathways with z score > 1.5 or < −1.5 are shown with their predicated activation state in the S3 tubules of THP−/− mice. SAPK, stress-activated protein kinase; IRF, interferon regulatory factor; PPAR, peroxisome proliferator–activated receptor. (C) Triplicate groups of HK-2 cells were treated with THP (1 μg/ml) or vehicle for 6 hours. Protein extraction was followed by quantitative label-free proteomics. Differentially expressed proteins were identified (shown in heat map). (D) Canonical pathway analysis of the proteomic data from (C) was performed, and pathways with z score > 2 or < −2 are shown with their predicated activation state in the THP-stimulated HK-2 cells. ARP-WASP, actin-related protein–Wiskott-Aldrich syndrome protein; GTPases, guanosine triphosphatases. fMLP, N-formyl-Met-Leu-Phe. (E) Left: Immunofluorescence confocal microscopy shows shifting of RAC1 (red) in THP−/− mice to the basolateral domain of S3 segments (right) compared to a cytoplasmic localization in THP+/+ (left). Insets show a higher magnification of the areas indicated by the arrowheads (the arrow points to basolateral RAC1). RAC1 shift is a surrogate for its activation. Right: THP−/− mouse kidneys (n = 5 per group) are shown. n.s, not significant. (F) Left: Active RAC1 was immunoprecipitated from total kidney lysates of THP+/+ and THP−/− kidneys (n = 5 to 6 per group; sample “a” of the THP−/− group bridges the two immunoblots). Right: Quantification of the immunoprecipitation experiment. (G) Phosphorylated JNK was assayed from nuclear extracts of total kidney lysates by immunoblot (left) and quantitated (right) by band densitometry. (H) Immunoblot of H2O2-dependent JNK activation after treatment with THP (1 μg/ml) in the left panel, quantitated in the right panel. Scatter plots are means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. MW, molecular weight; M-OS, medulla, outer stripe; CX, cortex.

  • Fig. 2 THP inhibits activation of p–c-JUN.

    (A to C) Sections from the outer medulla of kidneys from THP+/+ and THP−/− mice were stained for phosphorylated (active) c-JUN (p–c-JUN) (A). VTEA was used to quantify the percentage of cells positive for p–c-JUN staining (B). In (C), 3D rendering of a representative p–c-JUN stained image is overlaid with the VTEA gating. Scatter plot error bars represent means ± SD. *P < 0.05.

  • Fig. 3 THP−/− mice have increased oxidative stress and damage within the kidneys and systemically.

    (A) Live imaging with the ROS-sensitive dye H2DCFDA in kidneys of THP+/+ and THP−/− mice (inset: background fluorescence) in the left panel is quantitated in the right panel (n = 3 to 4 per group). (B) H2O2 concentrations in kidney lysates from THP+/+ and THP−/− mice (n = 5 per group). Values were normalized to the amount of protein present in each lysate. (C) Heat map of oxidized phosphatidylcholine (PC) and phosphatidylethanolamine (PE) concentrations in the kidneys of THP+/+ and THP−/− mice (n = 3 per group). (D to F) Concentration of 8OHdG, a marker of oxidative DNA damage, in the kidney [n = 7 to 15 per group (D)], serum (n = 15 to 17 per group (E)], and lungs (n = 8 per group (F)] of THP−/− mice compared to THP+/+ mice. Scatter plot error bars represent means ± SD. *P < 0.05 and ***P < 0.001 (statistical significance between the two groups).

  • Fig. 4 Serum THP decreases and oxidative DNA damage increases after ischemic AKI in THP+/+ mice.

    (A) Serum THP concentrations of THP+/+ mice before and 24 hours after IRI surgery (n = 15 mice). (B) Serum DNA damage concentrations of THP+/+ mice before and 24 hours after IRI surgery (n = 15 mice). (C) Serum concentrations of kidney injury markers cystatin C, neutrophil gelatinase–associated lipocalin (NGAL), and osteopontin in THP+/+ mice before and 24 hours after IRI surgery (n = 15 mice). Scatter plots are means ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 5 Plasma THP decreases and oxidative DNA damage increases in patients with AKI after transplant surgery.

    (A) Correlation of THP and 8OHdG concentrations from all time points for AKI cohort and control participants (n = 43). (B) Presurgery plasma THP concentrations compared to concentrations 18 hours after surgery (paired analysis, n = 34). (C) Presurgery plasma 8OHdG concentrations compared to concentrations 18 hours after surgery (paired analysis, n = 34). (D) Correlation of presurgery plasma concentrations of THP with plasma 8OHdG levels collected 18 hours after surgery (n = 34, R2 = 0.23, and P < 0.05). Scatter plot error bars are means ± SD. *P < 0.05 and ****P < 0.0001.

  • Fig. 6 Oxidative DNA damage is associated with mortality and need for dialysis in patients with AKI after transplant surgery.

    (A and B) Presurgery (A) and postsurgery (B) plasma concentrations of 8OHdG in patients who did or did not survive after transplant surgery and development of AKI (n = 34). (C and D) Presurgery (C) and postsurgery (D) plasma concentrations of THP in patients who did or did not survive after transplant surgery and development of AKI (n = 34). (E and F) Presurgery (E) and postsurgery (F) plasma concentrations of 8OHdG in patients who did or did not receive dialysis after transplant surgery and development of AKI (n = 34). *P < 0.05, **P < 0.01, and ***P < 0.001 (statistical significance between the two groups).

  • Fig. 7 THP inhibits TRPM2 calcium influx in vitro, and TRPM2 inhibition in vivo reduces oxidative damage.

    (A to D) HEK293 cells expressing a tetracycline-inducible TRPM2 channel were treated with doxycycline or vehicle control (vehicle control and no TRPM2) for 24 hours before loading with the calcium indicator Fluo-4. Cells were pretreated with THP, HSA, or N-(p-amylcinnamoyl) anthranilic acid (ACA), respectively, before activation of the TRPM2 channel with H2O2. Calcium influx was measured after addition of extracellular calcium (CaCl2). In (A), pretreatment with THP (1 μg/ml) is shown. In (B), pretreatment with THP (1 μg/ml) is compared to HSA (1 μg/ml). In (C), pretreatment with 0.01, 0.1, and 1 μg/ml THP are compared. In (D), treatment with THP (1 μg/ml) is compared to 25 μM ACA, a small-molecule inhibitor of TRPM2. Representative results are shown from a single experiment (n = 3 wells per treatment condition). (E) Concentration of oxidative DNA damage (8OHdG) in THP−/− mice treated with the TRPM2 inhibitor 2-aminoethoxydiphenyl borate (2-APB) (16 mg/kg) or vehicle (10% DMSO/90% sterile saline) for 3 days (n = 7 to 8 mice per group). (F) Concentration of oxidative DNA damage (8OHdG) in THP−/− and THP+/+ mice treated with TRPM2 inhibitor 2-APB (16 mg/kg) or vehicle (10% DMSO/90% sterile saline) for 3 days before performing a 22-min bilateral clamping of the renal artery followed by 6-hour recovery. (n = 7 to 8 mice per group). Scatter plots are means ± SD. *P < 0.05 and **P < 0.01.

  • Fig. 8 Inhibition of TRPM2 blocks JNK activation in HK-2 cells.

    (A) Immunoblot for TRPM2 in HK-2 cell lysate. (B) Immunoblot of H2O2-dependent JNK activation after treatment with 25 μM ACA in the left panel, quantitated in the right panel. Scatter plots are means ± SD. *P < 0.05 and ****P < 0.0001.

  • Table 1 Characteristics of the AKI cohort.

    Demographic and clinical data for a cohort of liver transplant recipients who developed AKI after transplant surgery.

    Sex
      Male56%
      Female44%
    Age (years)53.5 ± 11.3
    Race
      Caucasian85%
      African3%
      Hispanic9%
      Asian3%
    Body mass index (kg/m2)27.5 ± 5.03
    Presurgery plasma 8OHdG (ng/ml)11.8 ± 8.14
    Postsurgery plasma 8OHdG (ng/ml)16.3 ± 11.8
    Presurgery plasma THP (ng/ml)291 ± 258
    Postsurgery plasma THP (ng/ml)102 ± 93.6
    Presurgery eGFR (ml/min per 1.73 m2)67.8 ± 33.0
    Presurgery plasma creatinine (mg/dl)1.32 ± 0.807
    Postsurgery plasma creatinine (mg/dl)1.53 ± 0.689
    AKI stage
      Stage 115%
      Stage 229%
      Stage 356%
    Received dialysis47%
    Sixty-day survival77%

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/512/eaaw3639/DC1

    Fig. S1. Fluorescence-based laser microdissection and downstream transcriptomic and bioinformatic analyses.

    Fig. S2. THP treatment of HK-2 cells results in inhibition of multiple RAC1-dependent pathways.

    Fig. S3. Quantitation of membrane-associated and cellular RAC1 in kidney sections from THP+/+ and THP−/− mice.

    Fig. S4. Free radical scavenging proteins are down-regulated in the S3 tubules of THP−/− mice.

    Fig. S5. Molecules of the RAC1/JNK/c-JUN signaling network involved in free radical synthesis.

    Fig. S6. ROS concentrations are increased within the S3 proximal tubules of THP−/− mice.

    Fig. S7. Electron microscopy studies of mitochondrial morphology in S3 segments.

    Fig. S8. H2O2 activates IL-23 expression in HK-2 cells.

    Fig. S9. THP plasma concentrations in liver transplant recipients who did not develop AKI.

    Table S1. Demographic and baseline clinical data for liver transplant surgery group and healthy controls.

    Data file S1. Gene arrays S3.

    Data file S2. Proteomics HK-2 cell THP stimulation.

    Data file S3. Oxidized phospholipidomics from kidneys.

  • The PDF file includes:

    • Fig. S1. Fluorescence-based laser microdissection and downstream transcriptomic and bioinformatic analyses.
    • Fig. S2. THP treatment of HK-2 cells results in inhibition of multiple RAC1-dependent pathways.
    • Fig. S3. Quantitation of membrane-associated and cellular RAC1 in kidney sections from THP+/+ and THP−/− mice.
    • Fig. S4. Free radical scavenging proteins are down-regulated in the S3 tubules of THP−/− mice.
    • Fig. S5. Molecules of the RAC1/JNK/c-JUN signaling network involved in free radical synthesis.
    • Fig. S6. ROS concentrations are increased within the S3 proximal tubules of THP−/− mice.
    • Fig. S7. Electron microscopy studies of mitochondrial morphology in S3 segments.
    • Fig. S8. H2O2 activates IL-23 expression in HK-2 cells.
    • Fig. S9. THP plasma concentrations in liver transplant recipients who did not develop AKI.
    • Table S1. Demographic and baseline clinical data for liver transplant surgery group and healthy controls.
    • Legends for data files S1 to S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Gene arrays S3.
    • Data file S2 (Microsoft Excel format). Proteomics HK-2 cell THP stimulation.
    • Data file S3 (Microsoft Excel format). Oxidized phospholipidomics from kidneys.

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