Open Access, Peer-reviewed
pISSN 2671-5651
eISSN 2671-566X
    Korean J Phys Anthropol. 2017 Sep;30(3):87-98. Korean.
    Published online Sep 30, 2017.
    https://doi.org/10.11637/kjpa.2017.30.3.87
    © 2017 Korean Association of Physical Anthropologists
    Original Article

    Alteration of Akt, p-Akt, ERK, and p-ERK Proteins Expression in the Kidney of Hypokalemic Rat

    Choon Sang Bae, Hye Jung Cho and Kyu Yoon Ahn
      • Department of Anatomy, College of Medicine, Jeonnam University, Korea.
    • Correspondence to: Kyu Yoon Ahn. (Department of Anatomy, College of Medicine, Jeonnam University) Email: kyahn@jnu.ac.kr
    Received June 15, 2017; Revised August 10, 2017; Accepted August 10, 2017.

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    Hypokalemia causes metabolic alkalosis and morphological changes of the kidney. K+ balance is regulated not only by ion channels or pump gene, but also by various genes including NF-E2-related factor 2 (Nrf2). Previous study suggested the possibility that Akt and ERK kinase may be involved in Nrf2 transcriptional gene activation. In present study, we investigate the alterations of Akt, p-Akt, ERK, p-ERK protein in both normal kidney and K+-deficient diet kidney using Western blot analysis, and immunohistochemisrty. Our western blot data showed that the expression of Akt and p-Akt was increased gradually in K+-depleted diet (from 1W-3W) compared to normal group. The expression of ERK and p-ERK was markedly increased in K+-depleted diet 2W in comparison with normal group. Based on our immunostaining results, Akt protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K+-depleted diet 2 weeks. The localization of p-Akt proteins in K+-depleted groups was not different from normal group, but the immunoreactivity was significantly increased in distal convoluted tubule, macula densa and outer medullary thick ascending limb in K+-depleted diet 1 and 2 weeks groups. ERK protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K+-depleted diet 2 and 3 weeks. The localization of p-ERK proteins in K+-depleted groups was not different from normal group, but the immunoreactivity was prominently increased in the nucleus of outer medullary collecting duct especially in K+-depleted diet 2 weeks. Taken together, we suggest that the expression of p-Akt was gradually increased in K+-depleted groups of kidney, but the expression of p-ERK was markedly increased in K+-depleted diet 2 week group. Hence, the promotion of AKT and ERK phosphorylation in hypokalemic condition may be involved in the regulation of ion channels, ion transporters and subsequent intracellular signal transduction.

    Keywords
    Hypokalemia; Metabolic alkalosis; Akt; p-Akt; ERK; p-ERK; Immunohistochemisty

    Figures

    Fig. 1
    Western blotting showed the protein level of Akt, p-Akt, ERK, p-ERK in Low K+ (LK) diet rat kidney. (A) The protein level of Akt was gradually increased in LK3D, LK 1W, 2W, and 3W diet rat kidney in comparison with the normal diet rat kidney. (B) The protein level of p-Akt was gradually increased in LK 1W, 2W, and 3W diet rat kidney compared to the normal diet rat kidney. (C) The protein level of ERK was increased in LK 1W, 2W, 3W diet rat kidney compared to the normal diet rat kidney. (D) The protein level of p-ERK was markedly increased in LK 2W diet rat kidney compared to the normal diet rat kidney. Data are expressed as mean±S.E.M, and each experiment conducted 3 repeats per conditions. β-actin was used as control. Differences were considered significant at *p<0.05, **p<0.001 (ANOVA one way statistical analysis compared to the normal group) (Normal: normal diet rat kidney, LK3D: Low K+ diet rat kidney for 3 days, LK1W: Low K+ diet rat kidney for 1 week, LK2W: Low K+ diet rat kidney for 2 weeks, LK3W: Low K+ diet rat kidney for 3 weeks).

    Fig. 2
    Images showed the expression of Akt in Low K+ (LK) diet rat kidney. We checked the expression of Akt in kidney using 3,3′-Diaminobenzidine (DAB) staining. (A~C) Renal cortex, (D~F) outer medulla. In normal group, immunoreactivity of Akt was moderately detected in the distal convoluted tubule (DCT) and cortical thick ascending limb (CTAL) and outer medullary thick ascending limb (MTAL), and weakly in cortical collecting duct (CCD) and outer medullary collecting duct (OMCD). The immunoreactivity is prominently increased in outer medullary collecting duct especially in K+-depleted diet 2 weeks (Akt: Akt, CTX: renal cortex, OM: outer medulla, Normal: normal diet rat kidney, LK1W: Low K+ diet rat kidney for 1 week, LK2W: Low K+ diet rat kidney for 2 weeks. Scale bar: 30 µm, CTAL: cortical thick ascending limb (arrow), MTAL: outer medullary thick ascending limb (asterisk)).

    Fig. 3
    Images showed the expression of p-Akt in Low K+ (LK) diet rat kidney. We checked the expression of p-Akt in kidney using 3,3′-Diaminobenzidine (DAB) staining. (A~C) Renal cortex, (D~F) outer medulla. In normal group, p-Akt is moderately detected in the proximal convolute tubule (PCT) and outer medullary thick ascending limb, and weakly in distal convoluted tubule and macula densa (MD). The expressed localization of p-Akt proteins in K+-depleted groups was not different from normal group, but the immunoreactivity is significantly increased in distal convoluted tubule, macula densa and outer medullary thick ascending limb in K+-depleted diet 1 and 2 weeks groups (p-Akt: phosphorylation of Akt, CTX: renal cortex, OM: outer medulla, Normal: normal diet rat kidney, LK1W: Low K+ diet rat kidney for 1 week, LK2W: Low K+ diet rat kidney for 2 weeks. Scale bar: 30 µm, MD: macula densa (arrow), MTAL: outer medullary thick ascending limb (asterisk)).

    Fig. 4
    Images showed the expression of ERK in Low K+ (LK) diet rat kidney. We checked the expression of ERK in kidney using 3,3′-Diaminobenzidine (DAB) staining. (A~C) Renal cortex, (D~F) outer medulla. In normal group, ERK is moderately detected in the distal convoluted tubule and outer medullary collecting duct, weakly in cortical collecting duct, cortical thick ascending limb and outer medullary thick ascending limb. The immunoreactivity is prominently increased in outer medullary collecting duct especially in K+-depleted diet 2 and 3 weeks (ERK: ERK, CTX: renal cortex, OM: outer medulla, Normal: normal diet rat kidney, LK2W: Low K+ diet rat kidney for 2 week, LK3W: Low K+ diet rat kidney for 3 weeks. Scale bar: 30 µm. CTAL: cortical thick ascending limb (arrow)).

    Fig. 5
    Images showed the expression of p-ERK in Low K+ (LK) diet rat kidney. We checked the expression of p-ERK in kidney using 3,3′-Diaminobenzidine (DAB) staining. (A~C) Renal cortex, (D~F) outer medulla. In normal group, p-ERK is moderately detected in nucleus of cortical and outer medullary collecting duct and weakly in distal convoluted tubule and inner medullary collecting duct. The expressed localization of p-ERK proteins in K+-depleted groups was not different from normal group, but the immunoreactivity is prominently increased in the nucleus of outer medullary collecting duct especially in K+-depleted diet 2 weeks (p-ERK: phosphorylation of ERK, CTX: renal cortex, OM: outer medulla, Normal: normal diet rat kidney, LK1W: Low K+ diet rat kidney for 1 week, LK2W: Low K+ diet rat kidney for 2 weeks. Scale bar: 30 µm. DCT: distal convoluted tubule (arrow)).

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    MeSH Terms
    Alkalosis
    Animals
    Blotting, Western
    Diet
    Extremities
    Hypokalemia
    Ion Channels
    Ion Transport
    Kidney*
    NF-E2-Related Factor 2
    Phosphorylation
    Phosphotransferases
    Rats*
    Signal Transduction
    Transcriptional Activation
    Metrics
    Share
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