Anat Biol Anthropol.  2019 Dec;32(4):121-128. 10.11637/aba.2019.32.4.121.

Extraneural CGRP Induces Oxidative Stress in Kidney Proximal Tubule Epithelial Cells

• Moon D
• Yoon SP
• Jang HS
• Noh MR
• Ha L
• Padanilam
• Kim J

• KMID: 2467465
• DOI: http://doi.org/10.11637/aba.2019.32.4.121

Abstract

Calcitonin gene-related peptide (CGRP) is the most abundant neuropeptide in primary afferent sensory neurons. Exogenous CGRP can induce cell death in kidney tubular cells. The objective of this study was to determine whether exogenous CGRP could induce reactive oxygen species (ROS) production in kidney proximal tubule epithelial cells and whether CGRP-induced ROS production might contribute to cell death. In HK-2, LLC-PK1 and TCMK-1 cell lines derived from human, pig, and mouse respectively, administration of CGRP increased cell death in time- and dose-dependent manners, as demonstrated by decreased cell viability. Exogenous CGRP also increased ROS production levels in those cell lines. Treatment with CGRP receptor antagonist(CGRP(8-37)) significantly inhibited the increases in cell death and ROS production in CGRP-exposed cells. Furthermore, treatment with a ROS scavenger (MnTMPyP) markedly reduced kidney proximal tubule epithelial cell death after CGRP administration. Taken together, these data suggest that extraneural CGRP can induce cell death through excessive oxidative stress in kidney proximal tubule epithelial cells.

Keyword

CGRP; Kidney proximal tubule epithelial cell; Cell death; ROS; Oxidative stress; CGRP(8-37); MnTMPyP

MeSH Terms

Animals
Calcitonin Gene-Related Peptide
Cell Death
Cell Line
Cell Survival
Epithelial Cells*
Humans
Kidney*
Mice
Neuropeptides
Oxidative Stress*
Reactive Oxygen Species
Receptors, Calcitonin Gene-Related Peptide
Sensory Receptor Cells
Calcitonin Gene-Related Peptide
Neuropeptides
Reactive Oxygen Species
Receptors, Calcitonin Gene-Related Peptide

Figure

• 

  Fig. 1 Exogenous CGRP decreases cell viability in kidney proximal tubule epithelial cells. Cell viability was measured using MTT assay(n=9 wells from three independent experiments per group). In box plots, whiskers represent the minimum and maximum; bases represent the interquartile range between the first and third quartiles; and midlines represent the median.(a) HK-2, LLC-PK1, and TCMK-1 cells were treated with 0.1, 1, or 10 nM of CGRP in PBS (control) for 24 hours. *P<0.05, **P<0.01 versus control.(b) HK-2, LLC-PK1, and TCMK-1 cells were treated with 1 nM of CGRP in PBS for 0, 6, 24, or 48 hours. †P<0.05, ††P<0.01, †††P<0.001 versus 0 h.

• 

  Fig. 2 Exogenous CGRP induces ROS production in kidney proximal tubule epithelial cells. ROS production was measured using oxidative sensitive dye DCFDA(n=9 wells from three independent experiments per group). In box plots, whiskers represent the minimum and maximum; bases represent the interquartile range between the first and third quartiles; and midlines represent the median.(a) HK-2, LLC-PK1, and TCMK-1 cells were treated with 0.1, 1, or 10 nM of CGRP in PBS (control) for 24 hours. *P<0.05, **P<0.01, ***P<0.001 versus control.(b) HK-2, LLC-PK1, and TCMK-1 cells were treated with 1 nM of CGRP in PBS for 0, 6, 24, or 48 hours. †P<0.05, ††P<0.01, †††P<0.001 versus 0 h.

• 

  Fig. 3 CGRP receptor antagonism reduces ROS production and cell death induced by exogenous CGRP in kidney proximal tubule epithelial cells. HK-2, LLC-PK1 and TCMK-1 cells were treated with 1 nM of CGRP for 24 hours. One hour before treatment with CGRP, cells were treated with 1, 3, or 10 nM of CGRP8-37 in PBS (vehicle) (n=9 wells from three independent experiments per group). In box plots, whiskers represent the minimum and maximum; bases represent the interquartile range between the first and third quartiles; and midlines represent the median. #P<0.05, ##P<0.01, ###P<0.001 versus vehicle. (a) ROS production was measured using oxidative sensitive dye DCFDA. (b) Cell viability was measured using MTT assay.

• 

  Fig. 4 Correlation between the percentage of cell viability and ROS production in kidney proximal tubule epithelial cells after exposure to CGRP. HK-2, LLC-PK1, and TCMK-1 cells were treated with 0.1, 1, or 10 nM of CGRP for 0, 6, 24, or 48 hours(n=9 wells from three independent experiments per group). Pearson's correlation was used for parameter correlations between the percentage of cell viability and the level of ROS production at all CGRP-dose levels and time points shown in Fig. 1 and 2.

• 

  Fig. 5 ROS scavenging reduces ROS production and cell death induced by exogenous CGRP in kidney proximal tubule epithelial cells. HK-2, LLC-PK1, and TCMK-1 cells were treated with 1 nM of CGRP for 24 hours. One hour before treatment with CGRP, cells were treated with 10, 30, or 100 µM of MnTMPyP in PBS (vehicle)(n=9 wells per 3 independent experiments per group). In box plots, whiskers represent the minimum and maximum; bases represent the interquartile range between the first and third quartiles; and midlines represent the median. #P<0.05, ##P<0.01 versus vehicle. (a) ROS production was measured using oxidative sensitive dye DCFDA. (b) Cell viability was measured using MTT assay.

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