NADPH Oxidases Mediate Zinc Deficiency-Induced Oxidative Stress in Mouse Kidneys Open Access

Li, Mirandy Schiphen (2016)

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Zn2+ deficiency (ZnD) is comorbid with Chronic Kidney Disease (CKD). Experimental studies show that ZnD worsens kidney complications. Oxidative stress caused by increased reactive oxygen species (ROS) plays a role in the harmful effects of ZnD. However, the sources of oxidative stress continue to be identified. In the kidney, NADPH oxidases (Nox) are enzymes that promote oxidative stress. The objective of this study is to determine the role of Nox in ZnD-induced oxidative stress. We hypothesize that ZnD upregulates Nox resulting in sustained ROS levels and kidney damage. WT mice were pair-fed a ZnD- or control-diet for 6 weeks. After collecting kidneys and urine, kidney damage (urinary albumin:creatinine ratios), ROS levels (Amplex Red and 2',7'-dichlorofluorescein diacetate) and Nox expression (qRT-PCR and Western blot) were examined. To further investigate the effects of Zn2+ bioavailability on Nox, mouse tubular epithelial cells (mTECs) were exposed to the Zn2+ chelator N,N,N,N-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or vehicle (DMSO) for 24 hours ± Zn2+ supplementation for an additional 24 hours. Mice fed a ZnD-diet develop microalbuminuria that is accompanied by elevated renal Nox2 expression and ROS levels compared to control mice. In mTECs, Nox inhibition with diphenyleneiodonium (DPI) blocks TPEN-induced ROS levels. Furthermore, TPEN-induced Nox2 upregulation is attenuated with Zn2+ supplementation. Nox2 knockdown via si-RNA inhibits Zn2+ depletion-induced H2O2 generation. These findings identify Nox2 as a mediator of ZnD-induced oxidative stress and kidney damage. Understanding the mechanisms by which ZnD contributes to kidney injury may have important impact on the treatment of chronic kidney disease.

Table of Contents

Introduction: 1-3

Chronic Kidney Disease: 1

Zn2+ Deficiency in Chronic Kidney Disease: 1

Oxidative Stress in Chronic Kidney Disease: 1

Sources of Oxidative Stress: 2

NADPH oxidase in Oxidative Stress: 2

Study Objective: 3

Materials and Methods: 4-7

Zn2+ Deficiency Models: 4

Zn2+ Assessment: 4

Kidney Injury Assessment: 5

Reactive Oxygen Species Measurement: 6

NADPH Oxidase Expression: 6

NADPH oxidase Activation: 7

Statistical Analysis: 7

Results: 8-11

Dietary Zn2+ restriction promotes kidney injury in mice: 8

Dietary Zn2+ restriction stimulates renal reactive oxygen species

generation and NADPH oxidase upregulation in mice: 8

Intracellular Zn2+ modulates reactive oxygen species generation in mTECs: 8

NADPH oxidases mediate Zn2+ deficiency-induced oxidative stress in mTECs: 9

Intracellular Zn2+ modulates NADPH oxidases in mTECs: 10

Nox2- and Nox4-containing NADPH oxidases mediate Zn2+

deficiency-induced oxidative stress and cellular hypertrophy in mTECs: 10

Discussion: 20-22

References: 23-26

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