**1. Introduction**

Diabetes mellitus is currently a serious public health problem that affects millions of individuals worldwide. Diabetic nephropathy (DN), also called diabetic kidney disease, is one of the most important long-term complications in terms of morbidity and mortality for individual patients with diabetes. It is also accepted as the leading cause of end-stage renal disease in many countries around the world [1]. The latest report by the International Diabetes Federation (IDF) shows that half a billion people worldwide are diabetic, and more than 80% of cases of end-stage renal disease are caused by diabetes [2]. Thailand is among the countries in Asia with a high prevalence of diabetes. Currently, diabetes in the Thai population is estimated at 4.2 million cases, or 7–8% of the population, of which about 44% are experiencing diabetic kidney disease [2,3]. Global records, including Thailand, indicate that the number of diabetic patients who require renal replacement therapy has progressively increased over the last two decades [4]. The issue not only produced a significant burden on economics but also caused psychosocial problems in

**Citation:** Wongmekiat, O.; Lailerd, N.; Kobroob, A.; Peerapanyasut, W. Protective Effects of Purple Rice Husk against Diabetic Nephropathy by Modulating PGC-1α/SIRT3/SOD2 Signaling and Maintaining Mitochondrial Redox Equilibrium in Rats. *Biomolecules* **2021**, *11*, 1224. https://doi.org/10.3390/biom11081224

Academic Editors: Liang-Jun Yan and Juan Antonio Moreno Gutiérrez

Received: 23 May 2021 Accepted: 16 August 2021 Published: 17 August 2021

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terms of reduced quality of life and suffering of the patients. Importantly, data from the Thai National Health Examination Survey and the IDF atlas reveal that diabetes-related deaths are approximately 12–15% of total deaths in Thailand [2,5,6]. Finding strategies to prevent or slow down the onset and progression of kidney disease in people with diabetes remains an important and necessary task.

Rice is the seed of the grass species *Oryza sativa*. It is the agricultural commodity with the third highest worldwide production. Rice is the most widely consumed staple food in over half of the world's population, particularly in Asia and Africa [7]. Though rice can be cultivated worldwide, more than 90% is grown in Asia. Nowadays, the trend of health promotion with natural supplements is becoming increasingly popular. Rice, especially colored rice, is one of the popular choices, as it contains nutrients, vitamins, and phytochemicals. There is a variety of colored rice, including brown, red, and purple (or black) rice. Among these colored rice, purple rice has received the most considerable interest due to its anthocyanin content, which is higher by weight than that of other colored grains [8]. Studies have shown several health benefits of extracts from diverse parts of purple rice, i.e., rice grain and rice bran [9,10], and thus greatly increased the demand for this colored rice. Rice husks, a major organic source of waste of the rice milling process, are also accumulated. These wastes are often removed by burning, causing air pollution that directly affects climate change, quality of life, and human health. Therefore, turning these agricultural wastes into valuable stuffs is of interest and becomes challenging research. A study regarding rice husk, particularly from pigmented rice, indicated that rice husk is a good source of phytochemicals with antioxidant activity and suggested that it may have a great potential to turn into functional food or nutraceuticals to prevent diseases related to oxidative stress [11]. Recently, extract from purple rice husk has been shown to be a potent anticancer agent without any toxicity [12].

Mitochondria is well recognized as a major source of reactive oxygen species (ROS) production in the body, and thus, the maintenance of mitochondrial oxidative balance is essential. The peroxisome proliferator-activated receptor gamma coactivator 1-alphasirtuin 3-superoxide dismutase 2 (PGC-1α-SIRT3-SOD2) axis plays a significant role in the regulation of mitochondrial redox homeostasis [13,14]. PGC-1α is a nuclear-encoded transcriptional coactivator that regulates the expression of several nuclear-encoded mitochondrial proteins, including mitochondrial antioxidant and biogenesis genes [14]. SIRT3, a downstream target of PGC-1α, is a major mitochondrial deacetylase that directly deacetylates and activates numerous mitochondrial proteins, including the major mitochondrial antioxidant enzyme SOD2 [13–15]. SIRT3 is also being accepted as a global regulator playing a multifaceted role in the mitochondrial adaptive response to stress and is currently indicated as a new target for therapy aimed at improving end-organ damage and survival [14,15].

As reactive oxygen species (ROS) production, oxidative stress generation, and, particularly, mitochondrial dysfunction are central to the pathogenesis of diabetic nephropathy [1,16], it is assumed that purple rice husk extract (PRHE) may be able to prevent or reduce diabetes-induced renal deterioration. Herein, we examined the renoprotective potential of PRHE in a rat model of high-fat diet/streptozotocin-induced type 2 diabetes (T2DM) and explored whether the modulation of the PGC-1α-SIRT3-SOD2 axis contributes to protection by PRHE.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Preparation of PRHE*

Thai purple rice (*Oryza sativa* L. var. Indica) cv. Kum Doisaket was first identified by Associate Professor Dr. Dumnern Karladee, Faculty of Agriculture, Chiang Mai University, Thailand (Voucher Specimen No.: Rawiwan\_001) and planted at Mae Hia Agricultural Research, Faculty of Agriculture, Chiang Mai University. After harvested, the purple rice was confirmed again by comparing it to the known specimen identity deposited at the Faculty of Pharmacy, Chiang Mai University (Herbarium No.: 023252). Then, the rice husk

was separated from the rice paddy and soaked in 0.1% hydrochloric in absolute methanol for 48 h at room temperature. The procedure was repeated twice, and the entire solution was filtered through Whatman No. 1 filter paper. The supernatant was evaporated under reduced pressure and lyophilized to obtain PRHE, which was kept in tight-sealed dark containers and stored at −20 ◦C for further studies.
