Clinical Science: this is an Accepted Manuscript, not the final Version of Record. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date version is available at http://dx.doi.org/10.1042/CS20180148. Please cite using the DOI 10.1042/CS20180148
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Probiotic Lactobacillus paracasei HII01 protects rats against obese-insulin resistance-
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induced kidney injury and impaired renal organic anion transporter 3 (Oat3) function
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Keerati Wanchaia,g, Sakawdaurn Yasomb, Wannipa Tunaponga,c, Titikorn Chunchaia,c,h,
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Sathima Eaimworawuthikulc,e,h, Parameth Thiennimitrb, Chaiyavat Chaiyasutd, Anchalee
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Pongchaidechaa, Varanuj Chatsudthipongf, Siriporn Chattipakornc,e,h, Nipon Chattipakorna,c,h
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and Anusorn Lungkaphina,i,*
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a
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b
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c
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University, Thailand
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d
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Thailand
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e
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University, Thailand
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f
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g
School of Medicine, Mae Fah Luang University, Chiang Rai, Thailand
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h
Center of Excellence in Cardiac Electrophysiology Research, Chiang Mai University,
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Chiang Mai, Thailand
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i
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University, Thailand
Department of Physiology, Faculty of Medicine, Chiang Mai University, Thailand
Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University,
Department of Oral Biology and Diagnostic Sciences, Faculty of Dentistry, Chiang Mai
Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand
Center for Research and Development of Natural Products for Health, Chiang Mai
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Use of open access articles is permitted based on the terms of the specific Creative Commons Licence under 1 which the article is published. Archiving of non-open access articles is permitted in accordance with the Archiving Policy of Portland Press ( http://www.portlandpresspublishing.com/content/open-access-policy#Archiving).
ACCEPTED MANUSCRIPT
Department of Microbiology, Faculty of Medicine, Chiang Mai University, Thailand
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Running head: Probiotic, obesity and renal function
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Corresponding author
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Anusorn Lungkaphin, Department of Physiology, Faculty of Medicine,
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Chiang Mai University, Intravaroros Road, Chiang Mai, Thailand 50200
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Tel.: 6653-945362-4; fax: 6653-945365
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E-mail address:
[email protected];
[email protected]
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Clinical Perspectives
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i Obesity is a complex disease that involves the progression of complications in various
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organs. Gut microbiota has been found to be changed by obesity and also associated with the
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impairment of renal function.
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ii Obese rats showed significant increases in serum LPS, plasma lipid profiles, and insulin
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resistance. Renal Oat3 function was impaired in HF rats, this impairment being associated
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with a decrease in kidney function. We found the upregulations of inflammatory cytokines,
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ER stress and apoptotic markers in the kidney tissues of HF rats.
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gluconeogenesis was also increased in the kidney of HF rats.
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improved by Lactobacillus paracasei HII01 treatment.
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iii Probiotic supplementation alleviated kidney inflammation, ER stress, and apoptosis,
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leading to improved kidney function and renal Oat3 function in obese-insulin resistant rats.
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These benefits involve the attenuation of hyperlipidemia, low-grade systemic inflammation
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and insulin resistance.
In addition,
These alterations were
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3
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ABSTRACT
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The relationship between gut dysbiosis and obesity is currently acknowledged to be a health
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topic which causes low-grade systemic inflammation and insulin resistance and may damage
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the kidney. Organic anion transporter 3 (Oat3) has been shown as a transporter responsible
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for renal handling of gut microbiota products which involved in the progression of metabolic
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disorder. This study investigated the effect of probiotic supplementation on kidney function,
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renal Oat3 function, inflammation, ER stress and apoptosis in obese insulin-resistant rats.
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After 12 weeks of being provided with either a normal or a high-fat diet (HF), rats were
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divided into normal diet; normal diet treated with probiotic; high-fat diet; and high-fat diet
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treated with probiotic. Lactobacillus paracasei HII01 1x108 CFU/ml was administered to the
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rats daily by oral gavage for 12 weeks. Obese rats showed significant increases in serum
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LPS, plasma lipid profiles, and insulin resistance. Renal Oat 3 function was decreased along
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with kidney dysfunction in high-fat diet-fed rats. Obese rats also demonstrated the increases
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in inflammation, ER stress, apoptosis and gluconeogenesis in the kidneys. These alterations
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were improved by Lactobacillus paracasei HII01 treatment. In conclusion, probiotic
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supplementation alleviated kidney inflammation, ER stress, and apoptosis, leading to
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improved kidney function and renal Oat3 function in obese rats. These benefits involve the
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attenuation of hyperlipidemia, systemic inflammation and insulin resistance. This study also
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suggested the idea of remote sensing and signaling system between gut and kidney by which
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probiotic might facilitate renal handling of gut microbiota products through the improvement
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of Oat3 function.
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Keywords: Probiotic; Kidney function; Organic anion transporter 3; Obesity; Inflammation;
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ER stress
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Abbreviations: Akt, protein kinase B; Bax, bcl-2-associated protein; CFU, colony forming
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unit; CHOP, CCAAT-enhancer-binding protein homologous protein; COX2,
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4
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cyclooxygenase 2; ER, endoplasmic reticulum; GPR78, 78 kDa glucose-regulated protein;
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IL-1E, interleukin 1 beta; IL-6, interleukin 6; JNK, c-Jun N-terminal kinase; LPS,
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lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; NF-NBp65, nuclear factor
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NF-kappa-B p65 subunit; Oat3, organic anion transporter 3; PEPCK, phosphoenol pyruvate
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carboxy kinase; PI3K, phosphoinositide 3-kinase; PKCD, protein kinase C alpha; TNF-D,
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tumor necrosis factor-alpha; TNF-R1, tumor necrosis factor receptor 1
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INTRODUCTION Obesity is a complex disease that involves the progression of complications in various
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organs. Several lines of evidence, from human-based studies, have shown a relationship
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between obesity, and the development and the progression of chronic kidney disease (CKD)
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[1-4]. Obesity is defined as a chronic inflammatory disease and shows to increase the
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production and secretion of adipokines and pro-inflammatory cytokines [5]. Recently, it was
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shown that tumor necrosis factor (TNF)-D plays a role in obesity-induced kidney
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inflammation and oxidative stress leading to impaired kidney function in high-fat diet fed-
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mice [6]. It could be proposed that chronic inflammation might be a factor describing an
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underlying pathology of obesity-induced kidney injury. In addition, it has also been reported
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that obesity is associated with endoplasmic reticulum (ER) stress which could lead to
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inflammation in adipose tissues and induced insulin resistance [7]. This mechanism involved
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in obesity increased free fatty acid-mediated reactive oxygen species (ROS) production. ER
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is an essential organelle responsible for protein homeostasis, including protein folding, post-
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translational modification and protein secretion. In a condition which increase in the
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accumulation of unfolded proteins in the ER, resulting in ER stress, the cells have a
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mechanism called unfolded protein response (UPR) for maintaining ER function. However,
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the excessive ER stress leads to the failure of this system and ultimately activates apoptosis
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pathway [8]. It could be suggested that the occurrence of ER stress may be found in the
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kidney in case of obesity and causes kidney damage.
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In the past few years, studies have extensively shown that high-fat diet could
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modulate intestinal environment and the composition of gut microbiota, which shows the
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relationship in obesity and insulin resistance conditions [9, 10]. Gut dysbiosis causes the
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impairment of the gut barrier and leads to an increased level of gut-derived endotoxins, such
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as lipopolysaccharides (LPS), in the circulation. This endotoxin could circulate reaching
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various organs, activating the immune system in those organs, resulting in an induction of an
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inflammatory response [11]. It has been reported that LPS could circulate to the kidney and
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induce inflammation and the oxidative stress pathway that goes on to promote kidney injury
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[12]. A previous study found that a receptor for LPS, Toll-like receptor 4 (TLR4), was
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increased and was related to the high levels of NF-NB p65 and TNF-D in kidney tissues of
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diet-induced obese mice [13]. In a mouse model of LPS-induced acute kidney injury, it was
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also shown that LPS could induce TLR4 expression and NF-NB activation, followed by the
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increased production of TNF-D, IL-6 and IL-1E [14]. These studies demonstrated that a
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significant role of this gut endotoxin could promote kidney injury that might occur in the
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obese condition.
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Kidney is an important organ containing transporters called organic anion transporters
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(Oats) in the proximal tubule for excreting organic anion substances and drugs [15]. Beyond
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this common role of Oats, a number of metabolomic studies have been demonstrated an
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endogenous role of this transporter on transporting systemic hormones, signaling molecules
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and products by gut microbiota metabolism [16]. There are evidence showed that an
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alteration in systemic level of these molecules have been found and associated with
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pathophysiological stages such as metabolic syndrome, diabetes, liver disease, diabetic
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nephropathy and CKD [17, 18]. Among the Oat members, it has been shown that Oat3
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appeared to have an important role in modulating the levels of gut-derived uremic toxins
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[19]. Previous studies reported that expressions of Oat1 and Oat3 were reduced in the
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experimental model of renal failure resulting in increased serum uremic toxins [20, 21]. The
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regulation of Oat activity is depended on the activation of specific protein kinase C (PKC)
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isoform [22]. It has been demonstrated that down regulation of Oat3 is activated by PKCD
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that involved in internalization of Oat3 [23]. In addition, our previous study has been
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reported that impaired insulin signaling as shown by the decreases in Akt and PKC] activities 7
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could affect renal Oat3 trafficking and decrease Oat3 expression in the kidneys of type 1
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diabetic rats [24, 25]. However, the expression and function of this renal transporter under
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the condition of obesity and associated low-grade systemic inflammation have not been
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properly understood.
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A probiotic is defined as a live microorganism that when administrated in sufficient
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amounts can exert beneficial effects on host heath [26]. It has been reported that the
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modulation of gut microflora by probiotic supplementation is an effective way for attenuating
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gut dysbiosis [27]. The most commonly used probiotics are various strains of living bacteria
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including Lactobacilli and Bifidobacteria [28]. The anti-obesity effect and the improvement
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of the metabolic effects of probiotics have been documented. Previous studies demonstrated
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that Lactobacilli had a hypocholesterolemic effect by inhibiting enzyme 3-hydroxy-3-
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methylglutaryl-coenzyme A (HMG-CoA) reductase and enhanced bile salt hydrolase activity
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[29, 30]. Treatment with Lactobacillus plantarum has been shown to decrease body weight
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and the plasma lipid profiles in high-fat diet-induced obese rats. These findings were
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accompanied by the attenuation of systemic inflammation and a reduction in kidney injury
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and dysfunction [31]. Therefore, the link between obesity and gut dysbiosis could affect
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kidney function via the induction of systemic inflammation. Of note, the modulation of gut
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bacteria by the usage of probiotics, may be a targeted therapy for treatment of kidney injury
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in obesity.
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We designed experiments to investigate an intervention of a probiotic on kidney and
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renal Oat3 function in high-fat diet induced obese-insulin resistant rats. Our hypothesis was
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that high-fat diet could induce hyperlipidemia, low-grade systemic inflammation and insulin
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resistance leading to impaired kidney function and renal Oat3 function. Oral probiotic
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supplementation could restore lipid profiles, systemic inflammation and insulin resistance
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leading to a reduction in kidney inflammation, ER stress and apoptosis, resulting in the
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improvement of kidney function and renal Oat3 function.
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MATERALS AND METHODS
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Animals
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Experiments were carried out on six-week-old male Wistar rats (200-250g). They
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were purchased from the National Animal Center, Salaya campus, Mahidol University,
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Bangkok, Thailand and housed under temperatures of 25r1qC in a 12 hr light/dark cycle. All
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animal experiments were approved by the Laboratory Animal Care and Use Committee at
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Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand (Permit No: 13/2558).
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The high-fat diet included 59.28% energy from fat with an energy content of 5.35
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kcal/g was provided for the rats for 12 weeks. Control rats were fed with a normal standard
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pelleted diet (19.77% energy from fat, energy content of 4.02 kcal/g). The compositions of
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fatty acids in control diet are ~14% saturated (sFA), ~28% monounsaturated (MUFAs), and
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~58% polyunsaturated (PUFAs) from soybean oil. For the high-fat diet, additional fat from
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lard which is the source of fatty acid composition was added. The fatty acid profile of lard is
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~37% sFA, ~46% MUFAs, and ~17% PUFAs. Lard has specifically high amounts of
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palmitic, steric and oleic acid [32]. This high-fat diet composition has shown to induce
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insulin resistance and obesity in the rats after 12 weeks of diet providing [33, 34]. On week
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12, serum LPS was measured for the detection of systemic inflammation. After that, the rats
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in each group were randomly divided into 2 groups of six animals each: (1) vehicle normal
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diet (ND); (2) normal diet treated with probiotics (NDL); (3) vehicle high-fat diet (HF); (4)
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high-fat diet treated with probiotics (HFL). The probiotic used in this study was
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Lactobacillus paracasei HII01 which was prepared by Assoc. Prof. Dr. Chaiyavat Chaiyasut
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and his colleagues from the Faculty of Pharmacy, Chiang Mai University. This probiotic is a 9
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novel non-human origin-isolated strain of lactic acid-producing bacteria which has been
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approved by the Food and Drug Administration (FDA), Thailand. A live probiotic
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Lactobacillus paracasei HII01 in a safety dose, 1x108 colony forming unit (CFU), was
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administered to the rats daily by oral gavage for 12 weeks while vehicle control rats were
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given 2 ml of sterile phosphate buffered saline (PBS). This Lactobacillus strain has shown its
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positive effects on metabolic parameters including insulin resistance, hyperlipidemia, and
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systemic inflammation and oxidative stress in obese rat model [34, 35]. Moreover, treatment
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with probiotic for 12 weeks could improve systemic inflammation, gut microbiota
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disturbance and restore liver and kidney functions in rat models [36, 37].
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After 12 weeks of probiotic treatment, blood and urine were collected and an oral
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glucose tolerance test was performed. The animals were anesthetized using isoflurane
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inhalation, decapitated and then the kidneys were collected. The kidney tissues were used for
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the determination of Oat3 function and expression, histological study, TUNEL assay, and the
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measurement of protein expression.
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Biochemical and urinary analysis
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Blood samples were collected from the lateral tail vein after the animals were starved
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for 5-6 hours. Plasma glucose and triglyceride concentrations were determined by the
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enzymatic colorimetric method using a commercial kit (Biotech, Bangkok, Thailand,
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Bioversion). Plasma HDL was determined using a sensitive, colorimetric and fluorometric
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assay from a commercially available kit (Biovision Inc., Milpitas, USA) and plasma LDL
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was calculated using the Friedewald equation [38]. Plasma insulin was determined by ELISA
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method using a commercial assay kit (LINCO Research, MO, USA). Serum creatinine was
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measured by enzymatic colorimetric methods using commercial kits (Diasys Diagnostic
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Systems GmbH, Holzheim, Germany). Serum LPS level was determined using a Pierce
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limulus amebocyte lysate (LAL) chromogenic endotoxin quantitation assay kit (Thermo
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scientific, USA).
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For urinalysis, urine was collected by placing the animals in metabolic cages for 24
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hours. Urine microalbumin and creatinine levels were determined using an automatic
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biochemical analyser at the Clinical Laboratory, Maharaj Nakhon Chiang Mai Hospital,
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Chiang Mai, Thailand. Estimated glomerular filtration rate (eGFR) for the investigation of
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renal function was calculated by the measurement of creatinine clearance.
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Oral glucose tolerance test (OGTT) and HOMA index
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To measure glucose intolerance, an OGTT was performed at week 24. After fasting
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for 12 hours, the animals were given glucose solution (2 g/kg body weight), orally. A blood
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sample was collected to determine plasma glucose levels at 0, 15, 30, 60 and 120 min
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intervals. Total area under the curve (AUC) of OGTT was calculated. To determine the
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peripheral insulin resistance, Homeostasis Model Assessment (HOMA-IR) index was also
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calculated from the levels of fasting plasma insulin and plasma glucose as previously
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described [39].
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Histological study
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Kidney histomorphological assessment was performed using hematoxylin and eosin
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(H&E) staining. The kidney samples were cut and fixed in 10% neutral buffered formalin
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and embedded in paraffin. After that, 2 μm-thick of kidney sections in paraffin were
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mounted on microscope slides and stained with hematoxylin and eosin. The glomerular and
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tubular changes were observed under a light microscope by an observer blinded to the animal
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group. The kidney damage was scored in terms of the enlargement of Bowman’s capsule
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space around the glomerulus, tubular dilatation, sloughing of cells into the tubular lumen and
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infiltration by inflammatory cells. Each damage indicator was graded in a range between 0
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and 4, based on the percentage of the injury: 0 = 75%, as previously described [40]. The samples were scored using 5
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randomly accessed fields/rat and 5 rats/group. The mean score of each rat and each group
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were calculated and expressed as kidney injury score.
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Renal Oat3 transporter function
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The function of renal Oat3 was determined by the uptake of [3H] estrone sulfate (ES)
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into a renal cortical slice. Briefly, the kidney was decapsulated and cut into thin renal cortical
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slices 0.5 mm; 5-25 mg, wet weight) using a Stadie-Riggs microtome. The tissue slices
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were preincubated for 30 min in freshly-oxygenated ice-cold modified Cross and Taggart
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saline buffer pH 7.4, composed of 95 mM NaCl, 80 mM mannitol, 5 mM KCl, 0.74 mM
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CaCl 2 , and 9.5 mM Na 2 HPO 4 . The slices were then incubated in 1 ml of buffer containing
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50 nM [3H]ES ([3H]ES; Perkin Elmer, Waltham, MA, USA) for 30 min at room temperature.
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After that, 0.1 M MgCl 2 was used to wash the tissue slices. The tissues were weighed and
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shaken in a solution of 0.4 ml of 1 M NaOH overnight. The solution containing the tissues
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was then neutralized with 0.6 ml of 1 N HCl before radioactivity assessment using a liquid
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scintillation analyzer (Perkin Elmer, MA, USA). The percentage of [3H]ES uptake into the
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renal cortical tissue was calculated for demonstrating the function of Oat3.
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Apoptosis measurement by TUNEL assay
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Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
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(Calbiochem®DNA Fragmentation Detection Kits, Millipore, Billerica, MA, USA) was
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performed to detect DNA fragmentation as an indicator of kidney apoptosis. The paraffin-
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embedded kidney tissue sections were used to detect apoptotic nuclei using the principle that
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the terminal deoxynucleotidyl transferase (TdT) binds to exposed 3’-OH ends of DNA
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fragments. A brown insoluble colored substrate was seen at the site of DNA fragmentation
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after detection using a streptavidin-horseradish peroxidase (HRP) conjugated biotinylated 12
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nucleotides. These colored substrates were examined and calculated in five field areas per rat
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under a light microscope. The total TUNEL-positive cells are shown by the number of
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TUNEL positive cells per field.
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Western blotting
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Renal cortical protein was extracted using a lysis buffer (Sigma Chemical Co, St.
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Louis, MO, USA) with a protease inhibitor cocktail (Roche Applied Science, IN, USA). A
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BCA protein assay kit was used for total protein concentration measurement. Protein
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samples were mixed with a loading buffer and subjected to 10-15% SDS-PAGE and then
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transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking non-
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specific proteins with 5% non-fat dry milk in Tris-buffered saline (TBS) or PBS containing
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0.1% Tween-20, the membranes were incubated overnight at 4 qC with specific primary
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antibodies: Oat3 (KAL-KE035) from CosmoBio Co. Ltd., Tokyo, Japan; IL-6 (sc-1265, Lot
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F1015) and GRP78 (sc-1051, Lot E1215) from Santa Cruz Biotechnology, Santa Cruz, CA,
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USA; phosphorylated PKCD (9375, Lot 4), phosphorylated NF-NBp65 (3031, Lot 10), TNF-
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R1 (13377, Lot 1), COX2 (12282, Lot 2), CHOP (2895, Lot 10), Calpain2 (2539, Lot 2),
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phosphorylated Akt Ser473 (9271, Lot 13), PI3K (4292, Lot 4), Cytochome C (4272, Lot 6)
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and Bcl-2 (2870, Lot 5) from Cell Signaling Technology, Inc., Danvers, MA, USA; TNF-D
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(AB1837P, Lot 2839768), IL-1E (AB1832P, Lot 2828618), MCP-1 (AB1834P, Lot
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2761650), Caspase12 (MABC555, Lot Q2279643), Bax (04-434, Lot 2492230), Caspase 3
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(AB3623, Lot 2376423), SGLT1 (07-1417, Lot 2730935), JNK (06-748, Lot 2779430), E-
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actin (04-1116, Lot 2324657), GAPDH (ABS16, Lot 2794839) and Na+-K+ATPase (05-369,
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Lot 2384712) from Millipore, Billerica, MA, USA; and SGLT2 (ab37296, Lot GR173249-8)
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and PEPCK (ab87340, Lot 931569) from Abcam, Cambridge, UK. Secondary antibodies
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were used after incubating the membranes with a primary antibody. Signal of protein band
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expression was visualized using an enhanced chemiluminescent (ECL) reagent (Bio-Rad 13
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Laboratories, Inc., PA, USA) and imaged using the ChemiDoc Touch Imaging System (Bio-
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Rad Laboratories, Inc., PA, USA). The protein expression was quantified by Image J (Adobe
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Corp., CA, USA) normalized to ȕ-actin or GAPDH as a control.
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Statistical analysis
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Results were presented as mean ± SE. Data were analyzed using SPSS software,
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version 16 (SPSS Inc., USA). Differences between two and four groups were determined by
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an independent-samples t-test and a One-way ANOVA followed by Fisher’s Least
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Significant Difference test (LSD), respectively. Statistical significance was accepted at a p
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value less than 0.05.
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RESULTS
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The metabolic and kidney parameters after provision of a high-fat diet
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Obesity was induced in the rats by provision of a high-fat diet for 12 weeks. The
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metabolic and kidney parameters following the 12 weeks are shown in Table 1. Rats fed on a
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HF showed a remarkable increase in body weight when compared to the normal diet-fed rats
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(ND group) (p < 0.05). HF rats had about 60% increased in weight gain, while ND rats had
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about 40%. The increase in percentage of weight gain in HF rats was significantly higher
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than that of the ND rats (p < 0.05). Energy intake and plasma LDL levels were significantly
295
higher in the HF group when compared with the ND group (p < 0.05), these results indicated
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that obesity and hyperlipidnemia were developed in HF rats. Plasma HDL, triglyceride and
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fasting blood glucose levels, were not significantly different when compared between HF and
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ND groups. However, we found that HF rats showed a significant increase in plasma insulin
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level (p < 0.05) and exhibited signs of insulin resistance, as indicated by a significant increase
300
in HOMA-IR index, compared to the control group (p < 0.05). The provision of a high-fat
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diet to the HF group did not alter 24 hour-urine volume, water intake, urine creatinine, serum 14
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creatinine and eGFR when compared to those of ND group. However, microalbumin
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concentration in the urine was significantly increased in the HF group when compared with
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ND rats (p < 0.05), demonstrating possible glomerular injury in the HF rats. The results
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suggested that kidney injury was detected in HF rats after 12 weeks of high-fat diet
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consumption.
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The metabolic and kidney parameters after L. paracasei HII01 supplementation
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After 12 weeks of supplementation with the probiotic L. paracasei HII01 in HFL rats,
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body weight was not significantly reduced when compared to the HF group (Table 2). The
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increase in body weight was consistent with the increase in the percentage of weight gain
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which was not significantly different when compared between HF and HFL groups. When
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comparing the HF and HFL groups, the plasma HDL and fasting blood glucose levels were
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not significantly changed. Plasma insulin level and HOMA-IR index were greater in the HF
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group, indicating a greater propensity towards insulin resistance than that in the ND group (p
315
< 0.05), and these results were significantly recovered by probiotic supplementation in the
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HFL group (p < 0.05). In addition, hyperlipidemia was observed in the HF group as
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confirmed by the significantly increased plasma triglyceride and LDL levels when compared
318
to those of control group (p < 0.05). Oral supplementation with L. paracasei HII01 in HFL
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rats could significantly reduce these lipid profiles when compared to HF group (p < 0.05).
320
However, probiotic L. paracasei HII01 supplementation in NDL rats did not affect metabolic
321
parameters when compared to those of ND rats. These data suggested that although L.
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paracasei HII01 has no effect on the reduction in weight gain, it could attenuate the increase
323
in plasma LDL and triglycerides caused by high-fat diet consumption.
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At week 24, kidney weight to body weight ratio was significantly lower in the HF
325
group than that of the ND or NDL group (p < 0.05). Treatment with the probiotic did not
326
affect this parameter. Urine volume, serum creatinine and urine creatinine were not 15
327
significantly changed among 4 groups. However, eGFR was significantly decreased in the
328
HF group when compared to the ND or NDL group (p < 0.05), indicating that kidney
329
function was impaired in HF rats. Lactobacillus paracasei HII01 administration in HFL rats
330
resulted in the significant restoration of eGFR when compared to HF rats (p < 0.05). In
331
addition, severe microbuminuria was observed in the HF group when compared to the ND or
332
NDL group (p < 0.05). After supplementation with L. paracasei HII01 in HFL rats, the level
333
of microalbumin in the urine was significantly reduced when compared to the HF rats (p
