Effects of Lupinus luteus on hepatic and renal extracellular compounds turnover under diabetes in rat

Abstract Hepatic and renal extracellular matrix (ECM) turnover associated with diabetes and potential beneficial effects of yellow lupin extract (YLE) need further investigations. The aim of this study was to explore the effect of yellow Lupinus luteus extract (YLE) on renal and hepatic ECM under diabetes. Composition of YLE performed by LC‐ESI‐MS. Diabetes (DM) was induced in rats by alloxan (250 mg/kg, ip). Normal and diabetic rats received 100 mg/kg of YLE for 1 month. ECM was assessed by ELISA. Gelatinases and collagenases were analyzed by a colorimetric assay. Histology was performed on sections of liver and kidney. In the liver, diabetes increases collagen, laminin, and fibronectin contents, respectively, by 49% (p < .01), 56% (p < .01), and 67% (p < .05) compared to control rats. In the kidney, total collagen and laminin contents were increased by 91% (p < .01) and 35% (p < .01) in the DM group, while fibronectin content in diabetic animals and those treated with YLE remains similar to the control group. Collagenases and gelatinases activities were significantly increased by diabetes in liver and kidney. While YLE treatment abrogates diabetes‐enhanced MMPs activities in liver. In diabetic rats, the liver shows signs of diffuse dilatation of the sinusoid veins and steatosis. However, the liver of diabetic rats treated with yellow lupine extract showed a normal histological aspect similar to controls. Diabetes causes hepatic and renal ECM turnover. YLE can be useful to partially improve tissue disorders induced by diabetes.

The Yellow Lupin (Lupinus luteus) is an annual plant reaching 80 cm in height and cultivated in Mediterranean regions (Parra-González et al., 2012). Lupin was used in a variety of food applications such as to prepare bread for diabetic patients and also for animal feed (van de Noort, 2017). Several species of lupin have been studied for their therapeutic benefits and their antidiabetic effect such Lupinus albus and Lupinus mutabilis (Garmidolova et al., 2022).
However, there are no previous studies interested in the effect of L. luteus on diabetes.
In this context, the aim of this study is to investigate the involvement of diabetes in hepatic and renal ECM remodeling and the potential beneficial effects of L. luteus extract.

| Preparation of yellow lupine extracts
L. luteus seeds were ground using a food processor. Fifteen gram of the powder was extracted in 100 ml of ethanol 50% solvent and stirred for 24 h at room temperature in dark. The mixture was filtered on filter paper to remove the solid particles and solvents were removed using a rotary evaporator.

| Chromatography of phenolic acids and flavonoids by LC-ESI-MS
Separation of phenolic extracts, sugars, and vitamins was performed using LC-ESI-MS on Shimadzu UFLC XR system, equipped with SIL-20AXR autosampler, SCL-10A system controller, AC CTO-20 column, LC-20ADXR binary pump, and a 2020 quadrupole detection system. This instrument was equipped with a Discovery BIO Wide Pore C18 column (S250 × 4.0 mm id; 5 μm). The column temperature was set at 40°C and the injection volume was 5 μl with a flow rate of 0.5 ml/ min. Water with 0.1% formic acid and methanol with 0.1% formic acid were used as mobile phases A and B, respectively. Samples were analyzed using a linear gradient programmed as follows: 0-14 min,

| Determination of the content of total nitrogenous matter, total sugars, and vitamins
Total nitrogen was determined by the Kjeldahl method. The experimental approach consisted of mixing 0.5 g of ground and homogenized lupine powder with 12 ml of concentrated sulfuric acid and 1 g of selenium catalyst in a macro-Kjeldahl tube, which was then placed in the digester for at least 60 min at 450°C. After digestion, 50 ml of sodium hydroxide (35%) was added, and ammoniacal nitrogen was recuperated in a 4% boric acid solution.
Obtained distillate was titrated with a hydrochloric acid solution HCL (0.1 N).

| Animals
The animal protocols were approved by the University Animal Care and Use Committee of the University of Gabes, Tunisia, and were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Wistar rats were purchased from the Faculty of Sciences of Gabes, Tunisia. After an adaptation period of 2 weeks, all rats were maintained at the same conditions of 12-h light/dark cycle, temperature (23°C), humidity (70%), and fed a commercial pellet diet and tap water ad libitum.
CTRL group: Normal rat group (n = 8) has received an intraperitoneal injection of saline solution (0.9%) for 1 month.
Diabetes was induced in rats according to previous protocol (Dab et al., 2022). Briefly, a single intraperitoneal dose of alloxan (250 mg/ kg) from Sigma Chemical Co. (St Louis, MO, USA) dissolved in saline (0.9% NaCl) solution was used. Animals were injected after an 18-h fasting period and received 10% of glucose solution 24 h following injection. Blood glucose level was monitored daily with an electronic glucometer (ACCU-CHEK®, Roche, India) by performing a small puncture at the tail.
At the end of the various treatments, each rat was weighed and sacrificed by overdose of urethane. Blood was collected from the inferior vena cava, and the kidney and liver were removed, frozen immediately, and stored at −80 °C.

| Assay of serum parameters
The levels of urea, creatinine, triglycerides, cholesterol, HDL, AST, and ALT were assessed by an electronic automatic BA400 analyzer (Biosystems S.A., Spain) at the Habib Bourguiba University Medical Center, Medenine (Tunisia).

| Total protein extraction
Protein extraction from liver and kidney was performed according to a previous protocol (Dab et al., 2022). Organs were ground in a lysis buffer (0.25 M sucrose, 0.05 M Tris-HCL, 1 mM EDTA, pH 7.4).
After 24 h of incubation at −20°C, the samples were centrifuged at 9600 × g for 15 min. The supernatant was removed and stored at −60°C until use. The protein concentrations were measured in the supernatants using bovine serum albumin as a standard.

| Assay of total collagen, fibronectin, and laminin
The total collagen content in the samples as well as the level of fibronectin and laminin were determined by the commercial ELISA Kits Bio Vision (K218-100), Abcam (ab108850), and LifeSpan (LS-F6465), respectively, according to the manufacturer's protocol.

| Assay of matrix metalloproteinase activity
The measurement of collagenase activity (MMP-1, MMP-8, and MMP-13) is determined by a colorimetric method using a Bio vision kit and according to the manufacturer's instructions.

| Histological study
The histological study was carried out in the Department of Pathological Anatomy and Cytology of the Habib Bourguiba Hospital in Medenine. The liver and kidney were fixed in 10% formalin and stored at ambient temperatures. The organs are then cut into small fragments on a formalin absorbent table and placed in cassettes and then embedded in paraffin. The paraffin sections were cut into 5μm-thick slices and stained with hematoxylin and eosin (H&E), respectively, for 1 to 5 min and 5-10 min. After that slides were immersed in water and then in a 95% alcohol bath and followed by light green dye which stains ECM for 1 min, the preparations were observed and micro-photographed at 100×, 200×, and 400× at several levels under Leica microscope for reading by a pathologist. Representative images were taken using an optical microscope (Leica) equipped with a camera.

| Polyphenols and flavonoids content and LC-MS analysis
Our results show that the extraction yields of yellow lupine are 6.52%, 23.18%, and 31.45% for the three used solvents: water, ethanol (96%), and ethanol 50%, respectively.
The assay of total polyphenols was carried out according to the Folin-Ciocalteu method. Our results, reported in Table 1, show that the polyphenol content is about four times higher after extraction in ethanol 50% (119.55 ± 9.43 mg EAG/g) than in ethanol 96% (34.79 ± 3.26 mg EAG/g).
The phenolic compounds in the extracts obtained were analyzed by liquid-phase chromatography coupled with a mass spectrometry detector. The number of phenolic compounds identified are 4, 7, and 9, respectively, after extraction with water, ethanol 96%, and ethanol 50%. The concentration, in ppm, of the various compounds identified as well as their retention times are presented in Table 2 .

| Nitrogen, total sugars, and vitamins content
Total nitrogen is determined by the Kjeldahl method. The nitrogen content is estimated at 58.22% ± 5.96%. Analysis of the watersoluble vitamin composition showed that yellow lupine contains two water-soluble vitamins, retinol and k1. L. luteus seeds exhibited low levels of the following total sugars: fructose, glucose, sucrose, palatines, maltose, and lactose (Table 3).

| Serum parameters level
The biochemical parameters relating to liver (ASAT and ALAT) and renal function (urea and creatinine), lipid profile (total cholesterol, HDL, and triglycerides), and CRP were measured.
Analysis of lipid profile parameters showed that cholesterol levels increased in diabetic rats by approximately 31% (p < .05) compared to controls. Furthermore, the total cholesterol level of rats from the diabetic group which received yellow lupine extract decreased by 7% compared to the DM group. Our results show that the triglyceride level in the DM group increased by 8% compared to the controls.
Diabetes significantly increases the levels of the two transaminases by 134% and 120%, respectively, for AST and ALT (p < .05).
In addition, the association of diabetes and treatment with yellow lupine extract decreases the levels of both transaminases to levels statistically similar to controls.
Serum creatinine level was increased by 48% (p < .05) in the diabetic rats compared to the controls. On the other hand, the value of creatinine and urea in the YLE group was statistically similar to those of the control rats.
The uremia assay shows similar results to those of creatinine.

| ECM compound level
Extracellular matrix (total collagen, laminin, and fibronectin) assay was undertaken on liver and kidney homogenates. We demonstrate that in the liver, diabetes causes a significant increase in total collagen, laminin, and fibronectin contents, respectively, by 49% (p < .01), 56% (p < .01), and 67% (p < .05) compared to control rats. In diabetic animals treated with yellow lupine extract, the levels of the three components of the MEC remain statistically similar to the diabetic group ( Figure 1).
In the kidneys, the total collagen content was increased by 91% (p < .01) and 88% (p < .01), respectively, in the rats of the DM and DM + YLE groups compared to the control rats. Similarly, the laminin content increased significantly by 35% (p < .01) in the DM group and by 20% (p < .05) in the DM + YLE group (Figure 2).
Diabetes causes a significant increase in fibronectin content by 55% in the kidneys. In the group treated with the yellow lupine extract, the laminin level remains statistically similar to the CTRL group and also to the DM group.

| Activities of matrix metalloproteinases
The measurement of the activity of collagenases and gelatinases (MMP-1, MMP-8, and MMP-13) was undertaken on the homogenates from liver and kidney. In the liver, diabetes increases total collagenases and gelatinases activities, respectively, by 35% and 57% compared to the CTRL group. Therefore, treatment of diabetic rats with lupine extract decreased collagenases and gelatinases activities by 15% and 7%, respectively, when compared to diabetic rats ( Figure 3).
In the kidneys, diabetes causes an increase in total collagenases and gelatinases activities, respectively, by 25% and 47% compared to the CTRL group. However, no significant difference was observed for collagenases and gelatinases activities between the DM and DM + YLE groups (Figure 3).

| Histological study
Histological study was carried out on cross-sections of the liver stained with hematoxylin-eosin. In the control group and DM + YLE group, the liver had a normal appearance characterized by hepatocytes arranged in traves without any sign of necrosis or inflammatory cell infiltration. In the same group, the sinusoid veins look normal without signs of congestion (Figure 4a).
In diabetic rats, the liver shows signs of diffuse dilatation of the sinusoid veins associated with the presence of macroscopic and microscopic lipid vacuoles similar to hepatic steatosis (Figures 4b,c).  However, no signs of necrosis or inflammation were observed in the diabetic group.

| DISCUSS ION
In our previous study, we demonstrate that diabetes causes ECM remodeling and that some natural extracts can prevent several complications of diabetes. On the other hand, some species of lupin have been studied for their benefits on diabetes such as Lupinus albus and Lupinus mutabilis (Garmidolova et al., 2022). However, we are the first to investigate both (1)  In the present study, we used alloxan to induce diabetes in rats.
This technique is used to obtain diabetic rats for animal experimentation purposes. We used a dose of 250 mg/kg in the form of a single intraperitoneal injection in fasting rats in accordance with the doses used previously (Dab et al., 2022). Alloxan is a structural analog of glucose, which penetrates through the glucose transporters GLUT2 of pancreatic β-cells (Radenković et al., 2016) and causes rapid destruction of β-cells by the simultaneous action of reactive oxygen species and massive increase in intracytosolic calcium concentration. Alloxan is thus one of the most commonly used agents for the induction of diabetes mellitus (Rohilla & Ali, 2012).
The diabetes model undertaken in our present study was successful because the blood glucose level recorded on the third day following alloxan injection exceeded 200 mg/dl. Indeed, our results show average blood glucose values of 311 mg/dl on the third day after injection of alloxan. This value reached 325 mg/dl at the end of the experiment.
The success of the model was also confirmed by the presence of polyuria diabetic rats. Moreover, we showed that diabetes was associated with an increase in cholesterol, L-DL, and triglyceride levels in rats. Our results are consistent with previous studies in F I G U R E 1 Total collagen (μg/ml) (a), laminin (ng/mg) (b), and fibronectin (ng/ mg) contents (c) in the liver from control and treated rats. Data are mean ± SEM. *p < .05, **p < .01 vs. CTRL group; #p < .05, ##p < .01 vs. YLE group. CTRL, Control group; DM + YLE, Diabetic rats treated with 100 mg/kg of ethanol 50% extract of yellow lupine for 1 month; DM, Diabetic rats receiving injection of alloxan 250 mg/kg, ip; YLE, Control rats treated with 100 mg/kg of yellow lupine extract terms of the evolution of cholesterol level and triglyceride levels in human diabetes (Ahmad et al., 2008;Negreş et al., 2013;Patti et al., 2019).
Our results show that the ethanol 50% extract of yellow lupin is richer in total polyphenols than aqueous or ethanol 96% extracts.

F I G U R E 3 Gelatinases (MMP-2 and
Lipids play an important role in the pathogenesis of diabetes mellitus. The level of serum lipids is usually elevated in diabetes, and such an elevation represents a risk factor for coronary heart disease (Daisy & Kani, 2013). Insulin deficiency or insulin resistance is associated with symptoms of hypercholesterolemia and hypertriglyceridemia (Daisy et al., 2009). On the other hand, the treatment of rats with yellow lupine extract leads to hypolipidemia and improve triglyceride level.
We showed that AST, ALT, urea, and creatinine are similar in control and normal rats treated with lupin extract. These results confirm that yellow lupin is safe to use. On the other hand, dia- Our results show that in the liver and kidney, diabetes causes a significant increase in the content of total collagen, laminin, and fibronectin. Thus, this demonstrates a direct involvement of diabetes in the ECM remodeling in kidney and liver. Enhanced collagen level in the diabetic group was similar to previous investigations that report fibrosis installation in the lung of diabetic patients (Fariña et al., 1995). On the other hand, decreased fibroblasts adhesion, diminished response to growth factors and cytokines, and decreased production of collagens and fibronectin in wound healing were reported (Almeida et al., 2016;Hamed et al., 2010).
Laminin and fibronectin are widely present in the basal lamina which provide tissue cell-matrix adhesion. Several pathological processes as cancers and cardiovascular disorders were associated with changes in these two glycoproteins levels (Rohwedder et al., 2012).
In the study of the activity of MMPs in the liver and kidney, diabetes caused an increase in the activity of collagenases and gelatinases. On the other hand, the administration of the yellow lupine extract to diabetic rats slightly decreases MMPs activities. MMPs cover a large family of extracellular enzymes, which share common structural features, mainly regions involved in proteolytic activity.
Among these MMPs, we were interested in interstitial collagenases  which are the only enzymes in mammals with the ability to cut the triple helix of fibrillary collagen.
Interstitial collagenase (MMP-1) appears to have preferential activity against type III collagen. Polymorphonuclear collagenase  has an affinity for type I collagen and MMP-13 is the only collagenase able to cleave type 1, type II, and type III collagen (Malemud, 2006).
MMPs activation can be occurred also by reactive oxygen species or nonphysiological agents (Loffek et al., 2011).
The histological study of liver sections stained with hematoxylineosin shows that in the CTRL group, the liver has a normal appearance, whereas in diabetic rats, the liver shows signs of diffuse dilation of the sinusoidal veins associated with the presence of macroscopic and microscopic lipid vacuoles which testify to a stage of hepatic steatosis. These results are consistent with the dosages of total cholesterol which have increased by diabetes.
The study of the histological sections made at the level of the kidneys and stained with hematoxylin-eosin shows a normal structure of the renal tissue for the four groups of rats studied. These observations are not consistent with the results of the assays of the ECM molecules. Indeed, it is known that ECM disorders are difficult to detect histologically in young subjects like the age of the rats used in our present study.

| CON CLUS ION
In this study, we first started by testing the main hypothesis that diabetes could influence the distribution of ECM components as well as the activity of MMPs at the tissue level, and secondly, by studying the systemic effects of diabetes on the status of antioxidants and the various serum parameters. We identified nine compounds by LC-ESI-MS in yellow lupine extract. Our main results were a significant decrease in blood glucose after treatment of diabetic rats with yellow lupine extract compared to control diabetic rats. Thus, diabetes causes a significant increase in the content of total collagen, laminin, and fibronectin in the liver and kidney. Administration of yellow lupine extract in diabetic rats caused a slight decrease in collagenase and gelatinase activities compared to diabetic rats.

ACK N O WLE D G E M ENTS
We gratefully acknowledge all staff of Pathology Departement and Biochemistry Departement of the University Hospital Habib Bourguiba of Medenine, Tunisia.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available in article supplementary material.