Treatment Of Diabetes Induced Kidney Disease: Oleanolic Acid

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Preventing the onset of diabetes-induced chronic kidney disease during prediabetes: The effects of oleanolic acid on selected markers of chronic kidney disease in a diet-induced prediabetic rat model

Mlindeli Gamede, Lindokuhle Mabuza, Phikelelani Ngubane, Andile Khathi

1. Introduction

Approximately 30-40% of type 2 diabetes mellitus (T2DM) patients develop renal complications despite being on treatment with various conventional drugs for diabetes [1]. This can be partially attributed to the negative effects of these drugs on renal function [2. Diabetes is one of the leading causes of chronic kidney diseases(CKD), including diabetic nephropathy [3]. Renal complications that are predominantly found in diabetes include glomerular hyperfiltration, renal tubular disorders, and nephromegaly [4]. These complications are traditionally associated with overt T2DM, however recent studies have shown that diabetes-associated kidney complications commence during the prediabetic state [5. Previous studies in our laboratory have reported that a high-fat high carbohydrates diet-induced prediabetic rat model is characterized by systemic insulin resistance, impaired glucose tolerance (IGT), and oxidative stress [6. These complications are directly implicated in functional and structural abnormalities of the kidney 7I. The IGT may lead to sustained hyperglycemia that results in the non-enzymatic reaction of glucose with local renal proteins which form advanced glycation end-products(AGEs)and reactive oxygen species (ROS)[8]. The oxidative stress is associated with renal injury and the loss of the integrity of the glomerular basement membrane. This is normally characterized by the upregulation of kidney injury molecule-1 (KM-1)and loss of podocyte structure [7I. These structural abnormalities also impact the functionality of the kidney by increasing the glomerular basement membrane permeability(GBMP)which results in hyperfiltration [9. In addition, IGT can alter kidney function through increasing glomerular filtration of glucose and its subsequent reabsorption at the proximal tubule which increases sodium reabsorption [101. The increase of plasma sodium and augmented GBMP can lead to an increase in eGFR and urinary loss of macromolecules such as albumin [11]. Glomerular hyperfiltration is associated with podocyte loss and hence the presence of podocin in the urine [71. Diabetes is implicated in the alteration of renal electrolyte and water handling which is associated with the activation of the renin-angiotensin-aldosterone system (RAAS)to counteract the loss of sodium and water [2]. Diabetes-related renal complications are conventionally managed with pharmacological agents including insulin sensitizers and angiotensin-converting enzyme (ACE)blockers [13]. Moreover, dietary interventions are also recommended in cases where these complications arise due to T2DM [14]. However dietary intervention is often complex to implement and is normally associated with poor patient compliance [15]. Recent studies in our laboratory show that plant bioactive compounds such as oleanolic acid (OA) exhibit insulin-sensitizing and cardioprotective effects in diet-induced prediabetes[16]. However, the effects of OA (oleanolic acid) on renal function in prediabetes remain unknown. Hence, the present study was designed to evaluate the effects of OA (oleanolic acid) on selected markers of CKD (chronic kidney diseases), oxidative stress, and inflammation in the kidneys of diet-induced prediabetic rats.

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2. Methods and materials

21. Drugs and chemicals

All chemicals and reagents were sourced from trusted pharmaceutical suppliers and were of analytical grade.

2.2. Isolation method

OA (oleanolic acid) was isolated from Syzygium aromatic [(Linnaeus)Merill &Perry] [Myrcene](cloves)using an established protocol from Khathi et al. [17].

2.3. Animal care

All animal procedures and housing conditions were approved by the Animal Research Ethics Committee of the University of KwaZulu-Natal (ethics no: AREC/035/016M). Male Sprague-Dawley rats(130-160 g)were bred and housed in the Biomedical Research Unit of the University of KwaZulu-Natal. The animals were maintained under standard laboratory conditions of constant temperature (22± 2°C), CO2 content(<5000 ppm.), relative humidity (55±5%), and illumination(12 h light/dark cycle, lights on at 07h00 a.m.).The noise level was maintained at less than 65 decibels. The animals were allowed access to food and fluids ad libitum. The animals acclimatized to their new environment for one week while consuming standard rat chow and tap water before exposure to a well-established experimental high-fat, high carbohydrate (HFHC)diet

2.4. Induction of prediabetes and feeding program

The 36 male Sprague-Dawley rats weighing between 150 and 180 were randomly divided into two groups, the normal diet-fed group (n=6)and the high-fat high-carbohydrates diet-fed group (n = 30). The animals acclimatized to their new environment for one week while consuming standard rat chow and tap water before exposure to an experimental high-fat high carbohydrate(HFHC)diet. Animals that were induced prediabetes received an HFHC diet that was previously developed in our laboratory made by AVI foods, South Africa, while the normal control was fed normal rats' diet from the UKZN animal unit. The feeding program and prediabetes inductions lasted for 20 weeks. During this time, parameters including fasting blood glucose, glucose tolerance, and blood triglyceride were monitored every fourth week. For this study, prediabetes was defined as a significant increase in total body weight, fasting blood glucose,2-hour glucose tolerance (during oral glucose tolerance test), and blood triglycerides which were observed in the HFHC diet-fed animals).

2.5.Animals and groups

The overarching aims of the study were to determine whether OA (oleanolic acid) can ameliorate the prediabetes-induced risk factors of chronic kidney diseases in both the presence and absence of dietary intervention. In quest of investigating these aims, the normal diet-fed group was regarded as non-prediabetes control (NPC)(n=6). The animals that received HFHC diet and developed prediabetes were randomly subdivided into five groups according to their respective treatments, untreated prediabetic control group(PC), metformin-treated group(MET), metformin-treated with the dietary intervention group(MET+DI, oleanolic treated group (OA (oleanolic acid))and oleanolic acid-treated with the dietary intervention(OA (oleanolic acid)+DI). All groups were n= 6.

3. Treatment of prediabetic animals

The treatment period lasted for 12 weeks which renders enough time for glycation of hemoglobin. The animals were treated every third day where the MET and MET+DI groups received metformin(500 mg/kg p. o)while the OA (oleanolic acid) and OA+DIgroups were given oleanolic acid(80 mg/kg p.o). This dose is regarded as non-toxic and has more efficacy by the previous studies in our laboratory [15. Parameters, including fluid intake and urine output, were measured every fourth week in all groups for the duration of the treatment period. The code p.o is for the oral dose using oral gavage.

3.1. Blood collection and tissue harvesting

At the end of the 12-week treatment period, the animals were sacrificed, using the method of anesthetizing with Isoform(100 mg/kg)(Safeline Pharmaceuticals(Pty)Ltd, Roodeport, South Africa)via a gas anesthetic chamber(Biomedical Resource Unit, UKZN, Durban, South Africa)for 3 min followed by the decapitation and the blood was collected in all animals through cardiac punch method into pre-cooled heparinized containers. The blood was then centrifuged(Eppendorf centrifuge 5403, Germany)at 4 ℃C,503 g for 15 min, and the plasma was collected and stored at - 80 ℃℃ in a Bio Ultra freezer(Snijders Scientific, Holland)until ready for biochemical analysis. The kidneys were removed, rinsed with cold normal saline solution, and snap-frozen in liquid nitrogen before storage in a Bio Ultra freezer (Snijders Scientific, Tilburg, Netherlands)at -80°C until further biochemical analysis was performed.

3.2. Urine collection

Urine samples were collected from all experimental groups using a metabolic cage system, provided by the Biomedical Resource Unit. The urine samples were collected at various points of the treatment period, i. e. weeks 0, 4,8, and 12. The collected urine samples were stored at-80°℃C.

3.3. Biochemical analysis

Plasma and urine were sent to Global Clinical and Viral Laboratories (Amanzimtoti South Africa)for biochemical analysis. Urine analysis included quantification of electrolytes such as sodium, potassium, albumin, and creatinine. Kidney tissue analysis: The kidney tissue was homogenized with phosphate buffer saline (PBS) with a ratio of 1 g (tissue):9 mL(PBS) and the homogenate was used for the malondialdehyde (MDA)assay to quantify lipid peroxidation and oxidative stress using a well-established protocol. The MDA essay was accompanied by the quantification of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase(GPx) concentrations which were done using Elabscience ELISA kits [10.11].Plasma analytics: Plasma aldosterone and KIM-1 concentrations were also analyzed using their respective rat ELISA kits(Elabscience Biotechnology Co., Ltd) according to the manufacturer's instructions.

3.4. Quantitative real-time- PCR

The ribonucleic acid (RNA)was extracted from urine samples obtained during week 12 of the treatment period.RNA extraction was done with the ZR Urine RNA isolation kit (zymo research, United States of America) from Inqaba biotech(South Africa).RNA yield was determined using nanodrop, and standardization of RNA concentration was performed. The converted deoxyribonucleic acid(cDNA)was then synthesized using the cDNA synthesis kit (ThermoFisher).

The ThermoFisher light cycler SYBR Green I master mix was used for amplification according to the manufacturer's instructions on the Thermo fisher light cycler system. The cycling conditions were: Pre-incubation was carried out at 95 ℃ for the 60 s. Followed by a 3-step amplification of 45 cycles at 95 ℃C for 15 s,60 ℃ for the 30 s, and 72 ℃ for 30 s. Melting was effectuated at 95 ℃ for 10 s,65 ℃ for the 60 s, and 97 ℃C for1s. Furthermore, cooling was achieved at 37 ℃ for 30 s. Glyceraldehyde-phosphate dehydrogenase (GAPDH) as an internal control was used to normalize the data to determine the relative expression of the gene of interest. Gene expression values are represented using the 248c method. The below primers were used.

3.5. Calculation and equations

Urinary Albumin/Creatinine ratio(Alb/Cr-R) was calculated using albumin [UAlb] and creatinine [UCr] obtained from 24 hrs urine samples after week 12 of treatment. The units for Alb/Cr-R are in (mg/mgCr). ALB/Cr-R=albumin to creatinine ratio,[UALB]=urine albumin concentration and [UCr] =urine creatinine concentration

The quantification of RT-PCR was calculated using the comparative method

Fold change due to treatment = 2-△XR

ACT = CT(target gene )and CT(reference genes)

An ACT= ACT(target sample)-ACT(reference sample)

3.6. Traction excretion of electrolytes

Fraction excretion of Sodium（FENa）= WNex× 100 Fraction excretion of Potassium(FEK)= Wes× 100

UNaurnary sodium, PCR-plasma creatinine, UCR-urinary creatinine, PNa-plasma sodium, UK-urinary potassium, and PK- plasma potassium.

3.7. Statistical analysis

All data were expressed as means ± S.D. Statistical comparisons were performed with Graph Pad In-Stat Software (version 7.00, Graph Pad Software, Inc., San Diego, California, USA)using a one-way analysis of variance(ANOVA)followed by Bonferroni multiple comparison tests to simultaneously determine statistical differences between the means of two independent groups.

4. Results

4.1. Effects on fluid intake and urine output

Fluid intake and urine output for all experimental groups were monitored every four weeks during the treatment period. The results showed that from the start of the treatment period (week O), the PC group had a significantly higher fluid intake and urine output in comparison to NPC (PC vs NPC)(p<0.05). However, the administration of OA (oleanolic acid) with and without dietary intervention resulted in a significant progressive decrease in both fluid intake and urine output over the 12 weeks by comparison to PC. Treatment with MET had no significant difference with the PC in fluid intake throughout the four weeks. Whereas treatment with MET+DI decreased the fluid intake to the NPC range. In urine output, MET had an increased urine output in comparison to both NPC and PC, particularly in weeks 8 and 12(see Fig.1).

Fig. 1. The bar graph shows the effects of OA with and without dietary intervention and Met with and without dietary intervention (n = 6, per group) on fluid intake and urine output on all experimental groups from week 0 to week 12.

Values are presented as the standard deviation of mean ± SD. ★ =p < 0.05 denotes comparison with NPC; α = p < 0.05 denotes comparison with PC.

4.2. Effects on the fraction of sodium(FENa)and potassium(FEK)

The plasma and urine electrolytes concentrations were measured at the end of the treatment period (week12)while the fractional excretion of sodium and potassium was calculated. The PC had significantly lower FENa compared to NPC (p < 0.05). The administration of OA (oleanolic acid) with and without the dietary intervention (OA+DI and OA) significantly improved sodium excretion in comparison to PC(p< 0.05). The met-formin treated group (MET)showed overt sodium retention, however, when metformin was combined with dietary intervention (MET+DD, there was a significant improvement in sodium excretion by comparison with PC (p <0.05).

Interestingly, the PC had a significantly reduced FEK when compared to NPC. However, the administration of OA (oleanolic acid) with and without dietary intervention (OA and OA+DI)significantly improved potassium excretion. MET without dietary intervention had overt potassium retention which was comparable to PC. However, MET combined with dietary intervention(MET+DI)significantly improved potassium excretion in comparison with PC.

4.3. Effects on creatinine clearance(CRC)

The CRC was calculated from serum creatinine, week 12 urine creatinine, and volume for all experimental groups. The results showed that the PC group had significantly higher CRC in comparison to NPC. The administration of OA (oleanolic acid) with and without dietary (OA and OA+DD resulted in a significant decrease in CRC when compared to PC.MET had a significant increase in CRC when compared with the. Whereas MET+DI decreased the CRC to the NPC range (p <0.05)(see Fig.2).

Fig. 2. The bar graph shows the effects of OA with and without dietary intervention and Met with and without dietary intervention (n = 6, per group) on percentage fraction excretion of sodium and potassium of all experimental groups.

Values are presented as the standard deviation of mean ± SD. ★=p < 0.05 denotes comparison with NPC; α = p < 0.05 denotes comparison with PC.

4.4. Effects on proteinuria (albumin/creatinine ratio)

The proteinuria was examined using Alb/Cr-R, which was calculated with urine albumin and creatinine obtained from week 12 urine. The results showed that the PC group had a significantly increased Alb/Cr-R in comparison to NPC. However, the administration of OA with and without dietary (OA (oleanolic acid) and OA+DDshowed a significant decrease in Alb/Cr-R when compared to PC. MET and MET+DI also decreased the Alb/Cr-R in comparison to PC (p < 0.05)(see Figs.3 and 4).

Fig. 3. The bar graph shows the effects of OA with and without dietary intervention and Met with and without dietary intervention (n = 6, per group) on the creatinine clearance(CRC) rate of all experimental groups. Values are presented as the standard deviation of mean ± SD. ★ = p < 0.05 denotes comparison with NPC; α = p < 0.05 denotes comparison with PC.

Fig. 4. The bar graph shows the effects of OA with and without dietary intervention and Met with and without dietary intervention (n = 6, per group) on the albumin/creatinine ratio of all experimental groups. Values are presented as the standard deviation of mean ± SD. ★ = p < 0.05 denotes comparison with NPC; α = p < 0.05 denotes comparison with PC.

Fig.5. The bar graph shows the effects of OA (oleanolic acid) with and without dietary intervention and Met with and without dietary intervention (n=6, per group)on plasma aldosterone levels of all experimental groups. Values are presented as the standard deviation of mean ± SD.★=p<0.05 denotes comparison with NPC;α = p<0.05 denotes comparison with PC.

4.5. Effects on plasma aldosterone level

Plasma aldosterone concentrations were measured using the ELISA kit. The results showed that PC had significantly higher plasma aldosterone in comparison with NPC. However, the administration of OA (oleanolic acid) with and without diet intervention(OA and OA+DI)resulted in a significant decrease in plasma aldosterone in comparison to PC.MET had a significant increase in aldosterone concentration when compared with the NPC. Whereas MET+DI decreased the aldosterone concentration to the NPC range. (p <0.05) (see Fig.5).

4.6. Effects on kidney oxidative stress

Renal oxidative stress was evaluated through measurements of MDA SOD, and GPx in the kidney tissue. The results showed that PC had a significantly higher MDA concentration when compared to NPC (p <0.05). Furthermore, PC had significantly lower SOD and GPx concentrations when compared to NPC. However, the administration of OA (oleanolic acid) with and without dietary intervention resulted in significantly decreased MDA concentrations when compared to PC (p<0,05), while the levels of SOD and GPx were within the NPC range MET had a significant increase on MDA and reduced SOD and GPx concentrations in comparison to NPC. However, MET+DI increased the SOD and GPx to the NPC range. (p < 0.05)(see Table 1).

4.7. Effects on the expression levels of urinary podocin

Urinary podocin was quantified in urine obtained from week 12. The prediabetic control had increased urine podocin expression relative to standardized NPC. However, the administration of with and without diet intervention (OA and OA+DI)resulted in a significant decrease in urine podocin expression relative to PC. MET and MET+DI also decreased the urine podocin expression relative to PC. (p<0.05)(see Fig 6).

Table1: Effects of OA (oleanolic acid) and METon kidney MDA, SOD, and GPxof prediabetic, Values are presented as the standard deviation of mean ± SD.*=p< 0.05 denotes comparison with NPC;α=p<0.05 denotes comparison with PC.

Fig. 6. The bar graph shows the effects of OA with and without dietary intervention and Met with and without dietary intervention (n = 6, per group) on urinary podocin expression levels of all experimental groups. Values are presented as the standard deviation of mean ± SD. ★ = p < 0.05 denotes relative to NPC; α = p < 0.05 denotes relative to PC. NB. PC is relative to NPC which is = 1 and all other treatments are relative to PC.

4.8. Effects on kidney injury molecule-1(KIM-1)

Plasma KIM-1 concentrations were measured using the ELISA kit. The results showed that PC had a significantly higher plasma KIM-lin comparison with NPC. However, the administration of OA (oleanolic acid) with and without diet intervention (OA and OA+DDresulted in a significant decrease in plasma KIM-1 by comparison to PC. MET had a significant increase in KIM-1 when compared with the NPC. Whereas MET+DI decreased the KIM-1 to the NPC range. (p< 0.05)(see Fig.7).

Fig. 7. The bar graph shows the effects of OA with and without dietary intervention and Met with and without dietary intervention (n = 6, per group) on plasma KIM-1 of all experimental groups. Values are presented as the standard deviation of mean ± SD. ★ = p < 0.05 denotes comparison with NPC; α = p < 0.05 denotes comparison with PC.

5. Discussion

Previous studies have reported that the diet-induced prediabetic state is associated with an increased risk of developing cardiovascular diseases and early-stage CKD (chronic kidney diseases) [18]. Prediabetes is characterized by proteinuria, increased eGFR as well as electrolyte imbalance which are all risk factors of CKD (chronic kidney diseases). Conventionally, prediabetes is managed by the combination of pharmacotherapy and lifestyle modifications such as dietary intervention [19I.The lifestyle modifications are associated with low patient compliance which affects the efficacy of the pharmaco-therapy [20]. Studies in our laboratory have shown that the administration of OA (oleanolic acid) in diet-induced prediabetes restores insulin sensitivity and ameliorates the risk of cardiovascular complications associated with prediabetes in both the presence and absence of dietary intervention [21]. However, the effects of this plant-derived triterpene on the risk factors associated with the development of CKD (chronic kidney diseases) have not yet been investigated. Hence, this study sought to investigate the effects of plant-derived oleanolic acid (OA) on the selected risk factors of CKD (chronic kidney diseases) in both the absence and presence of dietary intervention in a diet-induced prediabetic rat model. The selected risk factors investigated in this study include kidney oxidative stress, glomerular filtration rate(GFR), a component of the renin-angiotensin-aldosterone system, albuminuria, renal fluid, and electrolyte handling across all the experimental groups.

Previous studies in our laboratory have reported that diet-induced prediabetic animals develop obesity with impaired glucose tolerance and impaired fasting glucose as a result of insulin resistance. The IGT increases the circulating glucose, which is increases the plasma osmo-larity[22. This may increase the plasma osmotic pressure and subsequently the thirst sensation resulting in fluid intake [23). In addition, obesity is also characterized by activation of the renin-angiotensin system which is also implicated in the development of T2DM. The current study revealed that prediabetic animals had increased 24 h fluid intake and urine volume. This may be attributed to the increased levels of angiotensin II as a result of obesity and insulin resistance. Interestingly prediabetic animals also had increased urine output, which is contradictory to the well-known effect of the RAS activation. Treatment with OA (oleanolic acid), with and without dietary intervention showed a progressive restoration of fluid intake and urine output, which were more pronounced during week 12. This might suggest that OA (oleanolic acid) can restore the regulation of the anti-diuretic hormone. However, this mechanism is yet to be established.

The activation of RAS was further confirmed by the elevated concentrations of plasma aldosterone in the prediabetic animals. Besides the increase of sodium reabsorption to the nephron, aldosterone also reduces peripheral insulin sensitivity leading to IGT, which is a risk factor for CKD (chronic kidney diseases).[24]. Interestingly, this study found that prediabetic animals had normal plasma sodium concentrations but reduced urine sodium concentrations. This may suggest that the increase in plasma aldosterone had an effect on sodium reabsorption from the nephron resulting in low urinary sodium or reduced fractional excretion of sodium (FENa)[25]. OA (oleanolic acid) treatment in both the presence and absence of diet intervention resulted in a decrease in plasma aldosterone concentration and improved sodium excretion. These findings concur with the previous studies, which reported that OA (oleanolic acid) reduces the mean arterial pressure through deactivation of the rennin-angiotensin-aldosterone system (RAAS)and reduction of sodium reabsorption [26]. The normalization of RAAS was evident by the decrease in the plasma aldosterone and urine sodium concentration[Nat][27. However, this may also be attributed to the reduction of succinate, a product of the citric acid cycle that is elevated during IGT. [28I. Succinate has been reported to stimulate the release of renin from macula densa cells which initiate the signaling that results in the activation of RAAS through interaction with G-protein receptor 91 (GPR91) metabolite [29]. The anti-hyperglycemic effects of OA could also have resulted in a decrease in the glucose substrate for glycolysis which then led to low production of pyruvate, which enters the citric acid circle and produces succinate [30J. Furthermore, dietary intervention may have played an important role in the reduction of glucosuria, succinate production, and renin secretion through the reduction of carbohydrate metabolites [31].

Under physiological conditions, the increase of plasma aldosterone increases in tubular Nat reabsorption through Nat/Kf ATPase channels resulting in K excretion [32. However, insulin resistance may be associated with hepatic ketones production, which raises the blood pH and excretion of hydrogen ion (H)through the sodium-hydrogen exchanger to Kexcretion [33I.Interestingly, this study observed that the increase in sodium reabsorption was associated with negligible potassium excretion on OA (oleanolic acid)-treated animals. This may be attributed to the stimulation of sodium-hydrogen antiport as a result of an increase in blood pH due to ketoacidosis production [6]. This may explain the increase in sodium reabsorption without any pronounced potassium excretion [34j. These observations concurred with findings from previous studies that have reported that OA (oleanolic acid) increases sodium excretion, and thus lowers systemic blood pressure [6]. However, the findings of this study suggest that OA promotes sodium excretion through sodium-hydrogen antiport excretion rather than the sodium-potassium ATPases [6]. Previous studies have also reported that diets that have more plant-derived proteins improve metabolic acidosis and slow down the progression of diabetic nephropathy [33I.This may also suggest that dietary intervention might have contributed to the improved sodium excretion.

The moderate hyperglycemia in prediabetes is independently implicated in mitochondrial dysfunction and increased the production of reacting oxygen species(ROS), which subsequently results in oxidative stress [35I. Previous studies have eluded that renal oxidative stress is associated with increased kidney injuries. loss of glomerular basement membrane integrity, and proteinuria [36]. Indeed this study showed that prediabetic animals had increased renal lipid peroxidation, elevated kidney injury molecule-1(KIM-1)urinary podocin, and increased albumin-creatinine ratio. This may be attributed to the high influx of glucose into the kidney due to peripheral insulin resistance that may result in ROS and advanced-glycosylation end products(AGEs)formation [371. Hyperglycaemia leads to the activation of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)oxidase enzyme, which catalyzes the production of superoxide (O2)from renal cells [37I. Moreover, oxidative stress and hyperglycemia are also strongly associated with the generation of advanced glycation end products(AGEs)through the non-enzymatic reaction of glucose with protein, fats or nucleic acids [38I. ROS is associated with disruption of cell-cell and cell-matrix adhesion junctions which lead to detachment of endothelial cells from the basement membrane resulting in the movement of KIM-1 into the blood [39].

The ROS and AGEs are associated with the damage of the podocyte layer of the glomerular basement membrane[40]. This may further compromise one of the primary functions of the nephron, which is to prevent the filtration of proteins leading to proteinuria. However, the administration of OA (oleanolic acid) with and without diet intervention resulted in a decrease in lipid peroxidation, reduction of KIM-1, reduction of urine podocin, and decrease of albumin-creatinine ratio. This may be attributed to the significant improvement in renal antioxidant enzymes superoxide dismutase(SOD)and glutathione peroxidase(GPx)that was observed in OA-treated groups. Superoxide(O2)is neutralized by the SOD and produces hydrogen peroxide (HzO2)[41. While HO2 is further converted to non-toxic HO and O, by the action of GPx[42. These results correlated with findings of Gamede et al.,2019 that reported that OA (oleanolic acid) improves the antioxidant availability in diet-induced prediabetic animals [21. This may further explain the reduction of kidney injury and podocyte damage seen in the animals that recited OA (oleanolic acid) with and without dietary intervention. The restoration of glomerular basement membrane integrity through prevention of podocytes loss may be credited to the reduction of albumin-creatinine ratio observed on the OA (oleanolic acid)-treated animals.

Renalhyperfiltration(increased CRC) has also been reported in newly diagnosed T2DM patients and it has been shown to precede the decline of renal function[43]. The impaired glucose homeostasis with hyperinsulinemia is implicated in the elevation of CRC, which is known to precede glomerular damage [44]. The current diet-induced prediabetic rat model has been previously reported to have cardiovascular complications such as systemic high blood pressure. Moreover, the current study also observed that the prediabetic group had increased CRC. The increase in CRCsignifies the elevation of glomerular filtration which may be attributed to various mechanisms such as afferent glomerular vaso-dilation as a result of elevated plasma insulin and renal AGEs [45I. Treatment with OA (oleanolic acid) resulted in the restoration of CRC which may be attributed to successful glycaemic control resulting in the reduction of plasma insulin and renal AGEs. In addition, OA has been previously reported to reduce hyperinsulinemia and improve glucose homeostasis [6]. A diet that is low on sodium is often recommended for a patient with increased GFR, and this has been regarded as one of the methods used to prolong kidney survival [6]. This study also found that OA (oleanolic acid) with and without dietary intervention has more potent effects in some risk factors of kidney disease such as KIM-1 and GFR in comparison to the standard drug metformin.

The use of metformin is not recommended at advanced stages of CKD (chronic kidney diseases) [46. This is due to the contraindications associated with metformin such as lactic acidosis, which may result in renal failure[47). In this study, we found that administration of metformin without diet intervention has no beneficial effect on factors such as renal fluid handling, eGFR, electrolyte handling, and renal oxidative stress diet-induced pre-diabetic animals. However, when metformin was combined with diet intervention, there was improved efficacy. This concurs with the previous studies that suggest that metformin is dependent on lifestyle modifications such as changes in diet and exercise.

Improve kidney function-cistanche

6. Conclusion and recommendations

The findings of this study suggest that administration of OA (oleanolic acid) in both the presence and absence of dietary intervention ameliorates markers associated with CKD (chronic kidney diseases), This was evident in the observed change to parameters such as water intake, urine output, and renal electrolyte handling, The improvement of renal electrolyte and water handling further explains the decrease in systemic blood pressure that was observed in untreated prediabetic rats. Furthermore, this study also reports that the administration of OA (oleanolic acid) in prediabetes prevents renal oxidative stress, kidney injuries, and subsequent proteinuria. Taken together, the findings of this study suggest that this plant-derived tri-terpene can be used as an alternative treatment as it possesses renoprotective properties in diet-induced prediabetes, However, more studies need to be conducted in investigating the exact mechanism by which this compound exerts its effects.

7. Study limitations

The GSH and GSSG, protein oxidation, total antioxidant capacity and gene expression of antioxidants genes can be analyzed to assess the redox imbalance that couldn't be analyzed dues to budget constraints. Furthermore, this study couldn't analyze all the electrolytes that may be involved in the alteration of renal function.

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