INTRODUCTION:

Chronic hyperglycemia and metabolic deviations in protein, fat, and carbohydrates are the clinical manifestations of diabetes mellitus, a group of metabolic disorders^1,2. Life-threatening chronic consequences such as diabetic kidney disease (DKD), retinopathy, and neuropathy are frequently associated with diabetes mellitus^3–5.

It is predicted that 15–40% of patients with diabetes may develop DKD during their lifespan, which is the primary origin of end-stage renal disease (ESRD) worldwide, particularly in those with diabetes^6–8. DKD is often associated with dyslipidemia, a significant risk factor for cardiovascular disease. Because individuals with DKD have a higher death risk from cardiovascular disease, managing dyslipidemia is crucial⁹. Moreover, it has been demonstrated that dyslipidemia is essential to the onset and course of DN¹⁰. Research suggests a strong association between diabetes and altered lipoprotein metabolism, as evidenced by elevated VLDL-C and LDL-C concentrations and decreased HDL-C levels¹¹. Apart from these quantitative alterations, lipoproteins in diabetes are more proatherogenic due to quality changes such as oxidized LDL (ox-LDL)¹². Diabetic nephropathy is characterized by a deterioration in the kidney's structural and functional qualities brought on by glomerular basement membrane build up, glomerular shrinkage, and extracellular matrix accumulation¹³. According to recent research, a number of molecular pathways may be activated by the overproduction of ROS, impairing the antioxidant defense systems and accelerating the course of the illness¹⁴.

Currently, focused treatment perspectives for diabetes include biguanides, thiazolidinediones, and sulfonylureas. Conversely, long-term use of these oral hypoglycemics is often associated with hypoglycemia, osteoporosis, gastrointestinal problems, and heart failure¹⁵. The burgeoning area of diabetic nephropathy therapeutics prioritizes the design of innovative, multi-modal interventions targeting synergistic pathways encompassing oxidative stress mitigation, inflammatory response regulation, and glycemic homeostasis, with the overarching objective of disease progression curtailment^16,17. Thus, using cutting-edge treatments with anti-inflammatory, antioxidant, and hypoglycemic properties may protect against diabetic nephropathy.

A native of tropical Asia and Africa, Hygrophila auriculata (K. Schum) Heine is an herbaceous medicinal plant in the Acanthaceae family that flourishes in wetlands. It is referred to as kokilaksha in India and has long been utilized for treating various ailments. Phytochemical analysis of Hygrophila identified a diverse spectrum of bioactive constituents. Preclinical studies suggest significant pharmacological potential notably nephro- and neuroprotection, antidiabetic, and antioxidant effects^18,19. The effectiveness of Hygrophila in conferring nephroprotective properties against non-diabetic (ND) nephropathy models, specifically in the context of gentamicin- and cisplatin-induced nephrotoxicity, has been firmly substantiated^20,21. While many studies have explored the potential of Hygrophila for treating various diseases, its impact on diabetic kidney disease (DKD) remains poorly understood. This research aimed to investigate whether Hygrophila extract could protect against DKD in a model using streptozotocin and nicotinamide.

MATERIALS AND METHODS:

Plant Procurement, Authentication, and Extraction:

Dr. Hemantkumar A. Thakur, affiliated with the Postgraduate Department of Botany at GES's HPT Arts and RYK Science Institution in Nashik, Maharashtra, India, has authenticated and verified the taxonomic identity of a complete Hygrophila auriculata (K. Schum) Heine specimen (herbarium specimen no. HPTRYK/342/2021–22) sourced from the indigenous Matori-Makhamalabad region. A pulverizer was used to coarsely powder the dried apical parts. The powder's methanol extract was made using Soxhlet's extractor. A rotary evaporator operating at lower pressure was used to concentrate the extract. The yield of the freeze-dried Hygrophila methanolic extract (MEHA) was computed. The presence of bioactive components was the main focus of the subsequent analysis of MEHA²².

Chemicals:

Streptozotocin (STZ), nicotinamide (NA) and metformin were acquired from Sigma-Aldrich CO, situated in St. Louis, Missouri, USA. All chemicals, reagents, and biochemical kits of analytical grade, aside from those explicitly specified, were procured from local suppliers. The reagents essential for antioxidant assays were synthesized within the laboratory prior to conducting the estimations.

Animals:

Mature male Wistar albino rats (150-200g), and Swiss albino mice (20-25g) were maintained under standard conditions of a stable temperature of 25°C±1°C, relative humidity within the 45-55% range, and adherence to a standard circadian cycle. Food pellets and filtered water were accessible ad libitum. Animals underwent a 7-10 day acclimation period before the experimental protocol commenced. The research proposal 24/BNCP/IAEC/2021 received approval from the ethical review committee at Bhupal Noble's University in Udaipur, Rajasthan to conduct the investigation in accord to the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)²³.

Methodology for Experimental Investigation:

Study on Acute Oral Toxicity:

An OECD Guideline 425-compliant investigation assessed the acute oral toxicity of MEHA in mice²⁴. Mice received varying oral doses of MEHA (suspended in 1% CMC) and were monitored for 72 hours for any signs of toxicity or mortality.

Induction of Diabetes and Experimental Procedures:

In a fasted state, male Wistar albino rats were injected with STZ (60mg/kg) intraperitoneally to induce type 2 diabetes mellitus (T2DM). To mitigate potential Glucose Transporter 2 (GLUT2) competition and hypoglycemic effects, NA (120mg/kg) was administered consecutively alongside a 5% glucose solution provided overnight. Following a 72-hour recovery period, diabetic rats (fasting blood glucose ≥200mg/dL) were identified via tail vein prick and selected for further investigation²⁵.

Investigational Protocol for a Multiple-Dose Study:

Adult male Wistar rats exhibiting hyperglycemia (FBG ≥200mg/dL) were segregated into six groups (n=6) following STZ/NA-induced diabetes. Group I (normoglycemic control) received oral saline gavage (1 mL/kg). Diabetic groups (II-VI) received normal saline (1mL/kg), graded doses of MEHA (100, 200, or 400 mg/kg), and metformin (180mg/kg) as a positive control. From the fifth to the eighth week of diabetes induction, MEHA and metformin were administered for four weeks via freshly prepared 1% (w/v) carboxymethyl cellulose (CMC) oral suspension²⁶.

Investigation of Diabetic Kidney Disease (DKD):

Quantification of Body Mass, Circulating Glucose, and Glycated Hemoglobin (HbA1c) Concentrations:

Following an eight-week intervention, the rats' body mass alterations (g) were evaluated. Blood was collected via tail venepuncture and aliquoted into Eppendorf tubes. After cold centrifugation at 3,000rpm and 6°C for 15 minutes, the serum was isolated and cryopreserved at -20°C for subsequent analysis. At the study endpoint, half of the isolated serum was analyzed for glycated hemoglobin (HbA1c) and glucose levels using a biochemical analyzer. The GOD-POD kit (Accurex, India) and the hemoglobin A1c (glycated) kit (Sigma Aldrich, USA) were employed for these measurements, respectively^27,28.

Estimation of Renal Functional Indices and Serum Lipid Profile:

Subsequent to initial analyses, residual serum from the samples was utilized for further biochemical assessment using assay kits procured from Arkray Healthcare Pvt. Ltd., Mumbai, India. This evaluation focused on key markers of renal function and lipid profile.

Estimation of Oxidative Stress Indicators:

After eight weeks of treatment, rats were euthanized under deep diethyl ether anesthesia. Their left kidneys were then surgically excised and homogenized in ice-cold phosphate-buffered saline (pH 7.4) using a Polytron PT 2500E probe homogenizer. The resulting homogenate was frozen at -20°C for subsequent biochemical analysis of lipid peroxidation quantifies through malondialdehyde (MDA)²⁹ levels, reduced glutathione (GSH)³⁰ and superoxide dismutase (SOD)³¹.

Histoarchitectural analysis of kidneys:

Right renal tissue from a diabetic animals was promptly preserved in 10% formalin and paraffin-embedded. Subsequently, 5 µm sections were generated using a Leica Biosystems microtome. Selected sections were deparaffinized and stained with hematoxylin and eosin (H and E) to evaluate histoarchitectural alterations under a 400x magnification microscope.

Data collection and analysis:

Data were presented as mean (± SEM) and subsequently analyzed using a one-way ANOVA in GraphPad Prism 9.0 software. To further elucidate potential group-wise differences, a post-hoc Tukey's multiple comparison test was employed. Differences were considered statistically significant at a p<0.05.

RESULT:

The percentage yield of the extract and bioactive constituents analysis:

Analysis of MEHA revealed the presence of bioactive components such as alkaloids, flavonoids, saponins, phenols, tannins, glycosides, terpenoids, and proteins. The freeze-dried MEHA exhibited a percentage yield of 8.95% w/w.

Acute oral toxicity study:

Administering 1% (w/v) MEHA suspension in 1% CMC solution orally for 72 hours did not cause any mortality or signs of toxicity in the studied animals. OECD recommends a maximum tolerated dose of 2000 mg/kg for MEHA, and dosages 100, 200, and 400 mg/kg were considered suitable for further investigation.

Body Mass, Circulating Glucose, and Glycated Hemoglobin (HbA1c) Concentrations:

Compared to group I, group II exhibited significant weight loss and elevated serum glucose and HbA1c concentrations. Conversely, groups IV, V, and VI supplementation demonstrated a substantial reversal of weight loss and improvements in glucose and HbA1c levels by the end of the 8^th week. These findings contrast with the sustained diabetic state observed in the group II receiving solely the STZ/NA (Table 1).

Table 2. Effect of MEHS on creatinine, blood urea nitrogen (BUN), and uric acid in diabetic rats.

Treatment	Creatinine (mg/dL)	BUN (mg/dL)	Uric acid (mg/dL)
I- Non-diabetic (ND)	0.81 ± 0.07	16.12 ± 1.26	5.04 ± 0.60
II- STZ/NA control	1.44 ± 0.06^###	42.17 ± 1.03^###	15.52 ± 0.53^###
III- MEHA (100)	1.35 ± 0.06^ns	40.87 ± 1.10^ns	13.78 ± 0.52^ns
IV- MEHA (200)	1.13 ± 0.06^**	35.72 ± 1.13^**	12.74 ± 0.54^**
V- MEHA (400)	1.07 ± 0.06^***	34.57 ± 1.19^***	11.66 ± 0.53^***
VI- MET (180)	1.06 ± 0.07^***	33.33 ± 1.26^***	11.56 ± 0.62^***

Note: The data were expressed as mean ± SEM (n=6) and analyzed by one-way ANOVA trailed by post-hoc Tukey’ multiple comparison test. ^###p<0.001 for STZ control Vs ND group. ^ns: non-significant for MEHA (100), ^**p<0.01 for MEHA (200), ^***p<0.001 for MEHA (400), and ^***p<0.001 for MET, Vs STZ control group.

Table 3. Effect of SME on serum lipid profiles (TG, TC, LDL-C, VLDL-C and HDL-C) in diabetic rats.

Treatment	Serum lipid profiles (mg/dL)
Treatment	TG	TC	LDL-C	VLDL-C	HDL-C
I- Non-diabetic (ND)	147.00 ± 11.22	149.57 ± 5.73	57.67 ± 4.34	29.40 ± 2.24	62.50 ± 2.83
II- STZ/NA control	316.83 ± 7.91^###	282.20 ± 7.43^###	187.83 ± 5.88^###	63.37 ± 1.34^###	31.00 ± 2.38^###
III- MEHA (100)	293.50 ± 7.99^ns	273.70 ± 6.21^ns	175.83 ± 4.83^ns	58.70 ± 1.60^ns	39.17 ± 2.50^ns
IV- MEHA (200)	273.17 ± 8.70^**	264.47 ± 5.16^ns	164.50 ± 4.19^**	54.63 ± 1.74^**	45.33 ± 2.53^**
V- MEHA (400)	259.17 ± 7.16^***	255.17 ± 5.23^*	156.83 ± 4.00^***	51.83 ± 1.43^***	46.50 ± 2.68^***
VI- MET (180)	178.17 ± 11.39^***	235.30 ± 6.06^***	151.00 ± 4.12^***	35.63 ± 2.28^***	48.67 ± 2.54^***

Note: The data were expressed as mean ± SEM (n=6) and analyzed by one-way ANOVA trailed by post-hoc Tukey’ multiple comparison test. ^###p<0.001 for STZ control Vs ND group. ^ns: non-significant for MEHA (100), ^**p<0.01 for MEHA (200) except for TC, ^***p<0.001 for MEHA (400) except for TC, and ^***p<0.001 for MET, Vs STZ control group.


Figure 1. Effect of MEHA on diabetes-induced alterations in the extent of MDA production in renal tissue.	Figure 2. Effect of MEHA on diabetes-induced alterations in GSH level in renal tissue.

Histoarchitectural analysis of kidneys:

Table 4 elucidates histoarchitectural characteristics of renal tissues in STZ/NA induced diabetic kidney disease.

Figures 4 A-F. Effect of chronic treatment of MEHA on renal histoarchitecture in STZ/NA-induced diabetic nephropathy.

Photomicrographs of sections of kidneys from rats stained with H and E. G: glomeruli, T: tubules, GS: glomerular space, VD: vacuolar degeneration. 4A ND group I, 4B STZ/NA control group II, 4C MEHA (100) group III, 4D MEHA (200) group IV, 4E MEHA (400) group V, and 4F MET group VI. Microscopic examination under 400× light microscopy.

DISCUSSION:

Diabetes is a severe endocrine disorder characterized by insufficient insulin production, leading to hyperglycemia and metabolic abnormalities. With over 300 million people affected globally by 2025, the prevalence is expected to rise³². Diabetic nephropathy, a dangerous sequel, is a common cause of end-stage renal failure. Type 2 diabetes has a higher risk of developing this condition³³. While pharmaceutical interventions exist, research is focusing on herbal remedies as preventive measures.

Previous studies have convincingly established the efficacy of Hygrophila in protecting kidneys against non-diabetic nephropathy induced by drugs like gentamicin and cisplatin. The research explores the potential of Hygrophila auriculata (K. Schum) Heine to offer dual nephroprotective benefits in a type 2 diabetes model. The study uses the plant's antioxidant, antihyperglycemic, and renoprotective properties, along with bioactive phytoconstituents like lupeol, β-sitosterol, betulin, apigenin-7-O-glucuronide, and apigenin-7-O-glucoside, to protect kidneys against non-diabetic nephropathy¹⁹.

Recent research has used streptozotocin (STZ)-induced diabetic rats and mice pretreated with nicotinamide (NA) to assess insulin secretagogues. STZ targets pancreatic beta-cells, leading to diabetes development³⁴. The mechanism is elusive, but nuclear DNA is a key site of action. STZ uptake triggers free radical generation, causing DNA damage and activating poly(ADP-ribose) synthetase (PARS), an enzyme involved in DNA repair^35–37. NA, a precursor of nicotinamide adenine dinucleotide (NAD), mitigates this toxicity by inhibiting PARS and preserving NAD levels^38,39. Studies show a 50% decrease in pancreatic insulin content and moderate hyperglycemia in NA-pretreated STZ-diabetic rodents, suggesting NA-mediated PARS inhibition can partially restore beta-cell function and insulin secretion^25,40.

STZ/NA-induced type 2 diabetes leads to a significant decrease in body weight due to factors like elevated blood glucose, diminished insulin secretion, muscle breakdown, and tissue protein depletion^41,42. Treatment with methanol extract of Hygrophila (MEHA) and metformin (MET) increased body mass, preventing damage to muscle tissue. Diabetes causes chronic hyperglycemia, leading to a steady rise in glycohemoglobin (HbA1c). Oxygen free radicals generated through glycation have been implicated as a primary driver of diabetic kidney disease (DKD)^43,44. Diabetic rats showed elevated plasma glucose and HbA1c levels, but treatment with MEHA and MET reduced both parameters, suggesting potential therapeutic promise for DKD by mitigating free radical effects.

DKD is linked to elevated blood creatinine levels and BUN, along with reduced urine creatinine excretion^33,45. In diabetic rats sensitized to STZ/NA, high creatinine, BUN, and uric acid levels were reduced dose-dependently by MEHA and MET.

Diabetes mellitus (DKD) is often linked to dyslipidemia, a significant risk factor for cardiovascular disease. Diabetics with disrupted lipoprotein metabolism often have an elevated atherogenic lipoprotein profile, including VLDL-C and LDL-C, while experiencing a reduction in cardioprotective HDL-C. This increases their cardiovascular disease risk. Lipoproteins in diabetes, such as oxidized LDL, are more proatherogenic^9–12. Animals with diet-induced hypercholesterolemia developed localized glomerulosclerosis⁴⁴, while lipid-lowering therapy was linked to a decrease in glomerular lesions in Zucker rats⁴⁶. The tubulointerstitium, a key component of diabetic nephropathy and a significant predictor of renal failure, is adversely affected by hyperlipidemia⁴⁷. A recent study showed that hyperglycemia and hyperlipidemia work together to cause renal damage in LDL receptor-deficient mice. Dyslipidemia, characterized by elevated triglycerides and LDL cholesterol and reduced HDL cholesterol, has been implicated in the progression of diabetic nephropathy through disruption of coagulation and fibrinolysis, endothelial cell damage, and accelerated atherosclerosis^48,49. Both MEHA and MET normalized serum lipid profiles, leading to significant elevations in HDL-C and reductions in total cholesterol, LDL-C, VLDL-C, and triglycerides.

Oxidative stress is a key factor in the progression of diabetic complications, including nephropathy⁵⁰. This is due to mechanisms like NAD(P)H oxidase, glycation end products, polyol pathway dysfunction, and mitochondrial respiratory chain defects. Excessive ROS exposure disrupts cellular homeostasis, leading to fibrosis and end-stage renal disease⁵¹. A study found that MEHA and MET can effectively mitigate STZ/NA-induced oxidative stress in diabetic rats, boosting glutathione and superoxide dismutase levels, suggesting a potential therapeutic role for these compounds in diabetic nephropathy. These findings align with previous research⁵².

The study found significant renal lesions in diabetic rat models, including dilated Bowman's capsules, congested glomeruli, and inflamed interstitial tissue. Treatment with MEHA and MET attenuated these lesions, reducing glomerular meningeal hyperplasia, basement membrane thickening, shrunken Bowman's capsules, capillary blockage resolution, and renal fibrosis decrease.

CONCLUSION:

This preclinical study evaluated the efficacy of MEHA in mitigating diabetic kidney disease (DKD) in a Wistar rat model of type 2 diabetes induced by STZ/NA. MEHA exhibits promising nephroprotective potential against DKD, likely due to its combined hypoglycemic, hypolipidemic, and antioxidant properties.

CONFLICT OF INTEREST:

The authors affirm the absence of any conflicting interests.

ACKNOWLEDGMENTS:

The authors express gratitude to the Department of Pharmacology, B. N. College of Pharmacy, B. N. University, Udaipur-313001, Rajasthan, India, for facilitating the essential infrastructure required for the execution of this study. Furthermore, the authors extend their sincere thanks to Dr. Hemantkumar A. Thakur, Head of the Postgraduate Department of Botany at GES's HPT Arts and RYK Science Institution, Nashik, Maharashtra, India, for providing assistance in the authentication of the plant materials employed in this research.

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Received on 22.12.2023 Modified on 13.03.2024

Research J. Pharm. and Tech 2024; 17(10):4873-4879.

DOI: 10.52711/0974-360X.2024.00750

Treatment	Body mass (g)	Serum glucose (mg/dL)	Hemoglobin A1c (%)
I- Non-diabetic (ND)	241.67 ± 5.58	163.13 ± 6.75	5.04 ± 0.39
II- STZ/NA control	149.17 ± 3.00^###	460.10 ± 5.62^###	12.35 ± 0.58^###
III- MEHA (100)	1⁵³.17 ± 3.00^ns	452.80 ± 5.62^ns	12.21 ± 0.50^ns
IV- MEHA (200)	171.50 ± 4.23^**	419.80 ± 5.62^**	9.97 ± 0.47^**
V- MEHA (400)	175.17 ± 3.00^***	393.80 ± 5.62^***	9.55 ± 0.40^***
VI- MET (180)	175.67 ± 5.58^***	333.13 ± 6.75^***	9.41 ± 0.44^***

Groups	Histoarchitectural characteristics	Figures
I- Non-diabetic (ND)	Normal glomerular morphology with intact Bowman's capsule and well-defined proximal and distal convoluted tubules. Normal basement membrane thickness.	4A
II- STZ/NA control	Glomerular hypertrophy (enlargement) with Bowman's space dilation, alongside glomerular congestion and atrophy. Tubulopathy (tubular degeneration) with enlargement and inflammatory cell infiltration in the interstitium.	4B
III- MEHA (100)	Dose-dependent attenuation of renal lesions. Reduced glomerular mesangial expansion, decreased basement membrane thickening, and a reduction in Bowman's capsule size. Capillary obstruction and interstitial fibrosis.	4C
IV- MEHA (200)		4D
V- MEHA (400)		4E
VI- MET (180)	Notable attenuation of renal lesions similar to MEHA-treated groups.	4F