2023 Impact Factor
2023 Impact Factor
Diabetic kidney disease is characterized by chronic proteinuria, a gradually falling glomerular filtration rate, arterial hypertension, and, in many cases (Viberti and Walker, 1991; Mestry et al., 2017). Chronic, microvascular issues also constitute the main factor in end-stage renal failure (Forbes and Cooper, 2013).
The complex pathophysiological alterations that cause diabetic nephropathy (DN), which results in renal injury, include vascular disease, glomerulonephritis, and modifications to the tubulointerstitium, such as tubular atrophy and glucose homeostasis (López-Novoa et al., 2011). DN is a symptom of a variety of diseases caused by an inadequate insulin secretory response (Karalliedde and Gnudi, 2016; Stefano et al., 2016). The pathophysiology of diabetic complications begins with hyperglycemia, which is caused by the drastic modification of carbohydrate metabolic enzymes in experimental diabetes.
Free radicals and oxidative stress have been linked to the progression of DN in several studies (Giacco and Brownlee, 2010). Recent research has demonstrated that diabetes increases lipid peroxidation, which can lead to tissue damage. It has also been established that hyperglycemia is a crucial factor in severe oxidative stress seen in diabetes (Ito et al., 2019). Hence, it follows that reducing lipid peroxidation and free radical generation may reduce diabetes-related complications (Asmat et al., 2016). The pathogenesis of DN involves a multifactorial interplay linking lipid problems, oxidative stress, anomalies in renal hemodynamics, inflammatory cytokines, signaling systems for mitogen-activated protein kinases, and polyol activation (Amalan et al., 2015; Sharma et al., 2019). Consuming foods rich in polyphenolic antioxidants has been demonstrated to reduce the risk of diabetic problems and boost the body’s own antioxidant defense system (Gomes et al., 2014; Dal and Sigrist, 2016; Hong et al., 2017). Experimental diabetes is frequently induced in animal models using streptozotocin (STZ) because it stimulates cellular damage, which leads to insulin dysfunction and hyperglycemia, both of which are important factors in DN (Amalan et al., 2016; Park et al., 2019). It induces hyperglycemia in 48-72 h by causing necrosis in cells and insulin-producing pancreatic endocrine cells specifically. It has been stated that flavonoids have effective antidiabetic activity. The glycemic control, lipid profile, and antioxidant status of diabetes are improved by flavonoids derived from vegetables and medicinal plants (Ghorbani, 2017). In fact, several flavonoids have been suggested as potential treatments for diabetes and the associated consequences (Shi et al., 2019).
Prunetin (PRU), an O-methylated isoflavone discovered in Pisum sativum, Prunus avium, and Trifolium pratense, is a phytoestrogen compound (Yang et al., 2013). It has several pharmacological effects, such as inflammation reduction, stress reduction, and the control of proteolytic activity (Vinayagam and Xu, 2015). PRU is widely known for its ability to inhibit the action of growth factors in vitro, as a strong tyrosine kinase inhibitor. Although PRU was reported to have a protective effect against dexamethasone-induced pancreatic beta cell apoptosis (Kooptiwut et al., 2020), effects of PRU on STZ-induced DN still remain uncertain. Insulin therapy is one of the therapeutic modalities used to treat diabetes. Several medications are presently available that can be used to lower hyperglycemia in diabetic individuals (Barnes, 2004).
Here, we examine PRU’s effect on DN in STZ-treated rats by comparing biochemical changes, enzymatic and non-enzymatic antioxidants, insulin receptor substrate 1 (IRS-1), glucose transporter 2 (GLUT-2) amplification, and histological changes, in a dose-dependent manner.
PRU, STZ, and other research chemicals were bought from Sigma-chemicals (St. Louis, MO, USA).
Albino male Wistar rats weighing 170-180 g were obtained from the Central Animal House of Rajah Muthiah Medical College, Annamalai University in Tamil Nadu, India. The rats were housed in cages made of polypropylene and heated to 23 ± 2°C, with free access to water, and were given a standard pellet diet. The animal ethical committee accepted the method for the test, which was executed according to the requirements of CPCSEA New Delhi, India, (IAEC PROPOSAL No. AU-IAEC/1291/8/21).
The STZ was dissolved in a buffer solution of 0.01 M citrate (pH 4.5), and the estimated dose of the fresh solution (45 mg/kg) was administered intraperitoneally (i.p.) to overnight-starved rats. Blood sugar levels were assessed seven days after induction using an Accu-Check Active Blood Glucometer Kit (Roche Diabetes Care Pvt Ltd., Mumbai, India). Diabetes was considered to exist in those with >250 mg/dL of blood glucose and was used as the basis for subsequent research (Thomasset et al., 2007). The nephropathic rats were given PRU orally for 28 days.
The rats were separated into the following groups: Group I were the control rats; Group II were the DN control rats and received STZ as a single dose; Group III rats were administered orally with PRU only (80 mg/kg); Group IV rats received PRU (20 mg/kg body weight) orally with single STZ i.p. for four weeks; Group V rats received PRU (40 mg/kg body weight) orally with a single STZ i.p. for four weeks; Group VI rats received PRU (80 mg/kg body weight) orally with a single STZ i.p. for four weeks.
The initial and final body weights (On the first day of the experiment body weight of the rats was measured which is the initial body weight. After the treatment with PRU 80 mg/kg for a period of 28 days the final body weight was measured which is final body weight) of the rats were measured, and all rats were then slaughtered at the end of the experiment, following a 24-h fast. Blood was drawn for further analysis. The tissue samples were removed and cleaned with physiological saline at 4°C. The Trinder technique was used to assess the plasma blood glucose level (Mutalik et al., 2003). The technique developed by Burgi et al. (1988) was used to assess insulin levels (Venkatesan et al., 2022). Hemoglobin (Hb) and glycated hemoglobin A1c (HbA1c) (Trinder, 1969), urea, creatinine, total protein, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, cholesterol, very-low-density lipoprotein (VLDL), serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), and C-reactive protein (CRP) were assessed in all test animals, utilizing commercially accessible tests obtained from the Egyptian Company for Biotechnology (Spectrum Diagnostics, Cairo, Egypt).
Phosphate buffer pH 7.4 (10% 0.1 M) was used to homogenize the tissue sample in this investigation. A component of the supernatant from the centrifuged homogenate was used to assess endogenous glutathione (GSH) (Burgi et al., 1988), superoxide dismutase (SOD) (Bisse and Abraham, 1985), and enzyme catalase (CAT) activities (Moron et al., 1979). The hydrogen peroxide (HP) and thiobarbituric acid reactive substances (TBARS) in kidney tissues were also assessed (Marklund and Marklund, 1974; Aebi, 1984).
Liver, kidney, and pancreatic tissues were removed from the rats in every experimental group, promptly rinsed with saline solution, and then put in a 10% neutral solution of formalin buffered for storage. Tissues were paraffin fixed and were then cut into 2- to 5-micron pieces and fixed with Hematoxylin and eosin (H&E) stain before being studied at high magnification (40X) (Amalan et al., 2021).
The total RNA isolation was carried out by the TRIR kit (Thermo Fisher Scientific, Bengaluru, Karnataka). Real-time PCR was performed with the aid of the Bio-Rad CFX96 Real-Time PCR detection system (Bio-Rad, Bengaluru, Karnataka). The following primer sequences were used: β-actin (as a housekeeping gene) – Forward:5′-AAGTCCCTCACCCTCCCAA AAG-3, Reverse:5′-AAGCAATGCTGTCACCTTCCC′; IRS-1 – Forward:5′-GCCAATCTT CATCCA GTTGCT-3′, Reverse:5′-CATCGTGAAGAAGGCATA GGG-3′; GLUT-2 – Forward-5′-CTCGGGCCTTACGTGTGTCCTTCCTT-3′, Reverse-5′-GTTCCCTTCTGGTCTGTTCCTG-3′ (Jiang et al., 1992).
The data (n=6) were expressed as the mean ± SD of the total number of experiments. IBM SPSS analyzing variation in one dimension (one-way ANOVA) (Version 23.0, SPSS Inc, Chicago, IL, USA) was used to determine individual comparisons, and statistical analysis in addition to Multiple-range Duncan’s test was used (DMRT). P values <0.05 were statistically significant (Bancroft et al., 1996).
The body weight of group II rats decreased compared to control group. After treatment with the three different doses of PRU (20, 40 and 80 mg/kg), the body weight considerably increased compared to group II. The administration of 80 mg/kg PRU alone considerably increased the body weight near the control group, as shown in Fig. 1.
Figure 1. Effect of PRU on body weight control and experimental rats. Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
STZ substantially elevated blood glucose levels and lowered insulin levels (Fig. 2A, 2B). When PRU was administered to the STZ-induced diabetic (group IV, V, and VI) rats, blood glucose was considerably decreased, and blood insulin levels were increased in a dose-dependent manner, whereas blood glucose and insulin levels were unaltered by PRU treatment in the control rats.
Figure 2. Effect of PRU on plasma blood glucose (A) and insulin (B) levels. Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
Group II rats seemed to have lower Hb and higher HbA1c levels than the control group. DN rats (group IV, V, and VI) treated with PRU had substantially (p<0.05) better Hb and lower HbA1c levels compared to the untreated nephropathic rats (Fig. 3A, 3B).
Figure 3. Effect of PRU on (A) Hb and (B) HbA1c. Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
Urea and creatinine levels were found to be statistically increased and the total protein levels were decreased in nephropathic rats (group II) (Table 1). When DN rats (group IV, V and VI) were given PRU, the levels of urea, creatinine, and total protein were considerably lower than those of the untreated nephropathic rats.
Table 1 Effect of prunetin on renal markers and inflammatory marker in the serum control and experimental rats
| Groups | Urea mg/dL | Creatinine mg/dL | Total protein gm/dL | CRP mg/dL |
|---|---|---|---|---|
| Control | 25.31 ± 1.93 | 0.99 ± 0.14 | 6.20 ± 0.47 | 3.65 ± 0.54 |
| STZ (diabetic control) | 90.25 ± 6.91* | 4.55 ± 0.36* | 3.01 ± 0.30* | 16.57 ± 2.23* |
| PRU 80 mg/kg | 26.01 ± 1.98 | 1.03 ± 0.100 | 6.19 ± 0.42 | 4.01 ± 0.62 |
| STZ+PRU 20 mg/kg | 82.38 ± 6.30# | 3.55 ± 0.42# | 4.18 ± 0.32# | 11.90 ± 1.38# |
| STZ+PRU 40 mg/kg | 64.35 ± 4.92# | 2.96 ± 0.37# | 5.02 ± 0.38# | 7.96 ± 0.61# |
| STZ+PRU 80 mg/kg | 30.01 ± 2.28## | 1.19 ± 0.77## | 5.80 ± 0.46## | 5.01 ± 0.72## |
Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
As shown in Table 2, the total cholesterol (TC) and triglyceride (TGL) levels were higher, and HDL was lower in nephrotic rats (group II) compared to the control group. However, after receiving PRU (20, 40, and 80 mg/kg), the TC and TGL levels were reduced, and the HDL level was significantly elevated; the 80 mg/kg dose of PRU was the most beneficial.
Table 2 Effect of prunetin on the levels of lipid profile in plasma control and experimental rats
| Groups | Total cholesterol mg/dL | TGL mg/dL | HDL mg/dL |
|---|---|---|---|
| Control | 130.03 ± 9.90 | 112.02 ± 8.52 | 59.03 ± 4.49 |
| STZ (diabetic control) | 236.11 ± 18.07* | 210.42 ± 16.10* | 34.16 ± 2.6* |
| PRU 80 mg/kg | 125.13 ± 9.52 | 105.16 ± 8.04 | 59.03 ± 4.44 |
| STZ+PRU 20 mg/kg | 202.31 ± 15.48# | 185.14 ± 14.09# | 39.16 ± 2.99# |
| STZ+PRU 40 mg/kg | 183.48 ± 13.97# | 172.67 ± 13.14# | 44.15 ± 3.35# |
| STZ+PRU 80 mg/kg | 134.12 ± 10.21## | 116.34 ± 8.86## | 61.01 ± 4.64## |
Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
The levels of liver marker enzymes (SGOT, SGPT, and ALP) considerably increased in nephropathic rats (group II) compared with the control group. However, PRU administration (20, 40, and 80 mg/kg) dramatically reduced the liver marker enzymes, especially at a dose of 80 mg/kg. There was no significant alteration in the liver marker enzymes in rats treated with PRU (80 mg/kg) alone (Table 3).
Table 3 Effect of prunetin on the levels of liver marker enzymes in serum control and experimental rats
| Groups | SGOT (U/L) | SGPT (U/L) | ALP (U/L) |
|---|---|---|---|
| Control | 26.68 ± 1.88 | 22.33 ± 1.83 | 73.73 ± 3.22 |
| STZ (diabetic control) | 96.85 ± 1.33* | 106.31 ± 4.38* | 174.39 ± 3.78* |
| PRU 80 mg/kg | 29.95 ± 0.83 | 21.78 ± 3.27 | 75.16 ± 4.10 |
| STZ+PRU 20 mg/kg | 83.53 ± 2.68# | 75.24 ± 1.70# | 142.06 ± 2.88# |
| STZ+PRU 40 mg/kg | 65.84 ± 3.72# | 56.27 ± 5.22# | 120.26 ± 3.30# |
| STZ+PRU 80 mg/kg | 30.65 ± 1.09## | 25.12 ± 1.35## | 76.08 ± 1.75## |
Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
CRP levels were higher in the plasma of nephropathic rats (group II) compared to the control rats. PRU treatment of nephropathic rats (group IV, V, and VI) led to a significantly lower level of inflammation, but no significant alternation was observed in the group of rats treated with PRU alone (Table 1).
Nephropathic rats (group II) had reduced levels of SOD, CAT, and glutathione peroxidase (GPx) activities compared to the control group. However, the SOD, CAT, and GPx activities were restored, compared to the group II, after an oral dose of PRU (Table 4). Non-enzymatic antioxidants were found in lower concentrations in the rats of the DN control (group II). PRU injection increased the amounts of non-enzymatic antioxidants compared to the control group rats (Table 5).
Table 4 Effect of prunetin on the changes in the activities of enzymatic antioxidant SOD, CAT and GPx kidney tissue control and experimental rats
| Groups | SOD | CAT | GPx |
|---|---|---|---|
| Control | 19.67 ± 1.86 | 37.40 ± 1.12 | 7.89 ± 0.50 |
| STZ (diabetic control) | 7.61 ± 1.05* | 20.45 ± 1.30* | 3.17 ± 0.28* |
| PRU 80 mg/kg | 19.46 ± 1.74 | 37.20 ± 0.98 | 7.98 ± 0.51 |
| STZ+PRU 20 mg/kg | 10.20 ± 0.51# | 25.48 ± 0.34# | 4.70 ± 0.41# |
| STZ+PRU 40 mg/kg | 13.28 ± 0.33# | 29.34 ± 0.45# | 6.12 ± 0.17# |
| STZ+PRU 80 mg/kg | 18.48 ± 0.86## | 36.69 ± 1.46## | 7.54 ± 0.28## |
Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
Table 5 Effect of prunetin on the changes in the activities of enzymatic antioxidant Vitamin C, Vitamin E and GSH kidney tissue control and experimental rats
| Groups | Vitamin C | Vitamin E | GSH |
|---|---|---|---|
| Control | 1.18 ± 0.89 | 1.05 ± 0.04 | 24.69 ± 2.70 |
| STZ (diabetic control) | 0.56 ± 0.43* | 0.35 ± 0.008* | 12.64 ± 1.24* |
| PRU 80 mg/kg | 1.18 ± 0.09 | 1.06 ± 0.04 | 24.88 ± 1.93 |
| STZ+PRU 20 mg/kg | 0.68 ± 0.51# | 0.40 ± 0.01# | 16.81 ± 0.60# |
| STZ+PRU 40 mg/kg | 0.94 ± 0.72# | 0.49 ± 0.26# | 20.82 ± 1.01# |
| STZ+PRU 80 mg/kg | 1.16 ± 0.85## | 1.04 ± 0.04## | 23.13 ± 0.73## |
Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
HP and TBARS were elevated significantly in the renal tissues of nephrotic rats (group II) (Table 6). After PRU (80 mg/kg) treatment, the HP and TBARS significantly decreased compared to the untreated nephrotic rats. However, rats that were only given PRU 80 mg/kg had no significant change compared to the control rats.
Table 6 Effect of prunetin on TBARS and HP activity in the kidney tissues of control and experimental rats
| Groups | TBARS | HP |
|---|---|---|
| Control | 1.40 ± 0.10 | 55.01 ± 4.18 |
| STZ (diabetic control) | 2.64 ± 0.20* | 124.06 ± 9.49* |
| PRU 80 mg/kg | 1.44 ± 0.11 | 53.21 ± 4.05 |
| STZ+PRU 20 mg/kg | 2.01 ± 0.15# | 108.05 ± 8.27# |
| STZ+PRU 40 mg/kg | 1.86 ± 0.14# | 89.12 ± 6.78# |
| STZ+PRU 80 mg/kg | 1.55 ± 0.11## | 60.02 ± 4.56## |
Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
The carbohydrate metabolic enzymes were considerably greater in DN rats (group II) than in the control rats. On the other hand, phosphoenol pyruvate carboxykinase, fructose 1,6-bisphosphatase, and glucose-6-phosphatase levels were lower in the liver, kidney, and skeletal muscle tissues of DN rats (group II) compared to the control rats. In contrast, the hexokinase and glucose-6-phosphate dehydrogenase levels were significantly decreased, and phosphoenol pyruvate carboxykinase, fructose 1,6-bisphosphatase, and glucose-6-phosphatase were dramatically increased following the administration of PRU (Table 7A-7E). However, no significant difference was seen in the levels of these enzymes in the group treated with PRU (80 mg/kg) alone and the control group.
Table 7 A Effect of prunetin on hexokinase in the tissues of control and experimental rats
| Hexokinase (µmol of glucose phosphorylated/min/g protein) | |||
|---|---|---|---|
| Groups | Liver | Kidney | Skeletal muscle |
| Control | 152.34 ± 11.60 | 143.78 ± 10.95 | 172.16 ± 13.11 |
| STZ (diabetic control) | 94.84 ± 7.26* | 78.35 ± 6.00* | 104.43 ± 7.99* |
| PRU (80 mg/kg b.w.) | 154.20 ± 11.74 | 145.41 ± 11.07 | 173.14 ± 13.18 |
| STZ+PRU (20 mg/kg b.w.) | 103.06 ± 7.89# | 89.71 ± 6.87# | 121.52 ± 9.30# |
| STZ+PRU (40 mg/kg b.w.) | 118.33 ± 9.01# | 114.77 ± 8.74# | 139.10 ± 10.59# |
| STZ+PRU (80 mg/kg b.w.) | 149.71 ± 11.46## | 139.58 ± 10.68## | 165.87 ± 12.70## |
Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
Table 7B Effect of prunetin on Glucose-6-phosphatase in the tissues of control and experimental rats
| Glucose-6-phosphatase (µmol of Pi liberated/min/mg protein) | |||
|---|---|---|---|
| Groups | Liver | Kidney | Skeletal muscle |
| Control | 0.14 ± 0.01 | 0.12 ± 0.01 | 0.20 ± 0.02 |
| STZ (diabetic control) | 0.45 ± 0.03* | 0.39 ± 0.03* | 0.49 ± 0.04* |
| PRU 80 mg/kg | 0.12 ± 0.01 | 0.09 ± 0.01 | 0.18 ± 0.01 |
| STZ+PRU 20 mg/kg | 0.38 ± 0.03# | 0.31 ± 0.02# | 0.40 ± 0.03# |
| STZ+PRU 40 mg/kg | 0.23 ± 0.02# | 0.26 ± 0.02# | 0.36 ± 0.03# |
| STZ+PRU 80 mg/kg | 0.19 ± 0.01## | 0.17 ± 0.01## | 0.22 ± 0.02## |
Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
Table 7C Effect of prunetin on Phosphoenol pyruvate carboxykinase in the tissues of control and experimental rats
| Phosphoenol pyruvate carboxykinase (nmoL/min/mg protein) | |||
|---|---|---|---|
| Groups | Liver | Kidney | Skeletal muscle |
| Control | 1.44 ± 0.11 | 0.23 ± 0.02 | 0.16 ± 0.01 |
| STZ (diabetic control) | 4.20 ± 0.32* | 0.98 ± 0.08* | 0.73 ± 0.06* |
| PRU 80 mg/kg | 1.40 ± 0.11 | 0.22 ± 0.02 | 0.14 ± 0.01 |
| STZ+PRU 20 mg/kg | 3.96 ± 0.30# | 0.74 ± 0.06# | 0.68 ± 0.05# |
| STZ+PRU 40 mg/kg | 2.63 ± 0.20# | 0.63 ± 0.05# | 0.51 ± 0.04# |
| STZ+PRU 80 mg/kg | 1.98 ± 0.15## | 0.36 ± 0.03## | 0.20 ± 0.02## |
Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
Table 7D Effect of prunetin on Fructose 1,6-bisphosphatase in the tissues of control and experimental rats
| Fructose 1,6-bisphosphatase (µmol of Pi liberated/h/mg protein) | |||
|---|---|---|---|
| Groups | Liver | Kidney | Skeletal muscle |
| Control | 0.27 ± 0.02 | 0.35 ± 0.03 | 0.56 ± 0.04 |
| STZ (diabetic control) | 0.73 ± 0.06* | 1.42 ± 0.11* | 1.29 ± 0.10* |
| PRU 80 mg/kg | 0.26 ± 0.02 | 0.32 ± 0.02 | 0.54 ± 0.4 |
| STZ+PRU 20 mg/kg | 0.68 ± 0.05# | 1.30 ± 0.10# | 1.01 ± 0.08# |
| STZ+PRU 40 mg/kg | 0.48 ± 0.04# | 0.95 ± 0.07# | 0.88 ± 0.07# |
| STZ+PRU 80 mg/kg | 0.31 ± 0.02## | 0.40 ± 0.03## | 0.60 ± 0.05## |
Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
Table 7E Effect of prunetin on Glucose-6-phosphate dehydrogenase in the tissues of control and experimental rats
| Glucose-6-phosphate dehydrogenase (×10-4 ml U/mg protein) | |||
|---|---|---|---|
| Groups | Liver | Kidney | Skeletal muscle |
| Control | 5.04 ± 0.38 | 4.65 ± 0.35 | 4.84 ± 0.37 |
| STZ (diabetic control) | 2.21 ± 0.17* | 1.49 ± 0.11* | 1.10 ± 0.08* |
| PRU 80 mg/kg | 5.13 ± 0.39 | 4.70 ± 0.36 | 4.88 ± 0.37 |
| STZ+PRU 20 mg/kg | 2.98 ± 0.23# | 1.83 ± 0.14# | 1.73 ± 0.13# |
| STZ+PRU 40 mg/kg | 3.49 ± 0.27# | 2.72 ± 0.21# | 3.06 ± 0.23# |
| STZ+PRU 80 mg/kg | 4.96 ± 0.38## | 3.80 ± 0.29## | 3.95 ± 0.30## |
Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
The IRS-1 and GLUT-2 gene expression was lower in DN-rats (group II) than in the control group. In contrast, following administration of PRU 80 mg/kg, the IRS-1 and GLUT-2 amplification was considerably upregulated in the liver and pancreatic tissue compared to untreated nephropathic rats (group II) (Fig. 4A, 4B).
Figure 4. mRNA expression of liver tissue IRS-1 (A) and pancreas tissue GLUT-2 (B). Values are expressed as means ± SD (n=6 rats for each group; test were performed in triplicate). *p<0.05 versus Control group; #p<0.05, ##p<0.01 versus STZ (diabetic control group).
Histopathological examination of the control groups and experimental groups administered with PRU (20, 40, 80 mg/kg) revealed the distinctive architecture of the tissues, as shown in Fig. 5A, 5B, and 5C. The control rats seemed to be normal. The cellular structure of the hepatic tissues of the rats in the control group was regular. Photomicrographs of the livers of the nephropathic rats (group II) revealed symptoms of the damage, including vascular congestion, sinusoidal dilatation, and degradation of liver tissue. PRU 80 mg/kg treatment improved the histological characteristics of the liver tissue. The control group rats showed normal proximal tubules and normal glomeruli, while the acute hemorrhage between the deteriorated tubules at the corticomedullary junction in the STZ-treated group (group II) demonstrated a gradual histological modification. PRU (80 mg/kg) therapy dramatically improved histological abnormalities in the renal tissues, as shown by the normal appearance and tubular diameter. Rats given PRU alone demonstrated paired kidney architecture, normal proximal tubules and no pathological alterations.
Figure 5. A comparison of the histopathology of various tissues. (A) Histopathological observations on the kidney tissues of DN rats. Kidney tissue sections stained with hematoxylin and eosin (100×). (a) Control, (b) Diabetic control, (c) PRU only oral administration (80 mg/kg), (d) Diabetic treated with PRU (20 mg/kg), (e) Diabetic treated with PRU (40 mg/kg), (f) Diabetic treated with PRU (80 mg/kg). (B) Histopathological observations on the liver tissues of Wistar rats. Kidney tissue sections stained with hematoxylin and eosin (100×). (a) Control, (b) Diabetic control, (c) PRU only oral administration (80 mg/kg), (d) Diabetic treated with PRU (20 mg/kg), (e) Diabetic treated with PRU (40 mg/kg), (f)Diabetic treated with PRU (80 mg/kg). (C) Histopathological observations on the pancreatic tissues of Wistar rats. Kidney tissue sections stained with hematoxylin and eosin (100×). (a) Control, (b) Diabetic control, (c) PRU only oral administration (80 mg/kg), (d) Diabetic treated with PRU (20 mg/kg), (e) Diabetic treated with PRU (40 mg/kg), (f) Diabetic treated with PRU (80 mg/kg).
Microscopic examination of pancreatic sections of the control rats revealed that the islets of Langerhans appeared normally. On the other hand, the nephropathic rats (group II) exhibited adverse pathological alterations in both exocrine and endocrine components. The acinar cells were enlarged, and virtually all of them had tiny vacuoles. On the other hand, PRU 80mg/kg-treated animals showed indications of cellular regeneration in the islets of Langerhans (Fig. 5C).
These studies demonstrated that PRU exerts renoprotective effects and could prevent the progression of DN, even without possessing any hypoglycemic effect. More than 300 million individuals are predicted to develop diabetes mellitus globally by 2025 (Amalan and Vijayakumar, 2015). End-stage renal failure is most frequently brought on by DN, a major side effect of diabetes mellitus (Bastaki, 2005).
The pancreatic β-cells are specifically destroyed by STZ to cause experimental diabetes mellitus (Wang et al., 2011). Due to elevated muscle wasting, hyperinsulinemia, hyperglycemia, and protein loss in the tissues, STZ-induced diabetes is linked to a considerable fall in weight (Brenna et al., 2003). The weight of STZ-induced diabetic rats (group II) gradually decreased, indicating the excessive breakdown of tissue proteins, while rats treated with PRU showed a great increase in body weight, indicating a reduction in the risk of hyperglycemia-induced damage to the muscle tissue.
Pancreatic β-cells that secrete insulin are the major factor affecting the pathophysiology of hyperglycemia; thus, pancreatic β-cells and insulin are crucial to the pathophysiology of diabetes (Cheng et al., 2013). The present findings show that the oral administration of PRU increases insulin levels while decreasing blood glucose levels, reducing the diabetogenic implications that are obvious and recognized. PRU may potentially cause the regeneration of insulin-secreting cells, which would raise insulin levels.
Elevated blood glucose levels and hemoglobin glycosylation typically affect red blood cell (RBC) characteristics, which reduces their flexibility and increases their propensity to clump, raising blood viscosity (Kahn, 2001). The decrease in oxygen-carrying capacity caused by the glycosylation of Hb also stimulates the adaptations and reactions associated with hypoxia, including systemic vascular vasodilation (Jadhav and Vaghela, 2022). Hb and HbA1c levels were both improved to near-normal levels in group IV, V, and VI after the oral administration of PRU 20, 40 and 80 mg/kg respectively.
The elevated urea and creatinine levels in the plasma brought on by diabetic hyperglycemia are regarded as important indicators of renal impairment (Peers et al., 2007; Duraisamy et al., 2022). The increased urea production in diabetes might be due to the enhanced catabolism of both liver and plasma proteins. Creatinine was increased in the serum in response to STZ treatment, suggesting an impairment of kidney functions. Blood urea, creatinine, and total protein levels are increased in the plasma of diabetics with hyperglycemia. However, the oral administration of PRU clearly showed an improvement in kidney functions, perhaps due to the antioxidant and anti-inflammatory properties of PRU gradually reducing the quantities of total protein, creatinine, and urea in the blood.
Fatty liver, elevated cholesterol, and increased triglycerides are all consequences of diabetes mellitus, and DN and excessive cholesterol are also associated conditions (Sivakumar et al., 2010). The abnormally high concentrations reverted to near normal levels after treatment with PRU 80 mg/kg, indicating the drug’s ability to improve lipid metabolism.
The role of the liver in the cholesterol metabolism process is crucial. Increased liver enzyme (SGOT, SGPT, and ALP) activity may also be a symptom of inflammation, which compromises insulin signaling. Hepatic diseases are evaluated using spleen enzymes, like SGOT, SGPT, and ALP. The membrane permeability that was altered because of cholestatic or active hepatic damage may be the subject here. These enzymes leak when the membrane permeability is altered, which disrupts the transfer of metabolites (Nesbitt, 2004; Zhang et al., 2010). In an assessment of crucial enzymes, untreated STZ-induced DN rats (group II) had significantly higher activities of the liver enzymes SGOT, SGPT, and ALP compared to the control rats. These enzyme activities were markedly reduced in the DN rats treated with PRU compared to the group II rats.
Type-2 diabetes and diabetic kidney complications are caused by CRP. Tubular epithelial cells can produce CRP in response to excessive blood glucose. CRP can directly activate Smad3 signaling via the TGF-1-dependent process, and indirectly through the interaction between Extracellular signal-regulated kinases (ERK) and p38 MAP kinase. mTOR, Smad3, and CD32b signaling may cause CRP to induce hepatic fibrosis, whereas the CD32b NF-ĸB signaling process may cause CRP to increase renal inflammation. In terms of preventing and treating diabetic kidney complications, CRP may provide an alternative strategy (You et al., 2016). In the STZ-injected rats (group IV, V, and VI) treated with PRU, diabetic kidney complications were significantly decreased.
Reactive oxygen species (ROS) formation leads to a wide range of DN problems. ROS weaken the cell’s antioxidant defenses, making oxidative damage more likely. Other alterations to cellular structure and function are also brought about because of the oxidation of proteins and lipids. In addition to maintaining a plasma antioxidant state, GSH performs a critical function as a free radical scavenger. Superoxide is changed by SOD into HP, and CAT further reduces the less reactive ROS to water. Thus, SOD is helped by CAT to completely neutralize ROS.
In our study, 7 days after administration of the STZ injection to Wistar rats, significant hyperglycemia and DN were noted. The generation of free radicals might harm renal cells (Haneda, 2006; Natesan et al., 2016), yet the situation deteriorates when people with diabetes mellitus have lower antioxidant levels (Townsend et al., 2003; Pasaoglu et al., 2004; Sharma et al., 2006). In contrast, current research indicated that STZ-induced (group II) diabetic kidney tissue homogenates had lower concentrations of enzymatic and non-enzymatic antioxidant levels, and these antioxidant levels were significantly raised when PRU was taken orally. Early in the illness, there may be a time of compensatory enhancement of free radical generation and antioxidant defense system activity, but this phase may not last, eventually leading to the deleterious consequences of unregulated oxidants that result in DM complications. In our study, when DN rats (group IV, V, and VI) were received PRU 80 mg/kg for 28 days, the levels of antioxidant enzymes appeared were near normal.
The cytotoxic effect of STZ is connected to ROS generation and eventually results in a decrease in insulin output. The synthesis of superoxide radicals, which can destroy pancreas β-cells, is thought to be enhanced because of the suppression of free radical scavenger enzymes (Amalan et al., 2016). Diabetes inhibits insulin secretion, which could cause lipid peroxidation in biological systems. The higher concentrations of TBARS and HP found in the nephrotic rats point to an overactive lipid peroxidative system and an excessive amount of free radical generation.
The first glycolysis enzyme hexokinase converts the phosphoryl group of ATP into glucose-6-phosphate, phosphorylating glucose in the process. The necrosis and degeneration of β-cells in the generation of diabetes by STZ in nephrotoxic rats results in a lack of insulin secretion, which impairs hexokinase activity, because glycolysis and the use of glucose for energy production are reduced (Alaofi, 2020). A modest increase in hexokinase activity was seen in PRU-treated nephropathic rats (group IV, V, and VI), despite the drug’s capacity to promote insulin secretion, which improved the utilization of glucose for energy generation and reduced kidney damage. The hexose monophosphate shunt (HMP shunt) may function less effectively in diabetic conditions due to the decreased activity of this enzyme, which may also create reducing equivalents like NADH and NADPH, which are needed for glutathione reductase activity and consequently affect the GSH levels of tissues (Díaz-Flores et al., 2006). In addition, our findings suggest that impaired insulin secretion contributes to the reduced activity of carbohydrate metabolic enzymes in STZ-induced nephropathic rats (group II).
Histopathological analysis of the kidney sections of group II rats revealed a thickening of the basement membrane, significantly increased glomerular space, glomerular vascular degeneration, and moderately intense periodic acid-Schiff (PAS) positivity in the glomeruli. PRU treatment considerably attenuated these alterations, demonstrating its preventative role in renal damage. Histological examinations of the pancreases of STZ-treated rats (group II) showed an increase in necrotic changes, a loss of the β-cell population, and a shrinking of the islets, followed by atrophy and fibrosis. When diabetic rats received PRU, the abnormal histological alterations were restored, and there was an increased islets of Langerhans β-cell population. Previous diabetes investigations have shown similar histology findings (Gandhi et al., 2011; Manogar et al., 2022).
It has been established that the glucose transporter 4 (GLUT4) regulatory mechanism is upstream of the IRS-1/PI3K/AKT signal pathway. Under physiological circumstances, IRS-1 stimulates PI3K phosphorylation. Following Akt phosphorylation, GLUT4 translocation from the inner vesicles to the membrane is accelerated by PI3K, which ultimately triggers glucose absorption (Wang et al., 2020). Earlier studies have shown that insulin resistance is linked to a drop in insulin sensitivity (Boura-Halfon and Zick, 2009). Interestingly, it has been suggested that diabetic nephropathy is related to GLUT2 deficiency (Berry et al., 2005). To further understand the molecular mechanisms, we examined the effects of PRU on IRS-1 and GLUT2, which are important elements of the insulin signaling cascade in insulin-resistant L6 myotubes. In the insulin-resistant L6 cells, PRU treatment (20, 40, and 80 mg/kg) increased the expression of GLUT2, as well as IRS-1. These findings suggest that PRU may help increase glucose absorption and GLUT2 translocation by raising the IRS and p-Akt phosphorylation levels, thereby turning on the insulin signaling pathway. However, further research is necessary to fully characterize the mechanism of PRU. Our results suggest that PRU causes preventive action against DN, with an effective dosage of 80 mg/kg.
The results of this dose-dependent investigation show that the oral administration of PRU normalized biochemical parameters, increased antioxidant activity, and upregulated IRS-1 and GLUT-2 mRNA expression. The antioxidant, antihyperglycemic, and nephroprotective ability of PRU (80 mg/kg) against DN was shown by the restoration of kidney functional parameters and reduced histological abnormalities. Therefore, we suggest that PRU (80 mg/kg) successfully protects STZ-induced DN rats.
The authors acknowledge the support from the Researchers supporting project number (RSP2023R393), King Saud University, Riyadh, Saudi Arabia.
Funding Information
King Saud University |
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