l-Arginine attenuates the ethylene glycol induced urolithiasis in ininephrectomized hypertensive rats: role of KIM-1, NGAL, and NOs

Abstract

Background: Ethylene glycol (EG) exposure caused formation of calcium oxalate crystal that led to renal failure, which is associated with higher prevalence of hypertension. l-Arginine is known to have an antioxidant and nephro-protective potential. Objective: To evaluate the effect of l-arginine against EG-induced urolithiasis in uninephrectomized hypertensive rats. Material and methods: Uninephrectomized male Wistar rats (180–200 g) were used to induce urinary calculi through oral administration of EG (0.75%) in distilled water. Rats were treated with either distilled water (10 mg/kg, p.o.) or telmisartan (10 mg/kg, p.o.) or Cystone (500 mg/kg, p.o.) or l-arginine (250, 500, and 1000 mg/kg, p.o.) for 28 days. Various hemodynamic, biochemical, molecular, and histological parameters were assessed in kidney and heart. Results: Rats treated with l-arginine (500 and 1000 mg/kg) significantly restored altered relative organ weight, urine output, urine density, urinary pH, and water intake. EG-induced alterations in electrocardiographic (QRS interval, HR, and ST height) and hemodynamic (SBP, DBP, MABP, and LVEDP) abnormalities were significantly restored by l-arginine (500 and 1000 mg/kg) treatment. It also significantly restored alteration in serum and urine biochemical parameters induced by EG. The elevated oxido-nitrosative stress was also significantly decreased by l-arginine (500 and 1000 mg/kg) treatment. It also significantly down-regulated EG-induced up-regulated renal KIM-1, NGAL, eNOS, and iNOs mRNA expressions. Histological aberrations induced in the renal and cardiac tissues were also ameliorated by l-arginine treatment. Conclusion: l-Arginine exerts its nephro- and cardio-protective potential in EG-induced urolithiasis in uninephrectomized hypertensive rats via modulation of KIM-1, NGAL, eNOS, and iNOs mRNA expression.

• Ethylene glycol
• eNOs
• iNOs
• KIM-1
• l-arginine
• NGAL
• uninephrectomy
• urolithiasis

Introduction

Nephrolithiasis or urolithiasis is a common disorder distributed worldwide that is estimated to occur in approximately 12% of the population, with a large recurrence rate of 70–80% in males and 47–60% females.1 Urolithiasis denotes the presence of stones anywhere in the urinary tract, which includes kidneys and bladder.2 The pathophysiological basis of the formation of kidney and bladder stones is entirely different. The majority of stones (up to 80%) which are found clinically and pre-clinically3 are composed of mainly calcium oxalate.4 It has been reported that high blood pressure is associated with abnormalities in calcium metabolism.5 Cross-sectional epidemiological surveys described an association between hypertension and kidney stone disease.6–8 However, a study carried out by researcher showed association between unilateral renal nephrectomy with hypertension.9

Experimental animal models of urinary calculi with hypertension have been widely used to establish a direct relationship between urinary calcium and blood pressure.6^,10 Increased urinary calcium concentration may be responsible for the formation of calcium stones.11 Research on the pathogenesis of calcium oxalate stone formation has reported that renal epithelial cell injury initiates the stone formation.12 Renal mass reduction or damage to one kidney, usually, induces morphological and functional hypertrophy, which increases filtered load and excessive urinary excretion of lithogenic substances in the remaining or alternative nephron units. This may cause the supersaturation of urine, leading to crystallization and urolithiasis as postulated by the hyperexcretion-crystallization hypothesis.13^,14 Angiotensin-II plays an important role in this process and production of local angiotensin-II by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes that have been identified as a major source of ROS generation. This elevated level of ROS causes the inflammatory response and accelerates oxidative stress.15–18

Recent studies have been suggested various functions of angiotensin type 1 receptor blockers (ARBs) other than reducing blood pressure. Reports have revealed that the anti-inflammatory effect is related to the renoprotective and anti-atherosclerotic effects of ARBs.19^,20 The suppression of angiotensin II activity by ARBs could protect the organs against inflammatory damage.

The medical management of urolithiasis mainly involves techniques like extracorporeal shock wave lithotripsy and percutaneous urolithotomy. However, the prevention of recurrence of stone formation is not assured. Moreover, these treatments cause undesirable side effects such as hemorrhage, hypertension, tubular necrosis and subsequent fibrosis of the kidney.21^,22 Therefore, it is necessary to look for an alternative for the management of urolithiasis in uninephrectomized patient, which not only give relief but also have minimal side effect. Therefore, an isolated bioactive moiety from plant source is being sought.

l-Arginine is a semi-essential nitrogen-rich amino acid and a vital precursor for the synthesis of various endogenous molecules, including creatine, urea, polyamines, and nitric oxide (NO). NO is an important signaling molecule involved in neurotransmission, vasodilatation, immune regulation, and host defense.23–25 l-Arginine has been reported to have many biologically important properties including anti-atherosclerotic, wound healing, gastro-protective,26 and platelet aggregation inhibitory potential.27 It is also known to have ameliorative effects against inflammatory bowel diseases,28 intestinal ischemia-reperfusion injury,29 and liver ischemia-reperfusion injury.30 It has been reported that l-arginine plays a protective role in STZ-induced nephrotoxicity,31 cisplatin-induced nephrotoxicity,32 and cyclosporine-induced nephrotoxicity.33 Administration of l-arginine showed significant effects against pulmonary hypertension in pre-clinical34 and clinical studies.35^,36 It also enhances liver regeneration after hepatectomy.37 However, very little is known about its possible attenuating effect in ethylene glycol (EG)-induced urolithiasis in uninephrectomized rats. Hence, the aim of this investigation was to evaluate the ameliorative effect of l-arginine against EG-induced urolithiasis in uninephrectomized hypertensive rats by assessing various hemodynamic, biochemical, molecular, and histological changes.

Materials and methods

Experimental animals

Adult male Wistar rats (180–200 g) were purchased from the National Institute of Biosciences, Pune (India). They were maintained at 24 ± 1 °C, with relative humidity of 45–55% and 12:12 h dark/Light cycle. The animals had free access to standard pellet chow (Pranav Agro-industries Ltd., Sangli, India) and water throughout the experimental protocol. All experiments were carried out between 09:00 and 17:00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Poona College of Pharmacy, Pune. All the experiments were performed in accordance with the guidelines of Committee for Control and Supervision of Experimentation on Animals (CPCSEA).

Drugs, chemicals, and kits

l-Arginine was purchased from Sigma Chemical Co., (St Louis, MO). The standard drug telmisartan was provided by Lupin Chemicals Limited, Pune as a gift sample. 1,1′,3,3′-Tetraethoxypropane, crystalline beef liver catalase, reduced glutathione (GSH), 5,5′-dithiobis (2-nitrobenzoic acid), EG were purchased from S.D. Fine Chemicals, Mumbai, India. Total RNA Extraction kit and One-step RT-PCR kit was purchased from MP Biomedicals India Private Limited, India.

Ethylene glycol induced urolithiasis

Surgical preparations

For uninephrectomy, rats were anesthetized with ketamine (80 mg/kg body wt, i.p.). A small incision was made over the left flank area, and the left kidney was removed after careful ligation of the renal vessels and ureter. After surgery, all uninephrectomized rats were treated with soframycin twice a day for prophylaxis of wound infection for 4 days.

Ethylene glycol induced urolithiasis in uninephrectomized rats

Rats were divided into following groups containing six rats in each.

• Group I: Normal group (N): Non-nephrectomized rats received distilled water (10 mg/kg, p.o.) for 28 days.

• Group II: Uninephrectomy control group (UC): Uninephrectomized rats received EG (0.75%) and distilled water (10 mg/kg, p.o.) for 28 days.

• Group III: Telmisartan (10 mg/kg) group T (10): Uninephrectomized rats received EG (0.75 %) and Telmisartan (10 mg/kg, p.o.) for 28 days.

• Group IV: Cystone (500 mg/kg) group C (500): Uninephrectomized rats received EG (0.75%) and Cystone (500 mg/kg, p.o.) for 28 days.

• Group V: l-arginine (250 mg/kg) group LA (250): Uninephrectomized rats received EG (0.75%) and l-arginine (250 mg/kg, p.o.) for 28 days.

• Group VI: l-arginine (500 mg/kg) group LA (500): Uninephrectomized rats received EG (0.75%) and l-arginine (500 mg/kg, p.o.) for 28 days.

• Group VII: l-arginine (1000 mg/kg) group LA (1000): Uninephrectomized rats received EG (0.75%) and l-arginine (1000 mg/kg, p.o.) for 28 days.

Ethylene glycol-induced urolithiasis in uninephrectomized rats was induced in each group (except normal) by 0.75% of EG that was provided in their drinking water. The doses of l-arginine (250, 500, and 1000 mg/kg) were selected based upon the previous study.38 Food intake, water intake, and urine output were recorded after placing individual animals in metabolic cages (Techniplast, Italy).

Invasive measurement of hemodynamic changes

Hemodynamic changes such as heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MABP), left ventricular end-diastolic pressure (LVEDP), and ventricular contractility assessment (dp/dt) were measured using a polyethylene cannula (PE 50) filled with heparinized saline (100 IU/mL) inserted into the right carotid artery. The cannula was connected to a transducer, and the signal was amplified by bioamplifier. Left ventricular systolic pressure was measured by means of a Millar micro-tip transducer catheter (Model SRP-320, Millar instrument, INC 320-7051, Houston, TX) inserted into the left ventricle via the right carotid artery and connected to a bioamplifier. The left ventricular functions like dP/dt_max, dP/dt_min and left ventricular end-diastolic pressure signals were obtained from primary signals (left ventricular systolic pressure and blood pressure) by means of an acquisition data system (AD Instruments Pvt. Ltd. with software LabChart 7.3; AD Instrument Pvt. Ltd, Gurgaon, India).39

Renal function

Immediately after hemodynamic measurements blood was collected from the animals by ROP (retro-orbital puncture) and centrifuged at 7500 rpm for 15 min at 4 °C, the serum was transferred using a micropipette in Eppendorf tubes and stored at 4 °C till analyzed. Serum samples were assayed for BUN, uric acid, creatinine, sodium, LDH, and calcium which were measured using a spectrophotometer (UV–visible spectrophotometer, Jasco V-530, Tokyo, Japan) using commercially available reagent kits according to procedure provided by manufacturer (Accurex Biomedical Pvt. Ltd., Mumbai, India). Whereas urine samples were assayed for BUN, uric acid, creatinine, sodium, calcium, citrate, and albumin using commercially available reagent kits according to procedure provided by manufacturer (Accurex Biomedical Pvt. Ltd., Mumbai, India).

Endogenous antioxidant enzymes in kidney and heart

Animals were sacrificed; kidney and heart were isolated and weighed. Kidney and heart samples from two animals were arbitrarily selected for histopathology. The kidney and heart samples from remaining animals were cut into small pieces, placed in chilled 0.25 M sucrose solution, and blotted on a filter paper. The tissues were then homogenized in 10% chilled tris hydrochloride buffer (10 mM, pH 7.4) by tissue homogenizer (Remi Motors, Mumbai, India) and centrifuged at 7500 rpm for 15 min at 0 °C using Eppendorf 5810-R high-speed cooling centrifuge. The clear supernatant was employed to estimate superoxide dismutase (SOD), reduced glutathione (GSH), lipid peroxidation (MDA content), and nitric oxide (NO) as described previously.18^,40–42

Determination of KIM-1, NGAL, eNOs, and iNOs by Reverse Transcriptase PCR in kidney

RNA isolation

The levels of mRNA were analyzed in kidney tissue using a reverse transcription (RT)-PCR as described previously.43^,44

cDNA preparation

Single-stranded cDNA was synthesized from 5 µg of total cellular RNA using reverse transcriptase kit (MP Biomedicals India Private Limited, India) as described previously.43 The primer sequence for Kidney Injury Molecule-1 (KIM-1), Neutrophil gelatinase-associated lipocalin (NGAL), endothelial nitric oxide synthase (eNOs), inducible nitric oxide synthase (iNOs), and β-actin are presented in Supplementary Material. Amplification of β-actin served as a control for sample loading and integrity. PCR products were detected by electrophoresis on a 1.5% agarose gel containing ethidium bromide. Size of amplicons was confirmed using a 100-bp ladder as a standard size marker. The amplicons were visualized, and images were captured using a gel documentation system (Alpha Innotech Inc., San Leandro, CA). Expression of all the genes was assessed by generating densitometry data for band intensities in different sets of experiments and was generated by analyzing the gel images on the Image J software, Version 1.33 (Wayne Rasband, NIH, Bethesda, MD) semi-quantitatively. The band intensities were compared with constitutively expressed β-actin. The intensity of mRNAs was standardized against that of the β-actin mRNA from each sample, and the results were expressed as PCR-product/β-actin mRNA ratio.

Histological evaluation of kidney and heart

At the end of experiment, kidney and heart were isolated and fixed for histopathological evaluation with 4% buffered paraformaldehyde solution and embedded in paraffin. Three to four micrometer thick paraffin sections were dewaxed and brought to the water through graded ethanol. Sections were stained with hematoxylin–eosin stain (H & E), then dehydrated through graded ethanol, cleared in xylene, and mounted with Distyrene Plasticizer Xylene. H & E stained sections were graded to verify morphological assessment of tissue damage. Photomicrographs were captured at 40 × magnification.

Statistical analysis

Data are expressed as mean ± standard error mean (SEM). Data analyses were performed using Graph Pad Prism 5.0 software (Graph Pad, San Diego, CA). Data of biochemical parameters were analyzed by one-way analysis of variance (ANOVA), and Dunnett’s tests were applied for post hoc analysis. Data of electrocardiographic parameters were analyzed by two-way ANOVA, and Bonferroni’s tests were applied for post hoc analysis. A value of p < 0.05 was considered to be statistically significant.

Results

Effect of l-arginine on body weight, relative organ weight, water intake, and urine output in EG-induced urolithiasis in uninephrectomized rats

There was significant (p < 0.001) decrease in the body weight, urine output, and water intake of UC rats as compared to the normal group. However, the administration of telmisartan (10 mg/kg) and Cystone (500 mg/kg) significantly (p < 0.001) increased the body weight, urine output, and water intake as compared to the UC group. l-Arginine (250, 500, and 1000 mg/kg) treatment significantly (p < 0.05, 0.01, and 0.001) and dose-dependently increased the body weight, urine output, and water intake as compared to the UC group (Table 1).

Table 1. Effect of l-arginine on body weight, urine output, water intake, urine density, relative organ weight, and urine pH in EG (ethylene glycol) induced urolithiasis in uninephrectomized rats.

Treatment	Body weight (gm)	Urine output (ml)	Water intake (ml)	Urine density (gm/ml)	Dry weight of urine (mg)	Urine pH	Heart/body weight	Kidney/body weight
N	262.65 ± 7.12	11.67 ± 0.33	53.00 ± 2.51	0.82 ± 0.031	7.83 ± 0.95	7.50 ± 0.29	0.31 ± 0.026	0.47 ± 0.012
UC	190.52 ± 6.52###	8.67 ± 0.33###	25.00 ± 1.52###	1.09 ± 0.014###	22.57 ± 1.24###	5.67 ± 0.34###	0.37 ± 0.021###	0.67 ± 0.024###
T (10)	250.66 ± 8.98***	11.33 ± 0.33***	57.33 ± 2.33***	0.89 ± 0.026***	8.53 ± 0.72***	8.34 ± 0.33***	0.33 ± 0.014***	0.58 ± 0.010*
C (500)	253.00 ± 11.11***	15.33 ± 1.76***	54.33 ± 2.72***	0.83 ± 0.014***	7.8 ± 1.04***	8.67 ± 0.33***	0.36 ± 0.017	0.47 ± 0.022***
LA (250)	230.68 ± 4.20*	9.33 ± 1.02*	29.52 ± 1.08*	0.87 ± 0.005	18.74 ± 0.21	5.78 ± 0.53	0.37 ± 0.019	0.68 ± 0.030*
LA (500)	248.78 ± 9.54**	10.11 ± 0.43**	38.65 ± 6.17**	0.92 ± 0.042**	12.20 ± 0.15**	6.03 ± 0.31**	0.37 ± 0.023	0.57 ± 0.024**
LA (1000)	259.10 ± 7.54***	10.87 ± 0.51***	48.20 ± 2.15***	0.95 ± 0.043***	8.12 ± 0.64***	7.21 ± 0.41***	0.32 ± 0.01***	0.53 ± 0.014***

Notes: Results are represented as mean ± SEM (n = 6) Data are analyzed by One-way ANOVA followed by post hoc Dunnett’s tests. ###p < 0.001 as compared with normal group. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the urolithiasis control (UC) group.

The heart weight to body weight ratio and kidney weight to body weight ratio was increased significantly (p < 0.001) in the UC group as compared to the normal group. Administration of telmisartan (10 mg/kg) significantly (p < 0.001) reduced the heart weight to body weight ratio and significantly (p < 0.05) reduced kidney weight to body weight ratio as compared to UC control rats. On the other hand, cystone (500 mg/kg) administration significantly reduced (p < 0.001) the kidney weight to body weight ratio, however failed to produce any significant reduction in heart weight to body weight ratio as compared to UC control rats. l-Arginine (250, 500, and 1000 mg/kg) showed significant (p < 0.05, 0.01, and 0.001) and dose-dependent decrease in kidney weight to body weight ratio as compared to the UC group. When compared with the UC group, the heart weight to body weight ratio was significantly reduced (p < 0.001) by l-arginine (1000 mg/kg) treatment (Table 1).

Effect of l-arginine on urine density, urine dry weight, and urine pH in EG-induced urolithiasis in uninephrectomized rats

There was significant (p < 0.001) increase in the urine density and dry weight whereas significant (p < 0.001) decrease in the urine pH in the UC group as compared to the normal group. This increase in the urine density, dry weight, and decrease in urine pH was significantly restored (p < 0.001) by telmisartan (10 mg/kg) and cystone (500 mg/kg) treatment as compared to UC control rats. Administration of l-arginine (500 and 1000 mg/kg) significantly and dose dependently (p < 0.01 and 0.001) restored the EG-induced altered urine density, dry weight, and urine pH as compared to UC control rats (Table 1).

Effect of l-arginine on ECG parameters in EG-induced urolithiasis in uninephrectomized rats

Treatment with EG in the uninephrectomized rats resulted in the significant increase (p < 0.001) in heart rate, QRS interval, QTc interval, and ST height in UC group as compared to normal. However, administration of telmisartan (10 mg/kg) and cystone (500 mg/kg) significantly (p < 0.001) reduced the heart rate, QRS interval, and ST height as compared to the UC group. Treatment with l-arginine (250, 500, and 1000 mg/kg) significantly (p < 0.05, 0.01, and 0.001) and dose-dependently reduced the heart rate, QRS interval, and ST height as compared to UC rats (Table 2).

Table 2. Effects of l-arginine on electrocardiographic abnormalities in EG-induced urolithiasis in uninephrectomized rats.

Treatment	QRS interval (ms)	Heart rate (BPM)	QTc interval	ST height
N	13.79 ± 0.26	363.96 ± 15.23	108.84 ± 7.60	1.50 ± 0.33
UC	18.86 ± 0.78###	413.25 ± 8.94###	132.05 ± 4.04###	7.24 ± 0.56###
T (10)	13.47 ± 0.36***	374.05 ± 6.52***	120.00 ± 2.41	2.52 ± 0.56***
C (500)	14.79 ± 0.22**	383.93 ± 18.79***	138.28 ± 5.14	3.99 ± 0.46***
LA (250)	17.08 ± 0.41*	412.20 ± 11.20*	130.18 ± 8.12	5.42 ± 0.63*
LA (500)	15.48 ± 0.38**	381.22 ± 12.00**	129.22 ± 1.84	4.84 ± 0.11**
LA (1000)	14.22 ± 0.22***	371.55 ± 12.41***	129.42 ± 3.15	1.89 ± 0.48***

Notes: Results are represented as mean ± SEM (n = 6) Data are analyzed by One-way ANOVA followed by post hoc Dunnett’s tests. ###p < 0.001 as compared with normal group. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the urolithiasis control (UC) group.

Effect of l-arginine on hemodynamic parameters in EG-induced urolithiasis in uninephrectomized rats

The SBP, DBP, MABP, LVEDP, and max dp/dt were increased significantly (p < 0.001), and LV Contractility Index and min dp/dt were decreased significantly (p < 0.001) in UC group as compared to the normal group. Administration of telmisartan (10 mg/kg) significantly (p < 0.001) restored the alteration in SBP, DBP, MABP, LVEDP, LV Contractility Index, max dp/dt, and min dp/dt as compared to the UC group. When compared with UC control rats, cystone (500 mg/kg) treatment significantly (p < 0.01) decreased SBP, DBP, MABP, and LVEDP, however failed to produce any restoration in altered LVEDP, LV Contractility Index, max dp/dt, and min dp/dt. Administration of l-arginine (500 and 1000 mg/kg) significantly (p < 0.01 and 0.001) and dose dependently restored the EC-induced altered SBP, DBP, MABP, LVEDP, LV Contractility Index, max dp/dt, and min dp/dt as compared to UC rats (Table 3).

Table 3. Effect of l-arginine on hemodynamic parameters in EG-induced urolithiasis in uninephrectomized rats.

Treatment	Systolic BP (mmHg)	Diastolic BP (mmHg)	MABP (mmHg)	LVEDP (mmHg)	LV Contractility Index (s^−1)	Max dp/dt (mmHg/s)	Min dp/dt (mmHg/s)
N	107.3 ± 7.31	74.01 ± 1.59	78.55 ± 2.38	2.48 ± 0.55	83.75 ± 3.89	3148 ± 180.7	−2332 ± 79.75
UC	127.3 ± 2.45###	85.95 ± 2.64###	94.35 ± 1.15###	7.45 ± 0.35###	67.35 ± 3.14###	4276 ± 161.3###	−3594 ± 86.03###
T (10)	113.6 ± 2.79***	76.98 ± 1.98***	69.15 ± 3.27***	3.20 ± 0.27***	79.89 ± 2.42***	3441 ± 247.6***	−2576 ± 52.35***
C (500)	118.4 ± 5.07**	78.03 ± 1.38**	87.64 ± 2.84*	5.48 ± 0.15**	69.60 ± 1.47	4197 ± 199.6	−3479 ± 24.59
LA (250)	128.0 ± 3.33	84.78 ± 3.01	93.55 ± 2.21	6.08 ± 0.20	68.44 ± 2.14	4251 ± 122.87	−3487 ± 144.2
LA (500)	117.5 ± 2.84**	80.10 ± 0.89**	87.92 ± 2.55**	5.84 ± 0.20**	75.22 ± 1.89**	3658 ± 178.55**	−2879 ± 142.9**
LA (1000)	111.40 ± 1.87***	74.44 ± 1.54***	81.22 ± 1.16***	3.41 ± 0.44***	79.11 ± 2.15***	2875 ± 145.6***	−2487 ± 98.78***

Notes: Results are represented as mean ± SEM (n = 6) Data are analyzed by One-way ANOVA followed by post hoc Dunnett’s tests. ###p < 0.001 as compared with normal group. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the urolithiasis control (UC) group.

Effect of l-arginine on serum biochemical parameters in EG-induced urolithiasis in uninephrectomized rats

The serum BUN, uric acid, creatinine, sodium, LDH, and calcium levels were increased significantly (p < 0.001) in the UC group as compared to the normal group. Administration of telmisartan (10 mg/kg) significantly (p < 0.001) decreased BUN, uric acid, creatinine, sodium, LDH, and calcium level as compared to the UC group. Cystone (500 mg/kg) treatment decreased BUN, uric acid, creatinine, sodium, and calcium level significantly (p < 0.001) as compared to the UC group. When compared with the UC group, administration of l-arginine (500 and 1000 mg/kg) significantly and dose dependently (p < 0.01 and 0.001) decreased the serum BUN, uric acid, creatinine, sodium, LDH, and calcium levels (Table 4).

Table 4. Effect of l-arginine on serum and urine biochemical parameters in EG-induced urolithiasis in uninephrectomized rats.

Treatment	BUN (mg/dL)	Uric Acid (mg/dL)	Creatinine (mg/dL)	Sodium (mg/g)	LDH (mg/dL)	Calcium (mg/g)	Citrate (mg/g)	Albumin (gm/dL)
N
Serum	3.08 ± 0.43	2.15 ± 0.07	0.184 ± 0.039	141.8 ± 2.54	1331 ± 82.95	19.92 ± 0.19	–	–
Urine	12.17 ± 1.63	1.87 ± 0.26	8.48 ± 1.39	443.2 ± 7.17	–	18.54 ± 1.09	3.97 ± 0.09	2.27 ± 0.30
UC
Serum	17.80 ± 2.43###	4.14 ± 0.20###	0.614 ± 0.043###	187.1 ± 1.89###	1985 ± 126.6###	27.59 ± 2.30###	–	–
Urine	66.5 ± 4.13###	4.22 ± 0.37###	43.27 ± 3.02###	332.4 ± 47.63###	–	34.37 ± 0.77###	2.03 ± 0.20###	0.53 ± 0.10###
T (10)
Serum	8.60 ± 1.74**	2.73 ± 0.20***	0.329 ± 0.034***	171.4 ± 3.99*	1608 ± 220.0*	22.97 ± 0.41***	–	–
Urine	20.42 ± 1.85***	2.09 ± 0.35***	19.44 ± 5.43***	348.3 ± 33.01	–	24.18 ± 0.30***	3.80 ± 0.21***	1.74 ± 0.07***
C (500)
Serum	8.43 ± 0.90***	2.71 ± 0.21***	0.399 ± 0.077**	155.2 ± 5.23***	1734 ± 79.55	19.31 ± 1.36***	–	–
Urine	16.06 ± 8.10***	1.68 ± 0.25***	20.18 ± 2.07***	381.9 ± 2.34***	–	19.70 ± 0.80***	3.64 ± 0.28***	1.20 ± 0.6**
LA (250)
Serum	16.77 ± 1.26	3.23 ± 0.53	0.486 ± 0.018	180.00 ± 3.66	2083 ± 226.8	29.52 ± 0.98	–	–
Urine	60.35 ± 2.62	3.98 ± 0.26	41.29 ± 4.21	358 ± 14.69	–	32.61 ± 11	2.11 ± 0.05	0.62 ± 0.01
LA (500)
Serum	9.14 ± 2.01**	2.93 ± 0.42**	0.380 ± 0.061**	165.2 ± 1.04**	1520 ± 140.5**	24.99 ± 1.16**	–	–
Urine	47.92 ± 1.15**	2.50 ± 0.24***	22.29 ± 1.48**	414.9 ± 19.93**	–	27.94 ± 0.36**	3.34 ± 0.02***	1.35 ± 0.09**
LA (1000)
Serum	2.98 ± 0.56***	2.73 ± 0.21***	0.266 ± 0.104***	149.9 ± 1.58***	1216 ± 66.03***	15.92 ± 0.49***	–	–
Urine	2.17 ± 0.17***	1.37 ± 0.07***	20.61 ± 3.83***	449.3 ± 9.54***	–	20.92 ± 0.09***	3.76 ± 0.03***	1.97 ± 0.14***

Notes: Results are represented as mean ± SEM (n = 6) Data are analyzed by One-way ANOVA followed by post hoc Dunnett’s tests. ###p < 0.001 as compared with normal group. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the urolithiasis control (UC) group.

Effect of l-arginine on urine biochemical parameters in EG-induced urolithiasis in uninephrectomized rats

BUN, uric acid, calcium, and creatinine level in urine of UC group was significantly (p < 0.001) increased whereas sodium, citrate, and albumin level in urine of UC group was significantly (p < 0.001) decreased as compared to the normal group. However, administration of telmisartan (10 mg/kg) significantly (p < 0.001) restored the BUN, uric acid, creatinine, calcium, citrate, and albumin as compared to the UC group. The increased level of BUN, uric acid, calcium, creatinine as well as decreased level of sodium, citrate, and albumin in urine after EG treatment was significantly (p < 0.001) restored by cystone (500 mg/kg) treatment. Administration of l-arginine (500 and 1000 mg/kg) significantly and dose dependently (p < 0.01 and 0.001) decreased the urinary BUN, uric acid, calcium, creatinine level whereas significantly (p < 0.05 and 0.01) increased the urinary sodium, citrate, and albumin level as compared to the UC group (Table 4).

Effect of l-arginine on renal oxido-nitrosative stress in EG-induced urolithiasis in uninephrectomized rats

There was significant (p < 0.001) decrease in renal SOD and GSH levels in UC as compared to the normal group. Administration of telmisartan (10 mg/kg) significantly (p < 0.001) increased the renal SOD and GSH whereas significantly (p < 0.001) decreased MDA and NO levels as compared to the UC group. While the administration of cystone (500 mg/kg) significantly (p < 0.05) increased renal SOD level, it significantly (p < 0.01 and 0.001) decreased renal MDA and NO levels as compared to UC control rats. Administration of l-arginine (500 and 1000 mg/kg) significantly (p < 0.01 and 0.001) and dose-dependently increased the activity of SOD and GSH, whereas the levels of MDA and NO were decreased significantly (p < 0.01 and 0.001) and dose-dependently as compared to the UC group (Figure 1).

Figure 1. Effect of l-arginine on cardiac and renal SOD, GSH, MDA, and NO in EG-induced urolithiasis in uninephrectomized rats. Results are represented as mean ± SEM (n = 6) Data are analyzed by One-way ANOVA followed by post hoc Dunnett’s tests. ###p < 0.001 as compared with normal group. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with urolithiasis control group.

Effect of l-arginine on myocardial oxido-nitrosative stress in EG-induced urolithiasis in uninephrectomized rats

The SOD and GSH levels were reduced significantly (p < 0.001), whereas MDA and NO levels were significantly (p < 0.001) increased in the heart of UC control rats as compared to the normal group. Treatment with telmisartan (10 mg/kg) significantly (p < 0.001) increased SOD and GSH levels in heart and significantly (p < 0.001) decreased MDA and NO levels in heart as compared to the UC group. Cystone (500 mg/kg) treatment significantly increased (p < 0.01) myocardial SOD level whereas, there was significant (p < 0.01) decrease in myocardial NO level as compared to the UC control rats. When compared with the UC control group, l-arginine (500 and 1000 mg/kg) treatment significantly (p < 0.01 and 0.001) and dose-dependently increased SOD and GSH levels whereas decreased MDA and NO levels (Figure 1).

Effect of l-arginine on renal KIM-1 and NGAL mRNA expression in EG-induced urolithiasis in uninephrectomized rats

There was significant (p < 0.001) up-regulation in the renal KIM-1 and NGAL mRNA expression of UC control rats as compared to normal rats. This up-regulation in the renal KIM-1 mRNA expression was significantly (p < 0.01) inhibited by the telmisartan (10 mg/kg) treatment as compared to UC control rats. However, telmisartan (10 mg/kg) failed to produce any significant down-regulation in the NGAL mRNA expression as compared to UC control rats. When compared with UC control rats, cystone (500 mg/kg) treatment did not show any down-regulation in KIM-1 and NGAL mRNA expression. Administration of l-arginine (500 and 1000 mg/kg) significantly (p < 0.05 and 0.01) and dose-dependently down-regulated EG-induced up-regulation in KIM-1 and NGAL mRNA expression as compared to UC control rats (Figure 2A–C).

Figure 2. Effect of l-arginine on renal KIM-1, NGAL, eNOs, and iNOs mRNA expression in EG-induced urolithiasis in uninephrectomized rats (A), quantitative representation of mRNA expression of KIM-1 (B), NGAL (C), eNOs (D), and iNOs (E). Results are represented as mean ± SEM (n = 4) Data are analyzed by One-way ANOVA followed by post hoc Dunnett’s tests. ###p < 0.001 as compared with normal group. *p < 0.05, **p < 0.01 as compared with urolithiasis control group.

Effect of l-arginine on renal eNOs and iNOs mRNA expression in EG-induced urolithiasis in uninephrectomized rats

The renal eNOs and iNOs mRNA expression was significant (p < 0.001) down-regulated and up-regulated in the UC control rats as compared to normal rats respectively. Treatment with telmisartan (10 mg/kg) and cystone (500 mg/kg) failed to produce any significant restoration in the altered mRNA expression of eNOs and iNOs as compared to UC control rats. l-Arginine (500 and 1000 mg/kg) treatment significantly (p < 0.01) up-regulated eNOs mRNA expression and significantly (p < 0.01) down-regulated iNOs mRNA expression as compared to UC control rats (Figure 2A, D, and E).

Effect of l-arginine on alteration in kidney histology in EG-induced urolithiasis in uninephrectomized rats

Kidney tissue from normal rats showed normal architecture without any changes like congestion, necrosis, and inflammatory infiltration. It revealed no calcifications in the kidneys. It showed the presence of intact glomerulus basement membrane and tubules (Figure 3A). Administration of EG showed the deposition of calcium oxalate crystal in the renal cortex and medulla of UC control rats. It also showed the renal damage with intrinsic lesions within the glomeruli and epithelium. Intracellular edema was also detected with the presence of intraluminal cell debris and inflammatory infiltration (Figure 3B). Telmisartan (10 mg/kg) and cystone (500 mg/kg) treated rats showed the reduction in the renal damage reflected by decreased calcium oxalate crystal deposition along with reduced inflammatory infiltration (Figure 3C and D). Structural restoration of the glomerulus basement membrane and tubules were observed in the rats treated with cystone (500 mg/kg; Figure 3D). Renal tissue from l-arginine (500 mg/kg) treated rats showed the presence of few calcium oxalate crystal and it showed moderate intrinsic lesions within the glomeruli and epithelium (Figure 3E). Renal tissue from (1000 mg/kg) treated rats showed mild calcifications with a moderate number of inflammatory cells with reduced necrosis and edema. Thickens of glomerulus basement membrane was intact (Figure 3F).

Figure 3. Effect of l-arginine on alteration in kidney histology in EG-induced urolithiasis in uninephrectomized rats. Photomicrograph of sections of kidney of normal (A), urolithiasis control rats (B), Telmisartan (10 mg/kg) treated rats (C), Cystone (500 mg/kg) treated rats (D), l-arginine (500 mg/kg) treated rats (E), and l-arginine (1000 mg/kg)-treated rats (F). H & E staining at 100 × .

Effect of l-arginine on alteration in heart histology in EG-induced urolithiasis in uninephrectomized rats

Histological analysis of heart tissue from UC rats showed marked myocardial damage revealed by the features like inflammation, necrosis, nuclear pyknosis, cytoplasmic vacuolization, and cytoplasmic eosinophilia (Figure 4A). However, the normal group did not show any tissue damage in myocardial fibers (Figure 4B). Histological sections of hearts from telmisartan (10 mg/kg) treated rats showed markedly improved histological changes indicated by reduced myocardial damage, inflammation, and nuclear pyknosis (Figure 4C). However, myocardial tissue from cystone (500 mg/kg) as well as l-arginine (500 mg/kg) treated rats showed altered myocardial vasculature, reflected by the presence of vascular congestion, myocardial inflammation, and nuclear pyknosis (Figure 4D and E). Treatment with l-arginine (1000 mg/kg) remarkably improved the histological changes in the heart tissue reflected in reduced myocardial damage, diminished extent of inflammation and nuclear pyknosis (Figure 4F).

Figure 4. Effect of l-arginine on alteration in heart histology in EG-induced urolithiasis in uninephrectomized rats. Photomicrograph of sections of heart of normal (A), urolithiasis control rats (B), Telmisartan (10 mg/kg) treated rats (C), Cystone (500 mg/kg) treated rats (D), l-arginine (500 mg/kg treated rats (E), and l-arginine (1000 mg/kg) treated rats (F). H & E staining at 100×.

Discussion

Urinary stone or calculi is the third prevalent disease of the urinary system that affects approximately 12% of the population worldwide and need serious medical attention throughout lifetime of patient.45 It has been reported that approximately 80% of urinary calculi are comprised of calcium oxalate and calcium phosphate which causes urinary obstruction, hydronephrosis, infection, and hemorrhage in the urinary tract system.46 Nowadays, extracorporeal shock wave lithotripsy (ESWL), percutaneous nephrolithotomy, and rigid and flexible urethroscopy are the newly developed methods that have been employed for the effective and safe removal of urinary calculi. However, 50–70% of the stones will recur after treatment if urolithiasis patients neglect the preventive measures.47 Moreover, this surgical treatment may cause serious side effect including reduction in renal plasma flow and glomerular filtration rate along with morphological changes in the treated kidney. In some patients treatment may exhibit severe renal damage in the ipsilateral kidney and therefore need to require surgical removal later. If urolithiasis patients, who have undergone uninephrectomy or whose unilateral kidney has lost its function, continue to be exposed to dietary and/or other lithogenic risk factors, the remnant kidney may carry a higher risk of stone formation and is therefore more prone to further damage. Moreover, urolithiasis has been reported to have a higher prevalence of hypertension and proteinuria.48 Hence, this study was undertaken to evaluate anti-urolithiasis and antihypertensive the effect of l-arginine in uninephrectomized-hypertensive laboratory rats.

In order to maintain homeostasis of body fluid composition, there was induction of compensatory hyperfunction of the remaining kidney in uninephrectomized animals. Hyperfiltration and hyperexcretion occurred to minimize the reduced filtration rate and solute as well as water excretion in uninephrectomy condition.49^,50 However, this compensatory hyperfunction by remnant nephron unit may lead to its pathological aberrations followed by glomerular dysfunction. Moreover, elevated urinary excretion of lithogenic substances in the remaining nephron units leads to earlier hypercalciuria followed by renal hypertension as postulated by the hypertension-crystallization hypothesis.13^,14 In this investigation, administration of lithogenic substances such as EG in the uninephrectomized rats showed impaired renal function with hypercalciuria and renal hypertension.

EG is rapidly absorbed and metabolized in the liver by alcohol dehydrogenase/aldehyde dehydrogenase to glycolic acid.51 EG has in itself a low toxicity, but in vivo it is broken down to four organic acids viz: glycoaldehyde, glycolic acid, glyoxylic acid, and oxalic acid. Glycolic acid is oxidized to glyoxylic acid, which in turn further oxidized to oxalic acid by glycolate oxidase or lactate dehydrogenase.52 These metabolites are toxic to cells that cause depression in central nervous system along with cardiopulmonary, as well as renal failure.53 High intake of EG ( >2500 mg/kg body weight), particularly when given as an oral bolus, cause the saturation dependent accumulation of glycolic acid in the plasma. Therefore, glycolate oxidase (GO) is one of the rate-limiting reaction in the metabolism of EG. Hence, EG has been chosen as an appropriate material for the induction of urolithiasis in uninephrectomized rats as previously developed models.54^,55

It has been documented that uninephrectomy alone unable to induced renal dysfunction as well as urolithiasis or crystal deposition in the remaining kidney.55 However, administration of lithogenic substance such as EG in uninephrectomized condition caused significant renal damage and calcium oxalate crystal deposition in the remnant kidney. Hence, hypercalciuria is considered as the important hallmark of stone formation.50^,56 Feeding with EG significantly elevated the wet and dry kidney weight in uninephrectomized rats that are directly associated with urinary calculi formation and calcium oxalate crystal deposition. The severity crystal deposition and stone formation is directly associated with renal calcium content.57^,58 In this investigation, EG administration in uninephrectomized condition caused significant hypercalciuria reflected by the increased wet and dry kidney weight which is in accordance with the findings of previous investigators.57^,58 However, administration of l-arginine significantly decreased renal wet and dry weight, indicating its inhibitory potential against calcium oxalate crystal deposition.

Administration of EG produced type I distal renal tubular acidosis leads to nephrolithiasis and nephrocalcinosis conditions.59^,60 These conditions are associated with the elevated urinary pH, urine density, and calcium excretion as well as decreased excretion of sodium, citrate, and albumin in urine reflected the EG-induced renal damage in uninephrectomized rats.55 Citrate has been reported to have negatively charged inhibitors of urinary calcium and decreased level of urinary citrate indicated the hypercalciuria. Moreover, uric acid is documented to have an inhibitory effect against the calcium oxalate solubility, and the elevated level of urinary uric acid reflected the increased calcium oxalate crystal deposition.61 It has been documented that citrate form complex with calcium in the urine thereby reducing the concentration of calcium oxalate and preventing its agglomeration.62 However, l-arginine treatment significantly restored the altered levels of ions, uric acid, and citrate thus prevented the risk of stone formation.

It has been reported that EG caused deterioration of renal function due to stone formation resulted in obstruction of urine outflow thus decreases glomerular filtration rate. This in turn leads to accumulation of nitrogenous waste products BUN, uric acid, and creatinine in serum. The elevated level of nitrogenous waste product in serum has been implicated as the hallmark of renal damage clinically.63 In this study, EG administration produced significant nephrotoxicities indicated by significant elevation in these nitrogenous waste products in serum whereas l-arginine treatment significantly attenuated these elevated nitrogenous waste products level, thus reflecting its nephro-protective potential. Result of this study is in line with the findings of previous investigators where l-arginine significantly restored the altered level of BUN and creatinine in renal failure rats.32^,33^,64

Lactate dehydrogenase (LDH), a bifunctional enzyme of the glycolytic pathway is considered as a most sensitive indicator of acute kidney injury. Rise in LDH level is associated with loss of proximal tubular cell brush border due to renal epithelial cellular necrosis by intoxicant. It is also played a vital role in oxidation of glycolic acid (an EG metabolite) to oxalic acid that induced toxicity in renal plasma membrane.53^,65 The elevated level of LDH suggests that EG induces nephrotoxicity and its restoration by l-arginine indicates its nephro-protective potential.

It has been reported that hypertension is associated with urolithiasis.66 The reports of previous experimental studies have suggested that hypertension occurred in the altered acid–base state.67^,68 In this investigation, alteration in the acid–base state occurred after EG administration in uninephrectomized rats along with alteration in hemodynamic and ECG parameters. Treatment with l-arginine significantly restored the altered levels of hemodynamic and ECG parameters. The result of this study corroborates the findings of previous investigators.69^,70

Crystal-induced renal epithelial injury to a significant degree mediated by oxidative stress was reflected by increased lipid peroxidation and cellular damage in renal tissue.30 In renal epithelial cells, toxic responses generated due to a higher concentration of oxalate leads to altered membrane surface properties, disruption of mitochondrial function, and the formation of ROS. The elevated concentration of MDA in UC group is indicative of oxidative tissue damage, as lipid peroxides and peroxidation products accumulate and are usually excreted in the urine.71–73 Elevated oxidative stress caused depletion of enzymes that played an important role in the electron transport chain. Moreover, oxidative stress can elicit inflammation, enhance vasoconstrictors accrual, and endorse tubular cell apoptosis and necrosis.74–77 This higher level of ROS diminishes the copper–zinc–SOD enzyme motion alongside GSH performance in cells, which causes consequential hammering of cell integrity and necrosis.78^,79 Concrete with this notion, we found that UC control rats drastically lessened SOD, GSH levels, and elevated MDA content. Activity of SOD and GSH enzymes increased in l-arginine treated groups which confirmed that l-arginine has potent anti-oxidant potential which prevents the cardiac and renal oxidative damage. It has been documented that l-arginine possesses potent free radical scavenging property.33^,38 The restoration in the decreased level of SOD and GSH further confirms the anti-oxidative potential of l-arginine in cardiac and renal tissue against EG-induced oxidative damage which is in line with the finding of previous workers.33^,38

Nitric oxide (NO) has been implicated in an array of disorders including diabetes, cardiomyopathy, nephropathy, neuropathy, arthritis, etc.80–84 Nitrite is hallmark of endogenous nitric oxide production, and their levels serve as the gold standard reflecting the increased oxidative stress resulting in cellular dysfunction.85–87 It has been documented that administration of the substance with NO scavenging or NOS (nitric oxide synthase) inhibitory potential prevented EG-induced toxicity via inhibition of renal microvascular constriction to improve hemodynamic abnormality.88 In the present investigation, UC control rats showed elevated NO levels after EG administration whereas l-arginine treatment decreased NO level in both renal and cardiac tissues resulting in the inhibition of renal microvascular constriction which might help in improvement of electrocardiographic and hemodynamic abnormalities.

Clinically, acute kidney injury (AKI) is an important issue for patients during critical care in the intensive care unit (ICU).89^,90 In AKI, the mortality rates remained high due to paucity in the sensitive and specific biomarkers of renal cell injury.89^,91 However, recently developed techniques have identified the two sensitive and specific biomarkers for early AKI, namely, kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL).92^,93 It has been reported that the sensitivity for NGAL to predict AKI is 0.815.94 NGAL has also been known as human neutrophil lipocalin (HNL) and widely expressed by renal tubular epithelium. Renal epithelial injury and inflammation leads to rapidly induction in the expression of NGAL in nephron. KIM-1 is a transmembrane tubular protein with an immunoglobulin and mucin domain, rapidly expressed in the proximal tubule after AKI.95 KIM-1 played a vital role in phagocytosis within proximal tubules. Up-regulated expressions of NGAL and KIM-1 in kidney are hallmark of AKI as it is detectable before a rise in serum creatinine or urinary N-acetyl-glucosaminidase concentrations. In this investigation, elevated ROS after EG administration leads to AKI reflected by the up-regulated KIM-1 and NGAL mRNA expression in renal tissue of UC control rats. However, treatment with l-arginine showed significant down-regulation in the renal mRNA expression of KIM-1 and NGAL.

It has been reported that eNOs played a vital role in the management of hypertension.96^,97 Elevated level of eNOs caused release of moderate NO that is beneficial for the vasodilator action. Whereas, high NO levels generated by iNOS resulted in the production of peroxynitrite anion via interacting with superoxide anion. This peroxynitrite induces leukocyte adhesion, infiltration of inflammatory cell, and cellular dysfunction.98 It has been documented that l-arginine is served as a precursor for NO synthesis, and the conversation is catalyzed by nitric oxide synthase (NOs).99 This synthesized NO was released by endothelial NOs (eNOs) from the blood vessels of endothelium resulting in vasodilation via cyclic guanosine monophosphate (cGMP).100^,101 In this investigation, oral administration of l-arginine significantly improved hemodynamic changes via amelioration of EG-induced decreased endothelial nitric oxide synthase (eNOS) activity. The result of this study is in line with the findings of previous workers where l-arginine has been shown to increase eNOS expression in pulmonary endothelial cells.38^,102

Currently, ARBs (Losartan, valsartan, telmisartan, and olmesartan) are used worldwide to treat hypertension and proteinuria. Several studies have confirmed the inhibitory effect of ARBs by suppressing oxidative stress and tubule-interstitial injury through inhibition of osteopontin expression.103 In this study, administration of telmisartan and cystone showed amelioration in the EG-induced urolithiasis in uninephrectomized hypertensive rats via modulation of ROS; however, it failed to produce any significant down-regulation in the KIM-1 and NGAL mRNA expression. In conclusion, l-arginine showed the nephro-protective and cardioprotective property against EG-induced urolithiasis in uninephrectomized hypertensive rats via modulation of KIM-1, NGAL, eNOs, and iNOs mRNA expression.

Supplementary material available online

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.