INTRODUCTION:

Nephropathy is considered as thickening of mesangial expansion and basement membranes with the development of glomerulosclerosis, interstitial fibrosis and tubular atrophy¹ and is related to constant elevated arterial blood pressure, albuminuria, fluid retention and declined glomerular filtration rate (GFR)².The gentamicin-induced nephrotoxicity occurs by selective accumulation of the drug in renal proximal convoluted tubules resulting loss of its brush border reliability³, renal free radical generation, glomerular congestion, and acute tubular necrosis, leading to reduction of glomerular filtration rate and thus producing renal dysfunction⁴.

Upreggulation of transforming growth factor-beta (TGF-β) leading to extracellular matrix expansion, overproduction of extracellular matrix molecules and renal hypertrophy^5,6, elevation of endothelin-1 leading to increased macrophage/ monocytes infiltration in renal cortex and medulla, induction of oxidative stress⁷, genetic vulnerability⁸ and necrosis^9,10 are some of the pathological mechanisms involved in gentamicin-induced nephrotoxicity. However, the choice of treatment for administration of gentamicin nephrotoxicity is inadequate due to partial understanding of the chief pathogenesis of nephropathy.

Cromolyn sodium, a mast cell stabilizer, inhibits the mast cell activation by confining transition of calcium over mast cell membrane, avoiding degranulation and arrival of vasoactive substances and thus are used to prevent eye inflammation and asthma¹¹. A marked role of mast cell stabilizers in streptozotacin [STZ] induced nephropathy¹² and cisplatin-induced kidney injury¹³ has also been reported but their role in combination with fenofibrate has not been studied so far in gentamicin-induced nephropathy. Hence, the current study has been proposed to observe the role of mast cell stabilizer (cromolyn sodium) and PPAR-α agonist (fenofibrate) in preventing the gentamicin-induced nephropathy in rats.

Peroxisome proliferator-activated receptor (PPAR)-α, plays an important role in the regulation of lipid metabolism¹⁴. Treatment with fenofibrate, a PPAR-α agonist, provide renoprotection by reducing albuminuria, blood urea nitrogen, serum creatinine and preventing glomerular lesions in nephrotoxicity¹⁵. Therefore, concurrent activation of mast cell stabilizers and PPAR-α administered together, could be a possible therapeutic choice to regulate the release of mast cell mediators and hyperlipidemia in gentamicin-induced nephropathy in rats.

MATERIAL AND METHODS:

Experimental design:

The experimental protocol was approved from CPCSEA certified Institutional Animal Ethical Committee (Protocol No.–MMCP-IAEC-26) for the purpose of control and supervision of experiments on animals. Healthy albino rats weighing about 170-200g of either sex were used for the study and were kept under standard laboratory conditions (22±3°C) in hygienic dry polycarbonate cages and fed with standard chow and water ad libitum during the experimental period. Serum isolated from collected blood samples was stored at 2-8°C temperature for biochemical parameters estimation. The kidneys of rats were excised for histopathological studies.

Experimental protocol:

Six groups of albino rats (each consisting of 6 rats) of either gender were used in the current study. Treatment with cromolyn sodium and fenofibrate was continued for 10 days in nephrotoxic rats (induced by injecting gentamicin constantly for 8 days). The parameters were assessed at 0th, 9th and end of the 14th day in normal as well as gentamicin-induced rats with or without treatment. The rats were grouped as

· Group I [Normal Control]: This group was maintained on food and water.

· Group II [Gentamicin control]: Rats were administered gentamicin (80mg/kg i.p constantly for 8 days).

· Group III [Cromolyn Sodium treated Group]: The rats after 8 days of gentamicin administered were treated with cromolyn sodium (50mg/kg p.o) for 14 days.

· Group IV [Fenofibrate treated Group]: The gentamicin administered rats after 8 days were treated with fenofibrate (32mg/kg p.o) for 14 days.

· Group V [Lisinopril treated Group]: The gentamicin administered rats after 8 days were treated with lisinopril (1mg/kg p.o.) for 14 days.

· Group VI [Cromolyn sodium and Fenofibrate treated Group]: The rats after 8 days of gentamicin administration were treated with cromolyn sodium (80mg/kg p.o) and fenofibrate (32mg/kg p.o) combination for 14 days.

Assessment of Nephropathy:

Nephropathy was assessed by estimating serum creatinine using alkaline picrate method¹⁶, blood urea nitrogen (BUN) using berthelot method¹⁷, urine protein using pyragallol red method¹⁸ with commercially accessible kit (ERBA Diagnostics Mannheim GmbH, Transasia Bio-medicals Ltd., Baddi, India). The concentration of serum creatinine and blood urea nitrogen was observed to be markedly increased in gentamicin-induced rats after 8 days as compared to normal rats (Table 1). Treatment with fenofibrate and cromolyn significantly prevented increase in serum creatinine (Fig. 1a), protein urea (Fig. 1b) and blood urea nitrogen (Fig. 1c) in gentamicin-induced rats. However, treatment with Feno-cromolyn sodium combination was found more effective in reducing serum creatinine, proteinurea and blood urea nitrogen level than individual drug-treated groups (Table 2).

Histopathological study:

The changes in glomeruli were measured histologically as per method reported by Crowell et al., 2004²². The kidney was instantly engaged in 10% formalin and then dehydrated in ranked concentrations of alcohol, wrapped up in xylene and then fixed in paraffin. Kidney sections (5μm in the thickness) were made and marked with hematoxylin and eosin to measure pathological modifications occurs in glomeruli using light microscopy (400x). The kidney of the gentamicin-induced rats increases pathological alterations in the glomeruli such as extracellular mesangial expansion and glomerular capillary size reduction in contrast to kidney of the normal rat. The combined administration of cromolyn sodium and fenofibrate clearly reduced these pathological alterations in glomeruli in contrast to treatment with either drug alone (Fig. 3).

Fig. 3: Effect of cromolyn sodium and fenofibrate on pathological modifications in glomeruli.

Assessment of renal oxidative stress:

Estimation of thiobarbituric acid reactive substances (TBARS) using method reported by Ohkawa et al., 1979²³ and reduced glutathione (GSH) using method reported by Davies et al., 1984²⁴ was used for the assessment of renal oxidative stress. The TBARS and GSH was calculated using formula

TBARS= Optical density of organic layer/Extinction coefficient × Protein conc. × Incubation time × Volume of sample

Extinction coefficient of chromophore =

1.56 x 105 M^-1cm^-1

GSH= Optical Density/ Extinction coefficient × Protein conc. × Volume of sample

Extinction coefficient of chromophore = 13600 M^-1cm^-1

A marked increase in tissue TBARS and decrease in reduced glutathione (GSH) was observed in kidney of gentamicin-induced rats as compared to normal rats (Table 3). Treatment with fenofibrate, cromolyn sodium and Feno-cromolyn sodium combination significantly prevented gentamicin-induced increase in TBARS level (Fig. 4a) and decrease in GSH level (Fig. 4b). In addition, fenofibrate and cromolyn sodium also prevented an increase in TBARS and decrease in GSH levels (Table 3).

Table 3: Effect of Fenofibrate, Cromolyn sodium, Lisinopril and Feno-Cromolyn Combination on reduced Glutathione (GSH) and Thiobarbituric Acid Reactive Substances (TBARS) at 15^th day in gentamicin-induced nephropathy in rats.

Groups	Parameters
Groups	TBARS (nmol/ml)	GSH (μM/ml)
Normal Control	1.167±0.225^***	6.981±0.091^***
Gentamicin Control (80mg/kg )	5.056±0.495	2.539±0.356
Lisinopril Treated Group (1mg/kg)	2.123±0.225^***	5.504±0.403^**
Cromolyn sodium Treated Group (50mg/kg)	3.653±1.307^**	4.960±0.442^**
Fenofibrate Treated Group (32mg/kg)	4.036±0.045	3.719±0.943^***
Cromo+ Feno (50+32mg/kg)	1.581±0.180^***	6.724±0.628^***

All values are represented as mean ± S.D. *=p<0.05 vs gentamicin induced nephrotoxic control; **=p<0.01 vs gentamicin induced nephrotoxic control; ***=p<0.001 vs gentamicin induced nephrotoxic control

Fig. 4: Effect of Cromolyn sodium, Fenofibrate, Lisinopril and Feno-cromolyn combination on TBARS (4a) and GSH (4b) at 15^th day in gentamicin induced nephropathy.

Statistical analysis:

All values were expressed as Mean± S.D. The data obtained from various groups were statistically analyzed using one way ANOVA, followed by Tukey's multiple comparison test. P value less than 0.05 was considered to be statistically significant and were of two tailed.

DISCUSSION:

The current study investigated the potential role of cromolyn sodium, a mast cell stabilizer and fenofibrate, a PPAR-α agonist halting the nephrotoxicity in gentamicin induced rats. Elevated blood urea nitrogen (BUN), serum creatinine and proteinurea have been recognized as an indicator of gentamicin-induced nephrotoxicity²⁴. The administration of gentamicin shows a significant (P<0.05) increase in the level of all these parameters which points towards kidney dysfunction. Also, the reduced concentration of glutathione and increase in lipid peroxidation are considered to be an index of development of oxidative stress²⁵which has been evaluated by measuring TBARS which has been enhanced significantly (P<0.05), along with subsequent decrease in GSH.

Mast cells are the principal source of chymase (liberated through mast cell degranulation) along with powerful chemical mediators, such as cytokines, macrophage, granulocyte macrophage colony-stimulating factor (GM-CSF), histamine, heparin, leukotrienes^26,27, which plays an important role in inflammatory tissues through its proteolytic activities to cause tissue remodelling, and is responsible for formation of Angiotensin II, alteration in lipid metabolism and activation of TGF leading to tubulointerstitial fibrosis and glomerulosclerosis^28,29 which signifies that mast cells are linked with the progression of kidney fibrosis²⁸. In addition, it has been reported that release of histamine appreciably enhance intracellular accumulation of gentamicin³⁰, the main cause of nephropathy. Gentamicin in lower dose (4mg/kg) for 7-14 days and at high dose (100mg/kg/day) decreased secretion of glomerular renin and ACE from kidney respectively. The increased generation of reactive oxygen species (ROS) in gentamicin-induced nephrotoxicity results inactivation of antioxidant enzymes such as GSH and SOD³¹ and worsen the function and structure of kidney. Therefore, mast cell stabilization may reduce the renal oxidative stress in gentamicin induced nephrotoxicity in rats. Cromolyn sodium decrease the quantity of residential mast cells which further inhibits the release of newly synthesized and pre-formed chemical mediators from mast cells involved in inflammatory and allergic responses^32,33. In the current study, histopathological examination revealed intracellular oedema, degeneration, necrosis and glomerulus narrowing in epithelial cells of the proximal tubules approving that kidney is extremely susceptible to gentamicin toxicity and treatment with cromolyn sodium reduced the gentamicin-induced enhance in mast cell degranulation, renal oxidative stress and prevented histopathological changes, thus halting the progression of gentamicin induced nephrotoxicity.

Hyperlipidemia has also been reported to be an autonomous risk factor for introduction and development of nephropathy³⁴. The receptors present on mesangial cells occupies oxidized LDL, resulting in generation of prostaglandin E2 and cytotoxic agents, such as TNF, numerous cytokines viz. platelet derived growth factor (PDGF), IL-6 and TGFβ which promotes an injury to glomerular endothelial and epithelial cells, causes sclerosis^35,36. Thus, the drug-induced, increase in circulating lipids takes part in the progression and generation of nephropathy. In the current study an increase in triglycerides and serum cholesterol and subsequent decrease in serum HDL and increase in serum LDL levels has been observed in gentamicin induced rats with nephropathy. Treatment with fenofibrate (PPAR-α agonist) prevented the development of gentamicin induced nephropathy through its anti-apoptosis effect in renal tubular cells³⁷. It improves lipotoxicity via activation of AMPK-PGC-1α-ERR-1α-FoxO3a signalling, showing its potential as a therapeutic modality for nephropathy³⁸. Reducing the circulating lipids in renal patients may provide a new therapeutic option in managing nephropathy¹⁴. Fenofibrate also provides renoprotection by reducing COX-2 expression along with nitrosative and oxidative stress³⁹. Stimulation of PPAR-α using fenofibrate has been accounted to generate renoprotective outcome by down regulating the kidney expression of TGF-β⁴⁰. Renoprotective effect of lisinopril as an ACE inhibitor has been well accounted in various studies⁴¹due to which it has been used as a standard drug. The renoprotective effect of combination of cromolyn sodium and fenofibrate observed in the current study was better to the result produced by Lisinopril in gentamicin-induced nephropathy.

CONCLUSION:

Thus it can be concluded that the combined dose of cromolyn sodium and fenofibrate may have halted the development of gentamicin-induced nephropathy by declining the renal oxidative stress (ROS), preventing the degranulation of resident kidney mast cells and reducing the altered lipid profile.

ACKNOWLEDGEMENT:

The authors are highly grateful to the management of Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana for providing the necessary facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 09.09.2019 Modified on 16.11.2019

Research J. Pharm. and Tech. 2020; 13(7): 3215-3220.

DOI: 10.5958/0974-360X.2020.00569.7

Groups	Parameters
Groups	Serum creatinine (mg/dl)	BUN (mg/dl)	Protein urea (g/dl)
Normal Control	1.71±0.035^***	8.69±1.97^***	5.60±0.378^***
Gentamicin Control (80mg/kg )	2.46±0.073	18.97±1.164	9.42±0.321
Lisinopril Treated Group (1mg/kg)	1.85±0.122^***	14.81±1.701^***	8.68±0.337^***
Cromolyn sodium Treated Group(50mg/kg)	1.96±0.052^***	16.38±1.113^*	10.37±0.100^**
Fenofibrate Treated Group (32mg/kg)	1.94±0.058^**	16.01±0.693^**	8.52±0.170
Cromo+ Feno (50+32mg/kg)	1.74±0.092^***	10.21±0.511^***	7.28±0.090^***

Groups	Parameters
Groups	Triglycerides (mg/dl)	Cholesterol (mg/dl)	HDL-Cholesterol (mg/dl)	LDL-Cholesterol (mg/dl)
Normal Control	136.12±3.980^***	135.33±3.777^***	41.51±2.250^**	93.82±4.75^***
Gentamicin Control (80mg/kg )	148.61±2.690	179.67±2.805	37.06±2.337	142.60±2.97
Lisinopril Treated Group (1mg/kg)	142.34±2.013^**	154.80±4.856^***	38.68±2.092	116.12±2.97^***
Cromolyn sodium Treated Group (50mg/kg)	145.26±1.915	168.92±5.730^**	40.38±1.617	128.54±5.94^**
Fenofibrate Treated Group (32mg/kg)	146.17±1.483	171.13±2.626^***	39.77±2.002	116.40±3.20
Cromo+ Feno (50+32mg/kg)	139.73±1.914^***	141.50±3.274^***	40.95±1.394^*	94.25±3.17^***