Investigation of Efficacy of Selected Drugs on Matrix Metalloproteinase (MMP-2 and MMP-9) and Histone Deacetylase 2 Modulation in Experimentally Induced Diabetic Nephropathy

Abstract

Diabetes mellitus is an endocrine disorder with hyperglycemia and chronic complications affecting the eyes, blood vessels, nerves, and kidneys, causing burdens to public health. Attempts to curb it have failed. Diabetic nephropathy (DN) emerges as the prevailing microvascular complication, leading to end-stage renal disease, affecting 30% of diabetic patients, further burdening public health. This disorder manifests as persistent albuminuria and a progressive decline in the glomerular filtration rate. In diabetic nephropathy, it is common to observe pathologies such as Kimmelstiel-Wilson nodules, thickening of the glomerular basement membrane, glomerular sclerosis, and inflammation. Matrix metalloproteinases (MMPs) represent a diverse family of proteolytic enzymes, possessing the ability to degrade nearly all extracellular matrix (ECM) components. Consequently, they assume a significant role in tissue remodelling. The excessive production of MMP-2 and MMP-9 plays a pivotal role in the development of diabetic nephropathy. The pathogenic features of diabetic nephropathy have been linked to a dysregulation of MMPs, which contributes to an imbalance between the production and degradation of the extracellular matrix, ultimately resulting in the development of fibrosis, a symptom commonly observed in diabetic nephropathy. Furthermore, histone deacetylase (HDAC) isoforms can regulate molecular activities in diabetes through epigenetic and post-translational regulation of transcription factors. HDAC-2 is upregulated in diabetic kidneys, making it potential therapeutic target for attenuating diabetic nephropathy. Serum creatinine and BUN levels, as well as urine albumin and creatinine levels, are used to diagnose DN. These indicators indicate the existence of structural and functional renal problems. MMPs and HDAC-2 levels are postulated in the literature as potential early markers for the development of early renal changes. The flavonoid luteolin (LT), known for its anti-inflammatory and antioxidant properties, has been proposed as a potential treatment for preserving kidney function in diabetic patients. Disulfiram (DSL), a drug employed in aversion therapy for individuals with alcohol addiction, has recently garnered considerable interest as a prospective approach for cancer treatment and cancer prevention. Recent preclinical investigations have revealed that disulfiram possesses a renoprotective effect. Alpha-cyperone (α-CYP) is an active ingredient found in Cyperus rotundus. It is known for its antioxidant, anti-inflammatory, antibacterial, and anticancer properties. The aim of the present study was to determine the effectiveness of drugs in protecting kidneys in DN by examining their impact on the expression of MMP-2, MMP-9, and HDAC-2. We employed in-silico methods, including docking and molecular dynamics simulations, in-vitro assessments to investigate the effects of high glucose (HG) on NRK-52E cells, as well as in vivo approach. We used molecular docking simulations and in silico methods to look into the possible interactions between the selected drugs and the binding pockets of MMP-9 and HDAC-2. In in-vitro assay, we performed MTT assay to assess cell viability and effect of selected drugs on expression of MMPs and HDAC-2 on high glucose induced NRK- 52E cells injury. Furthermore, we investigated the impact of the selected drugs on the modulation of MMP and HDAC enzyme expression in the kidneys of diabetic rats. This was accomplished by utilizing in-vivo model of streptozotocin-induced diabetic rats and inducing renal ischemia-reperfusion injury in diabetic and non-diabetic rats. Morphological changes were also studied. Our objective was to understand the impact of hyperglycemia on MMPs and HDAC-2 expression, as well as the effects of selected drugs on their expression. We examined if luteolin, alpha-cyperone, and disulfiram could interact with MMP-9 and HDAC-2. We performed docking simulations to investigate whether these three molecules shared a binding site. Luteolin exhibited an interaction with both MMP-9, with a binding free energy of -50.37 kcal/mol, and HDAC-2, with a binding free energy of -21.29 kcal/mol, as determined by MMGBSA after a 100 ns molecular dynamics simulation. On the other hand, alpha-cyperone and disulfiram interacted solely with HDAC-2, with binding free energies of - 17.44 kcal/mol and -20.6 kcal/mol, respectively. These interactions occur through the occupation of the catalytic site and the interaction with the Zn++ ion and amino acids present in the binding pocket. Our study illuminates the molecular underpinnings of luteolin, alpha cyperone and disulfiram as MMPs and HDAC-2 inhibitors. Although it was observed that luteolin is having promising action as compared to alpha-cyperone and disulfiram. Our cell line study revealed that treatment with different concentrations of the selected drugs significantly improved cell viability after exposure to high glucose (30 mM). HG induced cell damage in NRK-52E led to an increase in MMP-2, MMP-9, and HDAC-2 expression. All doses of luteolin, alpha-cyperone, and disulfiram, however, were able to counteract these effects, which may be due to their antioxidant and anti-inflammatory activities. In in-vivo studies, elevated MMP-2, MMP-9, and HDAC-2 expression were observed in the renal homogenate of streptozotocin-induced diabetic rats. However, the administration of disulfiram (50 and 75 mg/kg), luteolin (25, 50, and 75 mg/kg), and alpha-cyperone (50 and 100 mg/kg) significantly reversed these changes, indicating the potential of these drugs for preventing diabetic complications. Furthermore, as compared to the control group, the test drugs reduced oxidative stress, inflammation, and fibrosis in diabetic rats. We also observed improvements in body weight, renal damage repair, and hemodynamic changes, as well as decreased levels of malondialdehyde (MDA), myeloperoxidase (MPO), and hydroxyproline (HP) content in renal tissue. H&E and Masson trichome staining revealed morphological alterations, indicating that the test drugs relieve pathological damage. Further to evaluate therapeutic efficacy in acute renal injury or end-stage renal disease (ESRD), renal ischemia-reperfusion injury was induced in diabetic rats and non-diabetic rats. Luteolin, alpha-cyperone, and disulfiram at a dose of 50 mg/kg have demonstrated renoprotective activity by inhibiting metalloenzymes and reversing all the above parameters of nephropathy. This could be a result of the antioxidant and anti-inflammatory properties of the test drugs. These results show that early targeting of metalloenzymes by pre-treatment with selected drugs such as luteolin, alpha-cyperone, and disulfiram can prevent diabetic nephropathy via inhibiting the expression of MMP-2, MMP-9, and HDAC-2 and lowering oxidative stress indices and pro-inflammatory mediators. This is a safe and cost-effective way to halt the progression of diabetic nephropathy. Further studies are warranted to investigate the molecular mechanisms and signalling pathways involved in exerting the therapeutic effect of the selected drugs.