Aminophylline suppresses chronic renal failure progression by activating SIRT1/AMPK/mTOR-dependent autophagy

Abstract

Chronic renal failure (CRF) is a severe syndrome affecting the urinary system for which there are no effective therapeutics. In this study, we investigate the effects and mechanisms of aminophylline in preventing CRF development. A rat model of chronic renal failure is established by 5/6 nephrectomy. The levels of serum creatinine (SCR), urinary protein (UPR), and blood urea nitrogen (BUN) are detected by ELISA. Histological evaluations of renal tissues are performed by H&E, Masson staining, and PAS staining. Functional protein expression is detected by western blot analysis or immunofluorescence microscopy. Glomerular cell apoptosis is determined using the TUNEL method. Results show that Aminophylline significantly reduces the levels of SCR, UPR, and BUN in the CRF model rats. Histological analyses show that aminophylline effectively alleviates renal tissue injuries in CRF rats. The protein expression levels of nephrin, podocin, SIRT1, p-AMPK, and p-ULK1 are greatly increased, while p-mTOR protein expression is markedly decreased by aminophylline treatment. Additionally, the protein level of LC3B in CRF rats is significantly increased by aminophylline. Moreover, aminophylline alleviates apoptosis in the glomerular tissues of CRF rats. Furthermore, resveratrol promotes SIRT1, p-AMPK, and p-ULK1 protein expressions and reduces p-mTOR and LC3B protein expressions in CRF rats. Selisistat (a SIRT1 inhibitor) mitigates the changes in SIRT1, p-AMPK, p-ULK1, p-mTOR, and LC3B expressions induced by aminophylline. Finally, RAPA alleviates renal injury and apoptosis in CRF rats, and 3-MA eliminates the aminophylline-induced inhibition of renal injury and apoptosis in CRF rats. Aminophylline suppresses chronic renal failure progression by modulating the SIRT1/AMPK/mTOR-mediated autophagy process.

1. Introduction

Chronic renal failure (CRF), also known as chronic kidney disease (CKD), is a severe urinary syndrome characterized by constant changes in kidney structure and a gradual loss of kidney function, usually resulting in hypertension, cardiovascular disease, edema, tumors, and even death [1]. Recent epidemiological studies have shown that CRF affects>7% of individuals worldwide. CRF is associated with multiple risk factors, including genetic mutations, acute and chronic kidney injuries, exposure to toxic reagents, a low nephron number at birth, obesity, and diabetes mellitus [2]. CRF development is known to be mediated by a loss of nephrons, remnant nephron hypertrophy, impaired glomerular filtration, and interstitial fibrosis [3]. The current clinical treatments for CRF include dialysis, kidney transplantation, and other replacement therapies. However, these forms of therapies are not available or affordable for all patients, especially for elderly patients with other complications [4]. New strategies to inhibit nephron loss or accelerate nephrogenesis and regeneration are urgently needed to prevent disease progression and improve the quality of life of CRF patients.

Aminophylline is usually a compound composed of theophylline and ethylenediamine. Theophylline is a caffeine analogue that acts as a nonselective inhibitor of phosphodiesterases, which catalyze the hydrolysis of cyclic nucleotides to modulate intracellular cAMP and cGMP levels. Because cAMP and cGMP are important second messengers associated with disease pathogenesis, aminophylline has long been used for the treatment of lung disorders such as asthma and chronic obstructive pulmonary disease (COPD). The therapeutic effects of aminophylline on lung disorders can be partially attributed to its ability to induce mitochondrial biogenesis and the resulting improvement in epithelial mitochondrial function. A previous study in a mouse model showed that aminophylline exerts a substantial anti-inflammatory effect due to its regulation of cytokine release, which might be useful for preventing inflammation-induced preterm parturition [5]. Furthermore, aminophylline has also shown promise for treating other human diseases, such as postdural puncture headache [6], hypercapnia [7], and irritable bowel syndrome [8]. More importantly, aminophylline demonstrated renoprotective effects in a mouse model of renal ischaemia‒reperfusion (I/R) injury, where it greatly improved renal function and inflammation indices [9]. Previous research has also shown that aminophylline can decrease serum creatinine level and prevent renal tissue damage in young patients with acute kidney injury (AKI) [10]. However, the possible use of aminophylline for repressing CRF development has not been studied.

Autophagy is an essential cellular process responsible for the degradation of cytoplasmic car