| Objective: Type 2 diabetes mellitus(T2DM)is one of the biggest health hazards in the world today,and T2 DM is highly susceptible to a decline in muscle function,manifested by decreased muscle strength or physical fitness.It increases the probability of accidental occurrence such as falls and fractures,and increases the risk of caregiving efforts.Diabetic nephropathy is one of the most serious microvascular complications of type 2 diabetes mellitus(T2DM)and a leading cause of end-stage renal disease(ESRD).Combined type 2 diabetic nephropathy(T2DN)is a risk factor for muscle hypofunction in patients with T2 DM.Currently,studies on muscle hypofunction in T2 DN are mainly focused on the end-stage of T2 DN,while less attention has been paid to early-stage of T2 DN,which is the key period for the progression of T2 DN.Amino acid therapy is an effective means of improving renal and muscle function,but studies addressing muscle function in patients with early-stage of T2 DN are rarely reported.Therefore,it is of great significance to explore the muscle function decline and its mechanism in patients with early renal disease,and to propose targeted intervention measures to reduce the incidence of accidents and improve physical function.Methods: This research is divided into three parts.(1)Determination of muscle function and analysis influencing factors in patients with early stage of T2 DN.In this study,81 diabetic patients who were hospitalized in a tertiary grade A hospital in Wuxi and met the inclusion criteria were selected as the subjects.Patients’ general data(gender,age,disease duration,number of falls),blood test indicators(2h PBG,Hb Alc,serum creatinine,urea nitrogen,e GFR,ACR,serum albumin,HDL,LDL,TCHO)and physical measurement indicators(BMI,grip strength,gait speed)were collected to analyze the relationship between muscle dysfunction and early-stage of T2 DN in patients with T2 DM.(2)Basic experimental and clinical data were combined to explore the mechanisms underlying the development of muscle hypofunction in patients with early-stage T2 DN.C57BL/6J mice aged 6-8 weeks were selected to establish the early-stage T2 DN model by high-fat feeding for 4 weeks and continuing high-fat feeding for 8 weeks after intraperitoneal streptozotocin(STZ,120 mg/kg)injection.Muscle strength and physical performance were determined by grid and fatigue rotarod experiments;kidney and skeletal muscle morphological changes were observed using H&E staining;the expression levels of kidney injury factors and skeletal muscle function related factors were detected by q PCR;the inflammatory factor TNFαand IL-1β in mice and patients were determined by ELISA;the plasma differential amino acids of mice were screened by amino acid analyzer.And the levels of differential amino acids in patients were measured;Pearson correlation analysis was used to test the relationship between muscle function and different amino acids and inflammatory factors.(3)Based on ethical considerations,preclinical validation was performed with the help of an animal model to explore the effect of citrulline supplementation on muscle dysfunction in early-stage T2 DN mice.The second part of the study found that the decrease of arginine content was an important factor affecting muscle dysfunction in patients with early-stage T2 DN.Citrulline is the precursor of arginine synthesis in vivo,and its bioavailability and safety are much higher than that of arginine.Therefore,citrulline was gave to mice.The modeling and experimental methods were the same as the second part.Results:(1)According to the muscle function,the study subjects were divided into two groups: muscle dysfunction group and normal muscle function group.The results showed that increased age and proteinuria(increased ACR level)were independent risk factors for muscle dysfunction in T2 DM patients.Then,the subjects were divided into T2 DM group(ACR<30)and T2 DN group(30≤ACR<300)according to the ratio of urinary microalbumin to creatinine(ACR),The result showed that the incidence of muscle dysfunction in DN group was higher than that in T2 DM group(41.03% vs 19.05%).Furthermore,T2 DN group was divided into muscle dysfunction group and normal muscle function group according to muscle function.And the result showed that the level of ACR in patients with muscle dysfunction was significantly higher,and ACR levels were negatively correlated with muscle strength(r=-0.424)and gait speed(r=-0.496).Besides,the level of ALB in patients with muscle dysfunction was significantly lower,and the level of ALB correlated positively with muscle strength(r=0.461)and gait speed(r=0.471).(2)Animal experiments showed that:(1)The renal function of early-stage T2 DN mice was decreased,and the kidneys developed fibrotic lesions manifested by the higher levels of collagen1,TGFβ1 and αSMA;(2)Muscle function of early-stage T2 DN mice was decreased,manifested by decreased muscle strength and physical fitness,atrophy of muscle fibers,decreased MYOG expression in skeletal muscle,and significantly increased MURF-1 and BAX expression;(3)The levels of plasma TNFα and IL-1β increased in T2 DN mice,amino acid metabolism was disordered,and the decrease of arginine content was the most significant.Clinical analysis showed that:(1)Muscle strength(r=0.603)and gait speed(r=0.641)in patients with early-stage T2 DN were positively correlated with plasma arginine level;(2)Serum TNFα and IL-1β was negatively correlated with muscle strength(r=-0.497,r=-0.400)and gait speed(r=-0.468,r=-0.355)in patients with early-stage T2 DN.(3)The results showed that:(1)Citrulline improved the muscle strength and physical fitness of early-stage T2 DN mice,improved skeletal muscle fiber atrophy,reversed the low expression of MYOG and the high expression of MURF-1 and BAX;(2)Citrulline improved renal function and alleviated renal fibrosis in early-stage T2 DN mice;(3)Citrulline supplementation decreased plasma inflammatory factor TNFα and IL-1β and increased the content of arginine in early-stage of T2 DN.Conclusion: Our study found that reduced muscle function was more frequently detected in patients with early-stage T2 DN,and ACR level were negatively correlated with muscle function,ALB level were positively correlated with muscle function.We further elucidated that the development of muscle wasting in early-stage T2 DN was associated with reduced renal function,increased levels of inflammation,innutrition,and lower arginine content.In addition,we found that citrulline supplementation improved the muscle function of early-stage T2 DN mice,which was mainly related to reducing the level of inflammation,improving renal function,improving malnutrition and increasing arginine levels. |
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