CHRONIC ADMINISTRATION OF ALBUMIN MODIFIED BY ADVANCED GLYCATION (AGE) INDUCES EXPRESSION OF PRO‐FIBROTIC, PRO‐APOPTOTIC AND RENIN‐ANGIOTENSIN SYSTEM GENES ON RENAL TISSUE
Authors: Thieme Karina , Fabre Nelly Takashima , da Silva Karolline Santana , Catanozi Sergio , Monteiro Maria Beatriz , Machado Ubiratan Fabres , Passarelli Marisa , Correa‐Giannella Maria Lucia Cardillo 
Published: November 2023

Introduction

Diabetes mellitus (DM) is the major cause of chronic kidney disease in the world. Hyperglycemia participates in the development of diabetic nephropathy (DN), among other pathways, through the increased production of advanced glycation end products (AGEs). AGEs not excreted by the kidneys are distributed into the tissues, remain biologically active and can cause local inflammation, oxidative stress and stimulate other systems, such as the renin‐angiotensin system (RAS). The main hypothesis of this study is that AGEs contribute to DN regardless of hyperglycemia, by changing the expression of pro‐fibrotic, pro‐inflammatory, and anti‐oxidant genes and stimulating the RAS. The second hypothesis is that treatment with the antioxidant N‐acetylcysteine (NAC) could prevent the effects of AGEs.

Methods

Male Wistar rats were divided into four groups (n = 9–11 per group) and treated for 90 days: Control albumin (Control Alb, 20 mg/kg/day, i.p.), NAC (600 mg/L of water,ad libitum), Glycated albumin (AGE Alb, 20 mg/kg/day, i.p.), AGE Alb plus NAC. At the end of the treatment, the animals were allocated in metabolic cages for 24 hours for urine sample collection. Urinary variables were evaluated. One kidney of each animal was submitted to mRNA expression of the following genes by RT‐qPCR: transforming growth factor β (Tgfb1), tumor necrosis factor α (Tnf), collagen type III (Col3a1) and type IV (Col4a1), connective tissue growth factor (Ctgf), angiotensinogen (Agt), renin (Ren), angiotensin converting enzyme 1 (Ace), NADPH oxidase isoform 4 (Nox4), thiorredoxin interacting protein (Txnip) and apoptosis regulators Bax and Bcl2 and β‐actin (Actb; reference gene). The other kidney was perfused and submitted to morphological analyses.

Results

Renal variables analyses showed no differences in the body weight/kidney weight ratio, urine flow rate and Na+ and K+ excretion. There was a decrease in the protein/creatinin ratio in animals treated by AGE Alb plus NAC (vs AGE Alb; p=0.0047). There was an increase in the expression of pro‐fibrotic Tgfb1 (AGE Alb vs Control Alb, p= 0.0105) and Col4a1 (AGE Alb vs Control Alb; p= 0.0070) genes. Col4a1 upregulation was corrected by NAC (AGE plus NAC vs AGE Alb; p= 0.0152). Regarding RAS genes, there was an increase in the expression of Agt (AGE Alb vs Control Alb; p= 0.0335) and Ren, (AGE Alb vs Control Alb; p= 0.0122) genes, without changes in Ace mRNA expression. Ren upregulation was corrected by NAC treatment (AGE plus NAC vs AGE Alb; p= 0.0343). Nox4 expression was not changed among groups, however AGE‐treated rats exhibited an increase in Txnip expression (AGE Alb vs Control Alb; p= 0.0120). Bax/Bcl2 expression ratio also increased in AGE Alb‐treated rats (AGE Alb vs Control Alb, p= 0.0356) and was corrected by NAC treatment (AGE plus NAC vs AGE Alb; p= 0.0377). AGE Alb‐treated rats showed an increase in total glomerular area, which was not corrected by NAC treatment (AGE Alb vs Control Alb; p= 0.0436).

Conclusions

Chronic administration of albumin modified by glycation activates the expression of pro‐fibrotic, pro‐apoptotic and RAS genes and induces glomerular hypertrophy in normoglycemic animals. NAC administration only partially reverted these changes. These results suggest that AGEs exert renal deleterious effects per se.

Support or Funding Information

Fapesp (2014/17251‐9; 2012/04831‐1).