Box A of HMGB1 Maintains the DNA Gap and Prevents DDR-induced Kidney Injury in D-galactose Induction Rats

Abstract

Background/aim: Disability and mortality rates for renal failure are still increasing. DNA damage and oxidative stress intoxication from body metabolism, high blood glucose, or the environment cause significant kidney damage. Recently, we reported that Box A of HMGB1 (Box A) acts as molecular scissors, producing DNA gaps that prevent DNA damage in kidney cell lines and ultimately reverse aging phenotypes in aging rat models. The present study aimed to demonstrate the potency of Box A in preventing D-galactose (D-gal)-induced kidney injury.

Materials and methods: A Box A expression plasmid was constructed and administered to a rat model. D-gal was injected subcutaneously for eight weeks. Serum was collected to study renal function, and white blood cells were collected for DNA gap measurement. Kidney tissue was also collected for γ-H2AX and NF-κB immunostaining; Senescence-associated (SA)-beta-gal staining; and analysis of the mRNA expression of p16^INK4A, TNF-α, and IL-6. Moreover, histopathology analysis was performed using hematoxylin & eosin and Masson trichome staining.

Results: Pretreatment with Box A administration prevented the reduction of DNA gaps and the consequences of the DNA damage response, which include elevated serum creatinine; high serum BUN; an increased positive SA-beta-gal staining area; overexpression of p16^INK4A, NF-κB and senescence-associated secretory phenotype molecules, including IL-6, TNF-α; and histological alterations, including tubular dilation and collagen accumulation.

Conclusion: Box A effectively prevents DNA gap reduction and all D-gal-induced kidney pathological changes at the molecular, histological, and physiological levels. Therefore, Box A administration is a promising novel therapeutic strategy to prevent DNA-damaging agent-induced kidney failure.

Keywords: Box A of HMGB1; DNA damage; DNA durability; DNA protection; Kidney failure; youth-DNA gap.

Figure 1. Preventive effects of Box A on serum renal function in rats with D-gal-induced kidney injury. (A) Schematic diagrams for the groups pretreated with Box A plasmid/Ca-P nanoparticle (Box A) (100 μg/kg/week, i.p.) or PC/Ca-P nanoparticle (100 μg/kg/week, i.p.) on Day 0 and then treated daily with D-galactose (150 mg/kg/day, s.c.) beginning three days later and continuing for eight weeks. (B) The FLAG tag sequence was detected using immunofluorescence staining for representative plasmid delivery, represented in a green dot within the red circles (1,000×) in all groups (except for the negative control). Scale bar=20 μm. (C) Body weight (n=5). (D) Serum creatinine (mg/dl) (n=5). (E) Serum blood urea nitrogen (BUN) (mg/dl) (n=5). The PC+NSS, PC+D-gal, and Box A+D-gal labels represent the control group, the kidney injury (D-gal) group, and the group pretreated with Box A in kidney injury (Box A), respectively. The values represent the means±SEMs. **p≤0.01, ***p≤0.001.

Figure 1. Preventive effects of Box A on serum renal function in rats with D-gal-induced kidney injury. (A) Schematic diagrams for the groups pretreated with Box A plasmid/Ca-P nanoparticle (Box A) (100 μg/kg/week, i.p.) or PC/Ca-P nanoparticle (100 μg/kg/week, i.p.) on Day 0 and then treated daily with D-galactose (150 mg/kg/day, s.c.) beginning three days later and continuing for eight weeks. (B) The FLAG tag sequence was detected using immunofluorescence staining for representative plasmid delivery, represented in a green dot within the red circles (1,000×) in all groups (except for the negative control). Scale bar=20 μm. (C) Body weight (n=5). (D) Serum creatinine (mg/dl) (n=5). (E) Serum blood urea nitrogen (BUN) (mg/dl) (n=5). The PC+NSS, PC+D-gal, and Box A+D-gal labels represent the control group, the kidney injury (D-gal) group, and the group pretreated with Box A in kidney injury (Box A), respectively. The values represent the means±SEMs. **p≤0.01, ***p≤0.001.

Figure 2. Effects of Box A pretreatment on preventing cellular senescence-related histopathological changes in the kidneys. Histopathological examination of rat kidney tissue sections after eight weeks (H&E staining and Masson trichrome staining, 200×). Scale bar=100 μm. (A) Representative pictures of H&E staining in histological sections. The yellow arrow indicates the proximal renal tubular injury including tubular dilation with some areas representing karyolysis and thinned renal tubular epithelium. (B) Representative Masson trichrome staining pictures of histological sections. (C) The percentage of tubular dilation in histological sections (n=4) was calculated using Image J. (D) The percentage of positive area was analyzed to assess collagen accumulation (blue color, black arrow) in histological sections (n=4). The values represent the means±SEMs. *p≤0.05.

Figure 3. Effects of pretreatment with Box A on maintaining DNA gaps and preventing DNA damage. (A) The DNA gaps (DNA-GAP number) were quantitated in rat white blood cells (WBCs; n=4) and was considered 100% in PC + NSS rat WBCs. (B) The percentage of DNA damage-positive cells was analyzed using γ-H2AX immunostaining in histological sections (n=4). (C) Representative photographs of γ-H2AX immunostaining in histological sections (1,000×). The black arrow indicates the positive area of γ-H2AX-positive cells. Scale bar=20 μm. The values represent the means±SEMs. *p≤0.05, and **p≤0.01.

Figure 4. Preventive effects of Box A on cellular senescence markers, including SA-beta-gal activity and p16INK4A mRNA expression in rats with D-gal-induced kidney injury. (A) quantification of SA-beta-gal staining in rat kidney sections (n=3). (B) The mRNA expression of the p16INK4A gene was identified via RNA-seq analysis (n=4). Total RNA was isolated from kidney rat tissues at the end of the study and used for RT-qPCR analysis of the indicated genes normalized to the housekeeping gene (GAPDH). (C) Representative pictures of SA-beta-gal staining in rat kidney sections (200×). Scale bar=100 μm. The values represent the means±SEMs. *p≤0.05, and **p≤0.01.

Figure 5. Effects of Box A pretreatment on senescence-associated inflammatory responses in the kidneys. (A) The percentage of positive area was analyzed to assess NF-ĸB expression in histological sections (n=4) using immunohistochemistry. (B) mRNA expression of the TNF-α gene determined using RNA-seq analysis (n=4) in rat kidneys. (C) mRNA expression of the IL-6 gene determined using RNA-seq analysis (n=4) in rat kidneys. (D) Representative photographs of NF-ĸB immunostaining in histological sections (400×). Scale bar=50 μm. The black arrow indicates the positive area for immunostaining (brown color). The values represent the means±SEMs. *p≤0.05, and **p≤0.01.