#2853 MITOCHONDRIAL DNA DEREGULATED PATTERN PARALLELS INFLAMMATION IN EARLY DIABETIC KIDNEY DISEASE OF TYPE 2 DIABETES MELLITUS PATIENTS
Authors: Petrica Ligia , Vlad Adrian , Gadalean Florica , Muntean Danina , Vlad Daliborca , Dumitrascu Victor , Bob Flaviu , Milas Livia Oana , Suteanu-Simulescu Anca , Glavan Mihaela , Jianu Catalin , Ursoniu Sorin , Lavinia Balint , Mogos Maria , Ienciu Silvia , Cretu Octavian , Popescu Roxana 
Published: June 2023

Abstract

Background and Aims
Mitochondrial DNA (mtDNA) abnormalities, as well as complex inflammatory processes involved in the pathogenesis of DKD may be detected early in the course of DKD. The aim of the study was to evaluate a potential relation of mtDNA modifications in blood and urine with podocyte injury and proximal tubule (PT) dysfunction in early DKD of type 2 diabetes mellitus (DM) patients. The hypothesis according to which mtDNA changes may be related to a specific inflammatory response in normoalbuminuric DKD of type 2 DM patients was also questioned.

Method
A total of 150 patients [52 patients with normoalbuminuria, 48 patients with microalbuminuria, and 50 patients with macroalbuminuria)] and 30 age- and gender-matched healthy controls were enrolled in this case series study. All patients were assessed concerning urinary albumin/creatinine ratio (UACR), biomarkers of podocyte damage (synaptopodin, podocalyxin) and of PT dysfunction (kidney injury molecule-1-KIM-1, N-acetyl-D-glucosaminidase-NAG), serum and urinary interleukins (IL-17A, IL-18, IL-10), and eGFR (creatinine-cystatin C). MtDNA-CN and nuclear DNA (nDNA) was quantified in peripheral blood and urine by qRT-PCR (ABI 7900-HT–Applied BioSystem). TaqMan assays were utilized for the assessment of cytochrome b (CYTB) gene, subunit 2 of NADH dehydrogenase (ND2), and of beta 2 microglobulin nuclear gene (B2M). mtDNA-CN was defined as the ratio of the number of mtDNA/nDNA copies, through analysis of the CYTB/B2M and ND2/B2M ratio. DNA standards were created by plasmidial cloning vectors. Cloning was verified by PCR sequencing and validation.

Results
Serum mtDNA was decreased, while urinary mtDNA was increased in all study groups as compared to healthy controls. In univariable linear analysis, serum mtDNA correlated directly with eGFR and negatively with UACR, urinary synaptopodin, urinary podocalyxin, urinary KIM-1, and urinary NAG. Also, serum mtDNA correlated negatively with serum IL-17A and urinary IL-17A, and with serum IL-18 and urinary IL-18, and directly with serum IL-10 and urinary IL-10. Urinary mtDNA correlated directly with UACR, synaptopodin, podocalyxin, KIM-1, NAG, IL-17A, and IL-18, and showed an inverse correlation with IL-10, and eGFR. Multivariable linear analysis provided models in which serum mtDNA correlated directly with IL-10, and indirectly with UACR, IL-17A, and KIM-1 (R2 = 0.626; p = 0.0001). Urinary mtDNA correlated directly with UACR, podocalyxin, IL-18, and NAG, and negatively with IL-10 and eGFR (R2 = 0.631; p = 0.0001). This data suggests that increased urinary mtDNA levels may be attributed to increased filtration of circulating DNA and that this may derive from filtered circulating mtDNA due to lesions to the glomerular and tubular structures. The correlations of mtDNA with proinflammatory cytokines IL-17A and IL-18, as well as with the anti-inflammatory cytokine IL-10 could explain the association of these cytokines with mtDNA variations and with the expression of podocyte damage and PT dysfunction biomarkers in all studied groups, even in normoalbuminuric type 2 DM patients.

Conclusion
MtDNA abnormalities and inflammatory processes occur early in the course of type 2 DM. MtDNA displays a specific profile in relation to inflammation both at podocyte and tubular level in normoalbuminuric patients with type 2 DM. These observations are independent of renal function decline.