#5978 SUBCAPSULAR INJECTION OF EXTRACELLULAR VESICLES FROM MESENCHYMAL STEM CELLS, PROMOTED ADDITIONAL RENOPROTECTION IN AN EXPERIMENTAL MODEL OF CKD
Authors: Noda Paloma , Ornellas Felipe , Celestrino Giovanna , Teles Flavio , Noronha Irene L , Fanelli Camilla 
Published: June 2023

Abstract

Background and Aims
Chronic kidney disease (CKD) is considered a public health concern worldwide, both due to its insidious and highly debilitating feature and to its high global prevalence. Clinical management of CKD is often achieved by the blockade of the renin-angiotensin-aldosterone system (RAAS). However, this treatment alone is not efficient to prevent CKD progression completely, motivating the scientific community to search for alternative treatments to control CKD progression and to detain its evolution to the end-stage renal failure. In this context, we have recently shown that the association of a single renal subcapsular injection of 2 × 106 adipose-derived mesenchymal stem cells (ASC) to RAAS blockade with the AT1RB Losartan (LOS) promoted greater renoprotection when compared to LOS monotherapy, leading to the normalization of urinary protein excretion (UPE) and to the regression of established glomerulosclerosis (GS), in experimental CKD. Since there are methodological limits for the number of ASC that can be injected under the renal capsule, and based on the current literature, which suggests the main beneficial effects of ASC are not due to direct in situ cell differentiation, but to paracrine factors produced and released by the ASC; in the present study we aimed to investigate if the association of a renal subcapsular injection of extracellular vesicles (EV) derived from ASC, to the oral treatment with LOS would promote additional renoprotective effects in a model of experimental CKD.

Method
The present experimental protocol was approved by the Ethics Committee for the Use of Experimental Animals of the University of São Paulo Medical School (CEUA-FMUSP No 1761/2022). EV were obtained from ASC isolated from gonadal adipose tissue of 5 male Wistar rats. Cells were cultured until P4, characterized by flow cytometry and in vitro differentiation, and kept under serum deprivation for 24 h. The conditioned culture media, containing the EV released by these cells, was ultracentrifuged and the resulting EV pellets were diluted in 100 μL of sterile PBS and used for the renal subcapsular injections. Experimental CKD was induced in 25 male Wistar rats through the surgical ligation of 2 from the 3 branches of the left renal artery, followed by the total nephrectomy of the right kidney, resulting in a 5/6 renal ablation. Additional 10 Sham-operated rats were used as control. After 15 days from surgery, the animals submitted to renal ablation were stratified into 3 groups, with closely similar mean values of body weight (BW), systolic blood pressure (SBP), UPE and urinary albumin excretion (UAE), before the start of the different treatments. Animals from CKD group were kept untreated, LOS and LOS+EV animals received diary 50 mg/Kg/d of Losartan, diluted in drinking water and LOS+EV rats underwent a second surgery for the renal subcapsular application of EV. All animals were followed until 30 days after CKD induction, when BW, SBP, UPE and UAE were analyzed again. Animals where then euthanized for the assessment of serum creatinine (SCr) and blood urea nitrogen (BUN) concentration, as well as for histological studies to determine the percentage of GS and the renal interstitial infiltration by macrophages (CD68).

Results
The association of a single subcapsular injection of EV derived from ASC, to the oral treatment with the AT1RB LOS, significantly improved the effects of this antihypertensive drug. CKD+LOS+EV animals exhibited normal SBP, in spite of having only 1/6 of functioning renal mass. Moreover, the combined treatment significantly reduced UAE and numerically diminished de percentage of glomerulosclerosis, compared to LOS alone. Detailed obtained results are presented in Table 1. Data are presented as Mean ± SE. Differences among groups were analyzed by one-way ANOVA: p&lt;0.05 vs.*CONT, #CKD, †LOS. as Mean ± SE.

Conclusion
Despite the small number of animals in the association group, our preliminary results suggest EV from ASC to exert additional renoprotective effects when associated to LOS, especially regarding the control of SBP and the protection of the glomerular filtration barrier integrity.