Extracellular Vesicles (EVs) Derived from Mesenchymal Stem Cells (MSCs) as Adjuvants in the Treatment of Chronic Kidney Disease (CKD)

Abstract

Chronic kidney disease (CKD) is considered an important health issue worldwide. The renin-angiotensin-aldosterone system (RAAS) blockade through the administration of angiotensin II receptor blockers, such as Losartan (LOS), has been considered the best strategy for CKD treatment for decades. However, this approach promotes only partial detention of CKD progression and cannot reverse renal damage. The aim of the present study was to investigate whether the therapeutic administration of extracellular vesicles (EVs) derived from adipose stem cells (ASCs), associated to LOS treatment, would promote additional renoprotection in rats underwent the 5/6 renal ablation CKD model. ASC-derived EV were administered locally, in the renal subcapsular area, 15 days after CKD induction, when LOS therapy also began. Animals were followed for additional 15 days and our results demonstrated that subcapsular injection of ASC-derived EV associated with LOS significantly reduced glomerulosclerosis, renal interstitial infiltration by myofibroblasts, and macrophages in the 5/6 CKD model. Additionally, LOS + EV abrogated systemic hypertension, proteinuria, and albuminuria, and stimulated local gene overexpression of the endogenous anti-inflammatory Il-4. Although more studies are still required to establish the best EV dose and administration route, these findings point to therapy with ASC-derived EV as a potential adjuvant in CKD treatment.

Keywords: cell therapy; chronic kidney disease; extracellular vesicles; mesenchymal stem cells.

Figure 1

Characterization of ASCs at the 4th cell passage: (A) ASCs were immunophenotyped by flow cytometry, through which we observed that the cell population was 100% positive for CD29 and for CD44, and 99% positive for CD90, typical mSC biomarkers. Moreover, only 10% of these cells were positive for CD45, a surface protein expressed in hematopoietic cells and employed as a negative biomarker in mSC culture. (B) ASCs at P4 were also subjected to cell plasticity tests in order to assess the cell ability to differentiate when receiving appropriate culture media. As shown in the illustrative microphotographs, depending on cell media, ASCs can differentiate into adipogenic, chondrogenic, and osteogenic cell lineages, exhibiting lipid droplets stained in red, sulfated matrix proteoglycans stained in turquoise blue, or reddish calcium crystals, respectively.

Figure 2

Characterization of EVs obtained from ASCs: (A) The concentration and size distribution of EVs were analyzed using a NANOSIGHT 3 Nanoparticle Tracking Analysis (NTA) device (NanoSight Ltd.). After the dilution factor correction, we found that each EV inoculum was composed of approximately 3 × 10¹¹ particles, varying in size between 50 and 600 nm, with a predominant population of particles with an approximate diameter of 240 nm (mode)~270 nm (mean). (B) Obtained EVs were morphologically characterized by electron transmission microscopy, using Electron Transmission Microscope model JEM 1011-JEOL/MA/USA at 80 kV.

Figure 2

Characterization of EVs obtained from ASCs: (A) The concentration and size distribution of EVs were analyzed using a NANOSIGHT 3 Nanoparticle Tracking Analysis (NTA) device (NanoSight Ltd.). After the dilution factor correction, we found that each EV inoculum was composed of approximately 3 × 10¹¹ particles, varying in size between 50 and 600 nm, with a predominant population of particles with an approximate diameter of 240 nm (mode)~270 nm (mean). (B) Obtained EVs were morphologically characterized by electron transmission microscopy, using Electron Transmission Microscope model JEM 1011-JEOL/MA/USA at 80 kV.

Figure 3

Systolic blood pressure (SBP, mmHg): (A) Line graphs showing the time course of SBP in the different experimental groups, throughout the protocol. (B) Bar graphs of SBP after 30 days of CKD induction. (C) Delta bar graphs, obtained by subtracting the values observed at 30 days from those obtained at 15 days, before the beginning of treatments. Statistical differences are * p < 0.05 vs. sham, § p < 0.05 vs. basal CKD, # p < 0.05 vs. CKD, Φ p < 0.05 vs. CKD LOS, and † p < 0.05 vs. CKD EV.

Figure 4

Urinary protein (UPE, mg/24 h) and albumin (UAE, mg/24 h) excretion of the animals of each experimental group: Line graphs show the time course of UPE (A) and UAE (D) in the different groups, throughout the protocol. Bar graphs show UPE (B) and UAE (E) after 30 days of CKD induction. Finally, delta bar graphs for UPE (C) and UAE (F) were obtained by subtracting the values observed at 30 days from those obtained at 15 days, before the beginning of treatments. Statistical differences are * p < 0.05 vs. sham, § p < 0.05 vs. basal CKD, # p < 0.05 vs. CKD, Φ p < 0.05 vs. CKD LOS.

Figure 5

Glomerular damage: (A) Illustrative microphotographs of PAS-stained sections of each experimental group under final 400× magnification. (B) Bar graphs showing the percentage of glomerulosclerosis (GS%) in the different groups by the end of the protocol. Statistical differences are * p < 0.05 vs. Sham, # p < 0.05 vs. CKD. EV associated with LOS promotes further attenuation of renal fibrosis.

Figure 6

Interstitial fibrosis: (A) Illustrative microphotographs of Masson’s trichrome-stained sections of each experimental group under final 200× magnification. (B) Bar graphs showing the percentage of interstitial fibrosis (INT%) in the different groups by the end of the protocol. Statistical differences are * p < 0.05 vs. sham.

Figure 7

Interstitial myofibroblasts: (A) Illustrative microphotographs of immunohistochemistry for α-SMA, used to detect myofibroblasts, in renal cortical sections of each experimental group, under final 200× magnification. (B) Bar graphs showing the percentage of interstitial area occupied by α-SMA in the different groups by the end of the protocol. Statistical differences are * p < 0.05 vs. sham, § p < 0.05 vs. basal CKD, # p < 0.05 vs. CKD.

Figure 8

Renal interstitial macrophage infiltration: (A) Illustrative microphotographs of immunohistochemistry for CD68 in renal cortical sections of each experimental group under final 400× magnification. (B) Bar graphs showing the mean number of interstitial CD68+ cells/mm² of renal cortical area of animals from the different groups by the end of the protocol. Statistical differences are * p < 0.05 vs. sham.

Figure 9

Local renal inflammation—Interstitial PCNA+ proliferating cells: (A) Illustrative microphotographs of immunohistochemistry for PCNA in renal cortical sections of each experimental group under final 400× magnification. (B) Bar graphs showing the mean number of interstitial proliferating cells/mm² of renal cortical area of animals from the different groups by the end of the protocol. Statistical differences are * p < 0.05 vs. Sham.

Figure 10

Bar graphs illustrating the quantification of renal gene expression (RT-qPCR) of interleukins (A) Il-1β, (B) Il-2, (C) Il-4, (D) Il-6, and (E) Il-10. Statistical differences are * p < 0.05 vs. sham, § p < 0.05 vs. basal CKD, Φ p < 0.05 vs. CKD LOS.