Introduction:
Renal fibrosis is a hallmark of chronic kidney disease (CKD), characterized by excessive inflammation and fibrosis, and contributes significantly to progressive renal dysfunction. There is a pressing need for effective anti-fibrotic interventions, an area where options are lacking. One such promising treatment method is the employment of cell therapy with mesenchymal stromal cells (MSCs). MSCs have shown promise as anti-inflammatory and immunomodulatory agents, making them a promising opportunity for intervention. This study investigates two critical factors influencing treatment specificity 1) delivery methods as well as 2) preconditioning of adipose tissue derived MSCs, for the treatment of CKD-associated renal fibrosis.
Methods:
We investigated the effects of proinflammatory preconditioning on MSCs and their derived secretome using an in vitro fibrosis model. To this end, conditioned medium (CM) was collected from either preconditioned MSCs (Pr-MSCs) treated with TNF-α and IFN-γ or vehicle, or naïve MSCs. Furthermore, CM was processed using size exclusion chromatography, a soluble protein (SP) fraction and an extracellular vesicle (EV) fraction collected and employed in TGF-β stimulated HKC-8 cells. Analysis of gene expression of inflammatory and fibrotic markers was carried out using qPCR.
Additionally, in vivo experiments were conducted using a murine unilateral ureteral obstruction (UUO) model of CKD to evaluate the therapeutic efficacy of systemic versus local administration of preconditioned MSCs and their EVs. For this, C57BL/6j mice were anesthetized with isoflurane by inhalation and the left kidneys surgically obstructed with silk ligature or SHAM operated. Mice were concurrently treated with either local (subcapsular) or systemic (tail vein) delivered Pr-MSCs. Mice were sacrificed by collecting blood through the heart under anesthesia after five days. Analysis on gene expression levels with qPCR as well as immunohistochemical analyses and cytokine multiplex (V-PLEX Mouse Cytokine 19-plex, Meso Scale Diagnostics) screening of inflammatory cytokines were carried out.
Results:
Pr-MSC derived CM and SP demonstrated significant antifibrotic effects in vitro, reducing expression of fibrotic markers fibronectin (n=6, p<0.05) and collagen type 1α1 in TGF-β-stimulated HKC-8 cells (n=6, p<0.05) compared to vehicle-treated cells. EVs did not replicate these effects when compared to vehicle-treated cells (n=6, p>0.05), suggesting that SPs secreted by Pr-MSCs may be the primary mediators of antifibrotic effects. In vivo, both systemic and local delivery of Pr-MSCs were explored in a murine UUO model. Local administration of0020 showed a more pronounced impact on the cytokine profile of UUO compared to systemic administration. Particularly, local administration resulted in the upregulation of the mRNA of the inflammatory cytokine IL-10 (n=6, p<0.05) indicating anti-inflammatory properties of locally delivered Pr-MSCs in vivo. Additionally, local delivery, but not systemic delivery, showed a reduction in the number of myofibroblasts, as shown by decreased staining of the myofibroblast marker α-smooth muscle actin, compared to vehicle-treated UUO mice (n=5, p=0.059).
Conclusion:
This study provides critical insights into the therapeutic efficacy of MSCs and emphasizes the importance of delivery methods and preconditioning strategies in enhancing MSC-based therapies for renal fibrosis. Further optimization of MSC delivery strategies and mechanistic elucidation is warranted to fully exploit their potential in treating CKD.