Potentiating Renal Regeneration Using Mesenchymal Stem Cells

Abstract

Background: The potential of a mesenchymal stem cell (MSC) therapy to accelerate the repair of ischemically damaged human kidneys during 24 hours of warm perfusion was evaluated. The hypothesis was that by administering MSC directly to the renal tissue, there would be an improved opportunity for cellular repair mediated by intrarenal paracrine effects.

Methods: Studies were performed using the exsanguinous metabolic support (EMS) tissue-engineering platform. Five pairs of human kidney allografts from donation after circulatory death donors were studied. One human kidney was EMS perfused for 24 hours (control), whereas its paired kidney was EMS perfused with MSC (1 × 10). The kidneys were evaluated for DNA synthesis, cytokine/chemokine synthesis, cytoskeletal regeneration, and mitosis.

Results: Treatment with MSC resulted in reduced inflammatory cytokines synthesized by the kidneys. Mesenchymal stem cell treatment led to a significant increase in the synthesis of adenosine triphosphate and growth factors resulting in normalization of metabolism and the cytoskeleton. Toluidine Blue staining of MSC-treated kidneys demonstrated a significant increase in the number of renal cells undergoing mitosis (26%) compared with EMS perfusion alone.

Conclusions: To our knowledge, our work is the first to have demonstrated actual renal regeneration while ischemically damaged human kidneys are perfused ex vivo for 24 hours. The observed regeneration entails: increased synthesis of adenosine triphosphate, a reduced inflammatory response, increased synthesis of growth factors, normalization of the cytoskeleton and mitosis. The ability to regenerate renal tissue ex vivo sufficiently to result in immediate function could revolutionize transplantation by solving the chronic organ shortage.

Figure 1:. Pathways of Regeneration (ATP Synthesis)

ATP concentration was tested in human renal biopsies following 24 hours of Exsanguinous Metabolic Support. In test kidneys, mesenchymal stem cells were administered intra-arterially and compared to EMS treatment alone in the paired kidney. MSC treated human kidneys showed a significant increase in ATP concentration compared to controls in samples taken from the cortex and medulla (*denotes a (p<0.05)). (n = 5 pairs).

Figure 2:. Pathways of Regeneration (DNA Synthesis)

Human renal biopsies were taken following 24 hours of Exsanguinous Metabolic Support with or without the addition of 1×10⁸ mesenchymal stem cells. Tissue samples were fixed in 4% formaldehyde, processed and embedded in paraffin wax for sectioning. An indirect immunofluorescent assay was performed using a Mouse Anti-PCNA antibody (ABCAM) and Goat Anti-Mouse Alexa Fluor 488 secondary. A.) Represents PCNA expression after 24H of EMS perfusion B.) MSC treated human kidneys resulted in a significant increase in PCNA positive nuclei in the paired test kidneys (p<0.049) (n=5 pairs).

Figure 3:. Pathways of Regeneration (Mitosis)

Human kidney samples were taken following 24 hours of warm perfusion with or without the addition of 1×10⁸ mesenchymal stem cells. Tissue samples were fixed in 4% formaldehyde, processed and embedded in paraffin wax for sectioning. Toluidine Blue staining was performed to help visualize the presence of mitotic figures. A.) Mitotic figures after EMS perfusion. B. Mitotic figures after EMS with the addition of MSC had a 26% increase in prevalence in comparison to the paired controlled kidneys that were warm perfused alone (p<0.05) (n=5 pairs).