Effect of Everolimus versus Bone Marrow-Derived Stem Cells on Glomerular Injury in a Rat Model of Glomerulonephritis: A Preventive, Predictive and Personalized Implication

Abstract

Glomerular endothelial injury and effectiveness of glomerular endothelial repair play a crucial role in the progression of glomerulonephritis. Although the potent immune suppressive everolimus is increasingly used in renal transplant patients, adverse effects of its chronic use have been reported clinically in human glomerulonephritis and experimental renal disease. Recent studies suggest that progenitor stem cells could enhance glomerular endothelial repair with minimal adverse effects. Increasing evidence supports the notion that stem cell therapy and regenerative medicine can be effectively used in pathological conditions within the predictive, preventive and personalized medicine (PPPM) paradigm. In this study, using an experimental model of glomerulonephritis, we tested whether bone marrow-derived stem cells (BMDSCs) could provide better effect over everolimus in attenuating glomerular injury and improving the repair process in a rat model of glomerulonephritis. Anti-Thy1 glomerulonephritis was induced in male Sprague Dawley rats by injection of an antibody against Thy1, which is mainly expressed on glomerular mesangial cells. Additional groups of rats were treated with the immunosuppressant everolimus daily after the injection of anti-Thy1 or injected with single bolus dose of BMDSCs after one week of injection of anti-Thy1 (n = 6-8). Nine days after injection of anti-Thy1, glomerular albumin permeability and albuminuria were significantly increased when compared to control group (p < 0.05). Compared to BMDSCs, everolimus was significantly effective in attenuating glomerular injury, nephrinuria and podocalyxin excretion levels as well as in reducing inflammatory responses and apoptosis. Our findings suggest that bolus injection of BMDSCs fails to improve glomerular injury whereas everolimus slows the progression of glomerular injury in Anti-Thy-1 induced glomerulonephritis. Thus, everolimus could be used at the early stage of glomerulonephritis, suggesting potential implications of PPPM in the treatment of progressive renal injury.

Keywords: BMDSCs; anti-Thy1; apoptosis; everolimus; glomerular injury; renal inflammation.

Figure 1

Assessment of apoptosis (caspase 3) and necrosis (7-AAD) in glomeruli isolated from control and anti-Thy1-injected rats (1 mg/kg i.v.). Representative of flow cytometry analysis of apoptosis (caspase 3) and necrosis (7-AAD) in two-dimensional dot plot (A) and in histogram (B) in a comparative mode based on counted total cells. Injection of anti-Thy1 produced 9-fold increase in glomerular apoptosis vs. control.

Figure 2

Glomerular permeability to albumin (A) and albuminuria (B) in control and anti-Thy1-injected rats +/− everolimus (2 mg/kg daily) or single-dose administration of BMDSCs (10⁶ cells/200 µL of PBS, i.v.) after a week of injection of anti-Thy1. Only everolimus treatment significantly reduced the elevation in glomerular permeability to albumin and albuminuria in anti-Thy1-injected rats (n = 6–8, * vs. control and ^≠ vs. anti-Thy1-injected rats).

Figure 3

Representative H&E staining images for the kidney section (A) and creatinine clearance (B) from control and anti-Thy1-injected rats with or without everolimus or BMDSC treatment. Anti-Thy1-injected rats showed hypercellular glomeruli with loss of Bowman space and variable number of lymphocytes. Only everolimus treatment partially restored these pathophysiological changes (n = 4). There was no difference in creatinine clearance among rat groups (n = 6–8).

Figure 4

Representative images of immunohistochemical staining of glomerular nephrin expression in kidney sections (A), urinary nephrin excretion (B) and podocalyxin excretion (C) levels in control and anti-Thy-1-injected rats +/− everolimus or BMDSC treatment. Anti-Thy-1 injection markedly increased the loss of glomerular nephrin, nephrinuria and podocalyxin excretion. Only everolimus lowered nephrin and podocalyxin excretion levels as markers of glomerular injury and restored the loss of glomerular nephrin expression in anti-Thy1-injected rats (n = 6–8, * vs. control and ^≠ vs. anti-Thy1-injected rats).

Figure 5

Urinary TBARs excretion (A) as a marker of oxidative stress and MCP-1 excretion as a marker of inflammation (B) in control and anti-Thy1 rats with or without everolimus or BMDSC treatment. Only everolimus treatment significantly reduced the elevation in urinary TBARs and MCP-1 excretion levels in anti-Thy1-injected rats (n = 6–8, * vs. control and ^≠ vs. anti-Thy1-injected rats).

Figure 6

Representative flow cytometry plots (A) and % of total renal cells expressing CD3+ (B), IL-17+ (C) and CD68+ (D) as markers of naïve T cells, T helper cells and macrophages, respectively, in control and anti-Thy1-injected rats with or without everolimus or BMDSC treatment. Everolimus treatment prevented the elevation in T cells and IL-17 expression in anti-Thy1-injected rats. Everolimus provided better effect than BMDSCs in preventing the increase in renal macrophage infiltration in anti-Thy1-injected rats (n = 6, * vs. control, ^≠ vs. anti-Thy1-injected rats and ^λ vs. everolimus-treated rats).

Figure 7

Representative flow cytometry plots (A) and % of total renal cells expressing caspase 3+ (B) as indicative of apoptosis in control and anti-Thy1-injected rats with or without everolimus or BMDSC treatment. Everolimus was superior to BMDSCs in reducing renal caspase 3 expression in anti-Thy1-injected rats (n = 6, * vs. control, ^≠ vs. anti-Thy1-injected rats and ^λ vs. everolimus-treated rats). The result was further verified using TUNEL assay for renal apoptotic cells in the kidney sections from the same rat group (C) where number of renal apoptotic cells increased after anti-Thy1 injection and was only reduced with everolimus treatment.