Reversing Acute Kidney Injury Using Pulsed Focused Ultrasound and MSC Therapy: A Role for HSP-Mediated PI3K/AKT Signaling

Abstract

Acute kidney injury (AKI) is characterized by a sudden failure of renal function, but despite increasing worldwide prevalence, current treatments are largely supportive, with no curative therapies. Mesenchymal stromal cell (MSC) therapy has been shown to have a promising regenerative effect in AKI but is limited by the ability of cells to home to damaged tissue. Pulsed focused ultrasound (pFUS), wherein target tissues are sonicated by short bursts of sound waves, has been reported to enhance MSC homing by upregulating local homing signals. However, the exact mechanism by which pFUS enhances MSC therapy remains insufficiently explored. In this study, we studied the effect of bone marrow-derived MSCs (BM-MSCs), in conjunction with pFUS, in a mouse model of cisplatin-induced AKI. Here, BM-MSCs improved kidney function, reduced histological markers of kidney injury, decreased inflammation and apoptosis, and promoted cellular proliferation. Surprisingly, whereas pFUS did not upregulate local cytokine expression or improve BM-MSC homing, it did potentiate the effect of MSC treatment in AKI. Further analysis linked this effect to the upregulation of heat shock protein (HSP)20/HSP40 and subsequent phosphatidylinositol 3-kinase (PI3K)/Akt signaling. In summary, our results suggest that pFUS and BM-MSCs have independent as well as synergistic therapeutic effects in the context of AKI.

Keywords: acute kidney injury; apoptosis; focused ultrasound; heat shock protein; inflammation; kidney regeneration; mesenchymal stromal cell; signaling.

Graphical abstract

Figure 1

Physical Parameters and Biochemistry of AKI Mice following Cisplatin Injection (A) Animal body weight. (B) Plasma creatinine levels in animals. (C) Blood urea nitrogen levels in animals. Each group has n = 6, except in AKI, where n = 5. Error bars represent standard deviations. Significant differences include ^ap < 0.05 relative to untreated control; ^bp < 0.05 relative to AKI; ^cp < 0.05 relative to AKI + BM-MSCs; ^dp < 0.05 relative to AKI + BM-MSCs + pFUS; ∗p < 0.05 all time points relative to day 0.

Figure 2

Homing of BM-MSCs (A) Homing of BM-MSCs, shown by green due to expression of GFP in kidney sections. Each group has n = 6, except AKI, where n = 5 mice, 20 images per animal, was taken. (B) Quantification of GFP signal by flow cytometry in kidney homogenized cells. Each group has n = 6, except in AKI, where n = 5. (C) PCR showing the presence of human GAPDH in treated kidneys. Each group has n = 5 mice. (D) Cytokine expression within kidney lysate samples treated with pFUS, measured 2 days postsonication. Each group has n = 2 mice. Scale bar, 100 μm. Error bars represent standard deviations. Significant differences include ^ap < 0.05 relative to untreated control; ^bp < 0.05 relative to AKI; ^cp < 0.05 relative to AKI + BM-MSCs; ^dp < 0.05 relative to AKI + BM-MSCs + pFUS.

Figure 3

Histological Analysis of Kidneys from Untreated and Treated AKI Mice (A) Hematoxylin and eosin staining showing the different level of injury, cast formation, and fibrosis across different experimental groups. Scale bars, 100 μm. Each group has n = 6, except AKI, where n = 5, average of 20 images per animal, was taken. (B) Quantification of histological data. Each group has n = 6, except in AKI, where n = 5. Error bars represent standard deviations. Significant differences include ^ap < 0.05 relative to untreated control; ^bp < 0.05 relative to AKI; ^cp < 0.05 relative to AKI + BM-MSCs; ^dp < 0.05 relative to AKI + BM-MSCs + pFUS.

Figure 4

Immunohistochemistry and Assessment of Inflammation in Kidneys from Untreated and Treated AKI Mice (A) Immunohistochemical staining of inflammatory markers TNF-α, IL-6, and MCP-1 in AKI mice, which are untreated or treated with BM-MSCs alone or BM-MSCs + pFUS. (B) Serum cytokine measurement of inflammatory markers TNF-α, IL-6, and MCP-1 in AKI mice, which are untreated or treated with BM-MSCs alone or BM-MSCs + pFUS. (C) qPCR, gene expression of inflammatory markers TNF-α, IL-6, and IGF-1 in AKI mice, which are untreated or treated with BM-MSCs alone or BM-MSCs + pFUS. (D) Relative lactate dehydrogenase (LDH) expression compared to isotype control measured by flow cytometry in AKI mice, which are untreated or treated with BM-MSCs alone or BM-MSCs + pFUS. Scale bars, 100 μm. Each group has n = 6, except in AKI, where n = 5. Error bars represent standard deviations. Significant differences include ^ap < 0.05 relative to untreated control; ^bp < 0.05 relative to AKI; ^cp < 0.05 relative to AKI + BM-MSCs; ^dp < 0.05 relative to AKI + BM-MSCs + pFUS.

Figure 5

Apoptosis, Cell Death, and Protein Arrays in Kidneys from Untreated and Treated AKI Mice (A) Western blot showing the expression of BAX, PARP, Bcl-2, CASP3, and β-actin. (B) qPCR showing the expression of BAX, PARP, Bcl-2, and CASP3, normalized to GAPDH. (C) Western blot showing the expression of p-ERK1/2, p-AKT, and p-AMPK (targets of HSP20). (D) Quantification of western blot showing the expression of p-ERK1/2, p-AKT, and p-AMPK (targets of HSP20). Each group has n = 6, except in AKI, where n = 5. Error bars represent standard deviations. Significant differences include ^ap < 0.05 relative to untreated control; ^bp < 0.05 relative to AKI; ^cp < 0.05 relative to AKI + BM-MSCs; ^dp < 0.05 relative to AKI + BM-MSCs + pFUS.

Figure 6

Protein Arrays to Identify the Mechanistic Targets in Kidneys from Untreated and Treated AKI Mice (A) Quantification of protein arrays showing the comparative expression of various heat shock proteins taken from the protein array data. (B) Blot of protein arrays showing the expression of various heat shock proteins in the form of expression dots in the AKI mice group compared to treatment with BM-MSCs alone or BM-MSCs + pFUS. (C) Validation and quantification of heat shock protein by western blot, showing the expression of HSP20 in the AKI mice group compared to treatment with BM-MSCs alone or BM-MSCs + pFUS. Each group has n = 6, except in AKI, where n = 5. Error bars represent standard deviations. Significant differences include ^ap < 0.05 relative to untreated control; ^bp < 0.05 relative to AKI; ^cp < 0.05 relative to AKI + BM-MSCs; ^dp < 0.05 relative to AKI + BM-MSCs + pFUS.

Figure 7

Validation of HSP20 and Its Targets in Cultured Human Embryonic Kidney 293 (HEK293) Cells and HEK293-siRNA HSP20-Knockdown Cells (A) Western blot of HSP20 and HSP40 in cultured normal HEK293 cells, and HEK293-siRNA HSP20-knockdown cells. (B) Western blot of p-AKT in cultured HEK293 cells and HEK293-siRNA HSP20-knockdown cells. (C) qRT-PCR of HSP20, HSP40, and p-AKT in HEK293 cells and HEK293-siRNA HSP20-knockdown cells. (D) Beta-galactosidase staining, blue showing cell senescence in cultured normal HEK293 cells and HEK293-siRNA HSP20-knockdown cells. Error bars represent standard deviations. Significant difference includes ^ap < 0.05 HEK293 versus HEK293-siRNA cells.

Figure 8

Effects of pFUS on HEK293 Cells (A) Western blot of HSP20 and HSP40 in cultured HEK293 cells, untreated or induced with pFUS. (B) RT-PCR of HSP20 in cultured HEK293 cells, untreated or induced with pFUS. (C) Western blot of p-AKT in cultured HEK293 cells, untreated or induced with pFUS.