Pulsed focused ultrasound pretreatment improves mesenchymal stromal cell efficacy in preventing and rescuing established acute kidney injury in mice

Abstract

Animal studies have shown that mesenchymal stromal cell (MSC) infusions improve acute kidney injury (AKI) outcomes when administered early after ischemic/reperfusion injury or within 24 hours after cisplatin administration. These findings have spurred several human clinical trials to prevent AKI. However, no specific therapy effectively treats clinically obvious AKI or rescues renal function once advanced injury is established. We investigated if noninvasive image-guided pulsed focused ultrasound (pFUS) could alter the kidney microenvironment to enhance homing of subsequently infused MSC. To examine the efficacy of pFUS-enhanced cell homing in disease, we targeted pFUS to kidneys to enhance MSC homing after cisplatin-induced AKI. We found that pFUS enhanced MSC homing at 1 day post-cisplatin, prior to renal functional deficits, and that enhanced homing improved outcomes of renal function, tubular cell death, and regeneration at 5 days post-cisplatin compared to MSC alone. We then investigated whether pFUS+MSC therapy could rescue established AKI. MSC alone at 3 days post-cisplatin, after renal functional deficits were obvious, significantly improved 7-day survival of animals. Survival was further improved by pFUS and MSC. pFUS prior to MSC injections increased IL-10 production by MSC that homed to kidneys and generated an anti-inflammatory immune cell profile in treated kidneys. This study shows pFUS is a neoadjuvant approach to improve MSC homing to diseased organs. pFUS with MSC better prevents AKI than MSC alone and allows rescue therapy in established AKI, which currently has no meaningful therapeutic options.

Keywords: Acute kidney injury; Cellular therapy; Cisplatin; Kidney; Mesenchymal stromal cells; Renal; Renal failure; Stem cell transplantation.

Figure 1. Schematic of enhanced homing permeability and retention (EHPR) of MSC by pFUS and pFUS-induced EHPR of MSC during AKI

A) pFUS interacts with the parenchyma, interstitium and vasculature (Left panel). Levels of vascular integrins (ICAM and VCAM) and chemoattractants (soluble cytokines, chemokines, and trophic factors) increase in the treated volume (Center panel). Intravascularly injected stem cells (e.g. MSC) tether to cell adhesion molecules expressed on endothelial cell surfaces and extravasate in response to gradients of chemoattractants (Right panel). Schematic not shown to scale. B) Experimental timeline showing treatment schedules for two cohorts of mice. All mice received cisplatin on D0 (n=5 mice/group). On either D1 (black solid lines) or D4 (red dashed lines), mice received unilateral pFUS followed by i.v. human MSCs 4h later. Mice were euthanized 24h post-injection and MSC homing to pFUS-treated and untreated contralateral kidneys was quantified by immunofluorescence for human mitochondria. C) pFUS induced significantly greater MSC homing to kidneys on either D1 or D4. MSC homing to pFUS-treated kidneys was ~2-fold greater on either day compared to untreated kidneys on the same day. Significantly greater MSC homing was observed to contralateral kidneys on D4 compared to D1, possibly reflecting greater levels of signaling molecules shown in Fig 3A. Homing for all groups was compared by ANOVA (p<0.05) where like symbols indicate statistical similarity and different symbols indicate statistical differences (FOV; field of view). D) Representative human mitochondrial staining. Immunofluorescence imaging (10×) shows human MSCs in pFUS and control kidneys E) A high-magnification view of human mitochondrial staining (100×). MSCs are localized to peri-tubular spaces.

Figure 2. Kidney profile of cytokines, chemokines, growth factors, and integrins in kidneys during AKI with and without pFUS

A) Heat map showing fold changes in kidney signaling molecules over time during AKI (no pFUS, no MSC). Days indicated are time points post-cisplatin and “normal” represents saline-treated animals (no cisplatin). Few increases are detected at D1 and trends of sub-physiological expression for many factors are observed. Trends of increased expression of many factors are evident by D3 and persist until D5 (n=6 mice/group). B) Experimental timeline for C and D showing two cohorts of mice that received cisplatin on D0 and then pFUS either on D1 (black solid line) or D4 (red dashed line). C) pFUS-induced changes in kidney signaling molecules following unilateral pFUS on D1 or D4 (no MSC). Mice from both days were euthanized 4h post-pFUS (n=6 mice/group). Tissue levels in pFUS-treated kidneys were analyzed by multiplex ELISA and compared to untreated contralateral kidneys on the same day by t-tests (* p<0.05). Significant changes in molecular expression were evident following pFUS on either day—most factors increased following pFUS, but decreases were also observed for some. D) Changes in SDF-1α, ICAM, and VCAM expression during AKI and effects of pFUS. pFUS-treated and contralateral control AKI kidneys (no pFUS) from the indicated day, in addition to normal kidneys (n=6 mice/group), were analyzed by single-plex ELISA. Data for each group were compared to all other groups by ANOVA (* p<0.05). Control AKI kidneys (no pFUS) showed elevations in SDF-1α above normal kidneys at D1 and D4, but no additional increases were observed following pFUS of AKI kidneys. ICAM in control AKI kidneys was not elevated compared to normal kidneys on D1, but pFUS induced greater D1 ICAM expression. ICAM in control AKI kidneys was significantly greater than normal kidneys on D4, but pFUS failed to induce further increases. VCAM was not increased in control AKI kidneys at either day compared to normal kidneys, but pFUS at either day increased VCAM in AKI kidneys.

Figure 3. pFUS alone (no MSCs) does not alter AKI outcomes

A) Experimental timeline. Mice were given cisplatin on D0 and then either bilateral pFUS or sham pFUS (transducer power=0 W) on D1 (n=6 mice per group). Mice were euthanized on D4 and AKI was assessed by B) BUN and SCr levels, C) histological features such as necrotic tubules and tubular casts, and D) apoptosis (TUNEL) regeneration (pAKT), and proliferation (Ki67). These were unchanged by pFUS alone. E) Representative images for renal histology, TUNEL and Ki67 staining. Measurements between pFUS-treated and control kidneys were compared by t-tests and found not to be significant (p>0.05).

Figure 4. Coupling pFUS and MSC during early AKI leads to less severe disease

A) Experimental timeline. Normal mice, control AKI mice, and AKI mice treated with pFUS and/or MSC received appropriate treatment on D1 and were euthanized for analyses on D4. B) MSC with pFUS on D1 leads to better MSC retention in pFUS-treated kidneys at D4 compared to kidneys that received MSC alone (* p<0.05 following t-test). C) Coupling pFUS with MSC (Cis/pFUS/MSC) on D1 leads to better renal function at D4. BUN is significantly decreased by MSC alone and further decreased by pFUS with MSC. SCr is not improved by MSC alone, but is improved by pFUS+MSC. Groups with like symbols are statistically similar to each other and statistically different from groups with different symbols after ANOVA (p<0.05) (n=5–7 mice/group). D and E) Molecular and histological profiles of kidneys on D4 after pFUS and/or MSC on D1. Statistical summaries are shown in D, while representative microscopic images are shown in E. By H&E, the number of necrotic tubules was significantly decreased following MSC alone and further significantly decreased by pFUS+MSC, however no improvement in tubular cast number was observed for either group. pAKT and Ki67 expression were significantly increased only after pFUS with MSC. The number of apoptotic cells (red arrows) was reduced by MSC alone and further reduced by pFUS and MSC. Groups with like symbols are statistically similar to each other and statistically different from groups with different symbols after ANOVA (p<0.05) (n=5–7 mice/group).

Figure 5. Treating AKI with MSC alone after functional deficits manifest improves survival and coupling pFUS and MSC further improves survival and renal function

A) Experimental timeline. pFUS and/or MSC treatment was administered at D3 with renal functional measurements at D4 and D6. B) Kaplan-Meier curves of survival following treatment at D3. Survival is significantly increased following MSC alone (Cis/MSC) compared to untreated mice (Cis) (p=0.0292), while pFUS+MSC (Cis/pFUS/MSC) yields better survival compared to MSC alone (p=0.0247) or untreated mice (p<0.0001) using log-rank tests (n=14 mice/group). C) Renal function (BUN and SCr) measurements at D4 (24h post-treatment). BUN significantly improved after pFUS with MSC. The number of mice surviving to the measurement time point is indicated in boxes above each bar. Groups with like symbols are statistically similar to each other and statistically different from groups with different symbols after ANOVA (p<0.05). D) Renal function (BUN and SCr) measurements at D6 (72h post-treatment). Significant improvements in BUN clearance persist to D6 only after pFUS+MSC. The number of mice surviving to the measurement time point is indicated above each bar. Variance of the “Cis” group in panel D is omitted because n=2. Groups with like symbols are statistically similar to each other and statistically different from groups with different symbols after ANOVA (p<0.05).

Figure 6. Effects of MSCs with or without pFUS on immune cell populations and blood vessel density

The experimental timeline was Cisplatin on D0, pFUS and/or MSC on D1, euthanasia on D2. A) Statistical summaries of immune cell populations and blood vessels following treatment. CD11c and CD206 expression F4/80-positive cells. Macrophages appear more M2-like after MSC alone or pFUS+MSC (top row). CD11b expressed by natural killer (NK) cells and is reduced by MSC alone and further reduced by pFUS+MSC. Non-macrophage CD11c (e.g., on dendritic cells [DC]) is unchanged by MSC alone, but reduced following pFUS+MSC (middle row). CD31 expressed by endothelial cells is not significantly increased by MSC alone, but is significantly increased by pFUS+MSC (bottom row). Groups with like symbols are statistically similar to each other and statistically different from groups with different symbols after ANOVA (p<0.05) (n=4 mice per group) B) Representative IHC staining of AKI kidneys. The top row shows F4/80 (all macrophages; blue), CD11c (M1 macrophages; green), and CD206 (M2 macrophages; red) staining. The second row shows CD11b (red) expressed on natural killer (NK) cells. The third row shows non-macrophage CD11c (red) expressed by dendritic cells (DC) and the fourth row shows CD31 (red) expressed by endothelial cells.

Figure 7. Changes in human and mouse cytokines in the kidney and serum following MSC treatment or combination MSC and pFUS treatment

A) Experimental timeline showing treatment and euthanasia (n=6 mice per group). B) Greater human IL-10 produced by human MSC is present in the kidney following pFUS+MSC. Red line indicates assay signal from control AKI mice (no MSCs) that corresponds to background levels of signal. C) Lower levels of mouse TNFa and increased levels of mouse VEGF are present in mouse kidney following pFUS+MSC. D) Increased mouse VEGF is observed in the serum following pFUS+MSC.