Bryostatin-1 Attenuates Ischemia-Elicited Neutrophil Transmigration and Ameliorates Graft Injury after Kidney Transplantation

Abstract

Ischemia reperfusion injury (IRI) is a form of sterile inflammation whose severity determines short- and long-term graft fates in kidney transplantation. Neutrophils are now recognized as a key cell type mediating early graft injury, which activates further innate immune responses and intensifies acquired immunity and alloimmunity. Since the macrolide Bryostatin-1 has been shown to block neutrophil transmigration, we aimed to determine whether these findings could be translated to the field of kidney transplantation. To study the effects of Bryostatin-1 on ischemia-elicited neutrophil transmigration, an in vitro model of hypoxia and normoxia was equipped with human endothelial cells and neutrophils. To translate these findings, a porcine renal autotransplantation model with eight hours of reperfusion was used to study neutrophil infiltration in vivo. Graft-specific treatment using Bryostatin-1 (100 nM) was applied during static cold storage. Bryostatin-1 dose-dependently blocked neutrophil activation and transmigration over ischemically challenged endothelial cell monolayers. When applied to porcine renal autografts, Bryostatin-1 reduced neutrophil graft infiltration, attenuated histological and ultrastructural damage, and improved renal function. Our novel findings demonstrate that Bryostatin-1 is a promising pharmacological candidate for graft-specific treatment in kidney transplantation, as it provides protection by blocking neutrophil infiltration and attenuating functional graft injury.

Keywords: Bryostatin-1; ischemia reperfusion injury; kidney transplant; translational research.

Figure 1

Experimental outline. (A) An in vitro model of hypoxia and normoxia was used. For this, transwell systems were equipped with human umbilical vein endothelial cell (HUVEC) monolayers and treated with varying concentrations of Bryostatin-1 (1, 10, 100 nM) or a vehicle control. Next, HUVECs were incubated for 20 h at 37° under hypoxic (2% O₂, 5% CO₂, and 73% N₂) conditions (mimicking the ischemic phase in the ischemia reperfusion sequence), followed by 3 h under normoxic (21% O₂, 5% CO₂, and 74% N₂) conditions (mimicking the early reperfusion phase in the ischemia reperfusion sequence). Control cells were kept under normoxic conditions. Some experiments were conducted under hypothermia (4°); (B) Human neutrophils were obtained from healthy volunteers (CTR) or sickle cell patients (SCD), a disease known for its endogenous neutrophil activation; (C) To study endothelial barrier function, control and ischemically injured HUVEC monolayers were kept in transwell systems, and FITC-dextran (either 10, 40, or 70 kD) was added to the upper chamber. After 1 h incubation, FITC-dextran concentrations in the lower chambers were measured using a plate reader; (D) To study neutrophil trans-endothelial migration, control and ischemically injured HUVEC monolayers were kept in transwell systems, calcein-AM labeled neutrophils were added to the upper chamber, and the chemoattractant leukotriene B₄ (or vehicle control) was added to the lower chamber. After 3 h incubation, calcein fluorescence intensity was measured, and the migration index was calculated by dividing the number of neutrophils migrating toward LTB₄ by the number of cells migrating to the vehicle. Subsets of experiments were conducted with neutrophils from SCD patients or after pre-treatment with tumor necrosis factor alpha; (E) To study neutrophil activation, control and ischemically injured HUVEC monolayers were kept in transwell systems, and unlabeled neutrophils were added to the upper chamber, and the chemoattractant leukotriene B₄ (or vehicle control) was added to the lower chamber. After 3 h incubation, MPO activity was measured in the transmigrated neutrophils (this figure was created with BioRender.com, accessed on 25 February 2022).

Figure 2

Bryostatin-1 prevents hypoxia-elicited endothelial permeability and blocks neutrophil transmigration. Confluent human umbilical vein endothelial cell (HUVEC) monolayers were subjected to either normoxia (21% O₂, 5% CO₂, and 74% N₂) or hypoxia (2% O₂, 5% CO₂, and 73% N₂) followed by normoxia, and (A) FITC-dextran (10 and 40 kDa) permeability was measured. Hypoxia induced a significant increase in HUVEC permeability for FITC, irrespective of the molecular size. (B) HUVEC monolayers were treated with 100 nM Bryostatin-1 or vehicle (1 X PBS), and permeability for FITC-dextran (10, 40, and 70 kDa) was tested under (B) normoxic or (C) hypoxic conditions. (D) Neutrophil transmigration across confluent HUVEC monolayers toward leukotriene B4 (LTB₄, 10⁻⁶ M) was measured under normoxic and hypoxic conditions, showing a hypoxia-elicited increase in neutrophil transmigration. (E) Treatment with Bryostatin-1 (100 nM) blocked neutrophil transmigration under normoxic and hypoxic conditions. The migration index was calculated by dividing the number of calcein-AM labeled neutrophils that migrated to LTB₄ by the number of cells that migrated to the vehicle. All results are representative of at least three independent experiments (n = 3–7). Graphs present mean ± SEM, and data were analyzed with ANOVA with Bonferroni post-tests (A–C,E) or Student’s t-test (D). Significance is indicated by the following symbols: (A,D) **** p < 0.0001 and *** p < 0.001 versus the respective normoxia group; (E) #### p < 0.0001 versus vehicle control.

Figure 3

Dose-dependent effects of Bryostatin-1 in altering neutrophil activation and transmigration. Bryostatin-1 (Bryo) showed a dose-dependent (1, 10, 100 nM) effect on neutrophil activation as well as transmigration across confluent human umbilical vein endothelial cell (HUVEC) monolayers toward leukotriene B4 (LTB₄, 10⁻⁶ M) under (A,C) hypoxic and (B,D) normoxic conditions. The migration index was calculated by dividing the number of calcein-AM labeled neutrophils that migrated to LTB₄ by the number of cells that migrated to the vehicle. Neutrophil activation was determined by measuring myeloperoxidase (MPO) activity. All results are representative of at least three independent experiments (n = 5–7). Graphs present mean ± SEM, data were analyzed with ANOVA with Bonferroni post-tests, and significance is indicated by the following symbols: * p < 0.05 versus vehicle control, *** p < 0.001 versus vehicle control, **** p < 0.0001 versus vehicle control.

Figure 4

Bryostatin-1 reduces neutrophil transmigration under clinically relevant conditions. (A,B) Bryostatin-1 (Bryo, 100 nM) blocked hypoxia-elicited transmigration of stimulated neutrophils across confluent human umbilical vein endothelial cell (HUVEC) monolayers toward leukotriene B4 (LTB₄, 10⁻⁶ M). (A) Neutrophils were stimulated in vitro by a 30-min treatment with tumor necrosis factor alpha (TNF-α). (B) Neutrophils were obtained from patients with sickle cell disease (SCD) and compared to neutrophils from control (CTR, healthy donors). (C,D) Bryostatin-1 (100 nM) remained effective in blocking hypoxia-elicited neutrophil transmigration across confluent HUVECs monolayers towards LTB₄ even when (C) Bryostatin-1 was suspended in organ preservation solution (University of Wisconsin solution) or when (D) experiments were conducted under hypothermic conditions (4°). The migration index was calculated by dividing the number of calcein-AM labeled neutrophils that migrated to LTB₄ by the number of cells that migrated to the vehicle. All results are representative of at least three independent experiments (n = 3–7). Graphs present mean ± SEM. Data were analyzed with ANOVA with Bonferroni post-tests (A,B) or Student’s t-test (C,D). Significance is indicated by the following symbols: * p < 0.05 versus vehicle control, ** p < 0.01 versus vehicle control, **** p < 0.0001 versus vehicle control.

Figure 5

Bryostatin-1 acts by altering neutrophil–endothelial cell crosstalk pathways, rather than directly affecting the vascular endothelium. Confluent human umbilical vein endothelial cell (HUVEC) monolayers were treated with Bryostatin-1 (100 nM) or vehicle (1 X PBS), subjected to hypoxia or normoxia, followed by the addition of calcein-AM labeled neutrophils and leukotriene B₄ (LTB₄, 10⁻⁶ M). After three hours, neutrophils were removed, FITC-dextran (70 kDa) was added, and permeability was measured. Bryostatin-1 significantly reduced permeability for FITC-dextran. The migration index was calculated by dividing the number of calcein-AM labeled neutrophils that migrated to LTB₄ by the number of cells that migrated to the vehicle. All results are representative of at least three independent experiments (n = 7). Graphs present mean ± SEM, data were analyzed with ANOVA with Bonferroni post-tests, and significance is indicated by the following symbols: *** p < 0.0001 versus the respective normoxia group; ## p < 0.001 versus vehicle control.

Figure 6

Bryostatin-1 attenuates neutrophil transmigration in renal autografts. All analyses were conducted eight hours after reperfusion in a porcine model of renal autotransplantation with graft-specific treatment with placebo or Bryostatin-1 during 20-h static cold storage of the renal autografts. (A) Myeloperoxidase (MPO) activity (in units per gram renal tissue) in renal autografts was measured as an index for tissue neutrophil content and was significantly reduced in Bryostatin-1 treated renal autografts. (B) Systemic protein concentration (pg/mg) of interleukin-8 (IL-8) was significantly reduced in Bryostatin-1 treated animals, while (C) protein concentration (pg/mg) of tumor necrosis factor alpha (TNF-α) was comparable in placebo and Bryostatin-1 groups. To correlate systemic protein levels with local tissue changes, gene expression of (D) IL-8 and (E) TNF-α was analyzed in renal biopsies. Gene expression of cellular adhesion molecules (platelet endothelial cell adhesion molecule-1 (PECAM-1) (F) and intercellular adhesion molecule 1 (ICAM-1) (G)) was tested and found to be comparable between groups. All data are presented as mean values ± SEM from 6 (placebo) or 7 (Bryostatin-1) individually analyzed animals per group. Data were analyzed with Student’s t-test, and significance is indicated by the following symbol: * p < 0.05 versus placebo.

Figure 7

Protective effect of Bryostatin-1 following renal autotransplantation. All analyses were conducted eight hours after reperfusion in a porcine model of renal autotransplantation with graft-specific treatment with placebo or Bryostatin-1 during 20-h static cold storage of renal autografts. (A–D) Representative histopathologic images of hematoxylin and eosin (H&E), as well as periodic acid-Schiff (PAS), stained renal biopsies after eight hours of reperfusion: Samples from placebo-treated renal autografts (A,C) display striking glomerular shrinkage, distinct inflammatory cell infiltrates (arrowheads), and extensive tubular damage (arrows and (C) right panel), all of which were markedly reduced in the Bryostatin-1 group (B,D). g: glomeruli, bars: 50 µm. Ultrastructural injury score (consisting of structural integrity of glomeruli, basement membrane, podocytes, and endothelium) was significantly improved in Broystatin-1 treated renal autografts eight hours of reperfusion. Representative electron micrographs of (E–H) placebo and (I–L) Bryostatin-1 treated renal autografts: (E) Transmission electron microscopy in placebo treated renal autografts reveals ultrastructural changes of the endothelium (em) with massive disruptions of the endothelial integrity. Note that the endothelial fenestrations are not distinguishable due to tissue disintegration. In addition, (F,G) podocytes (po) and foot processes (f) showed irregular contours with destruction and flattening of the foot processes. As seen in (H), the basement membrane (bm) shows an irregular thickness. In comparison to control samples (placebo), (I) capillaries from Bryostatin-1 treated samples revealed an intact endothelium with (J,K) regular contours and multiple clearly distinguishable fenestrations. Podocytes showed a normal morphology with an intact cytoplasm and clearly visible organelles. (L) Foot processes with slit diaphragms and basement membranes reveal all characteristics of a normal glomerular filtration unit. Scale bar for E, G, I, J, K 500 nm, for F, H, 200 nm and for L 1 µm; c = capillary lumen, ery = erythrocyte, u = urinary space, arrow = slit diaphragms; arrowhead = fenestration.

Figure 8

Bryostatin-1 protects renal autografts from ischemia-reperfusion injury and improves kidney function following renal autotransplantation. All analyses were conducted eight hours after reperfusion in a porcine model of renal autotransplantation with graft-specific treatment with placebo or Bryostatin-1 during 20-h static cold storage of renal autografts. (A) Histological injury score (consisting of glomerular damage (shrinking), inflammatory cell infiltrates, edema, and tubular damage) was significantly reduced in Broystatin-1-treated renal autografts. (B) Ultrastructural injury score (consisting of structural integrity of glomeruli, basement membrane, podocytes, and endothelium) was significantly improved in Broystatin-1 treated renal autografts eight hours of reperfusion. Urine levels of (C) kidney injury molecule-1 (KIM-1) and (D) neutrophil gelatinase-associated lipocalin (NGAL) were measured eight hours after reperfusion in a porcine model of renal autotransplantation with graft-specific treatment with placebo or Bryostatin-1 during 20-h static cold storage of renal autografts and found to be comparable between the two groups. (E) Plasma levels of Cystatin C were measured after 8 h of reperfusion, with a non-significant reduction in animals receiving Bryostatin-1 treated renal autografts. (F) When the individual delta (difference between pre-transplant and eight-hour reperfusion sample) was calculated, Bryostatin-1 elicited a significant reduction in ischemia-associated rise in plasma Cystatin C. All data are presented as mean values ± SEM from 6 (placebo) or 7 (Bryostatin-1) individually analyzed animals per group. Data were analyzed with Student’s t-test, and significance is indicated by the following symbols: * p < 0.05 versus placebo, ** p < 0.01 versus placebo, *** p < 0.001 versus placebo.