Kidney histopathology in lethal human sepsis.

PURPOSE: The histopathology of sepsis-associated acute kidney injury (AKI) in critically ill patients remains an understudied area. Previous studies have identified that acute tubular necrosis (ATN) is not the only driver of sepsis-AKI. The focus of this study was to identify additional candidate processes that may drive sepsis-AKI. To do this we immunohistochemically characterized the histopathological and cellular features in various compartments of human septic kidneys. METHODS: We studied the following histopathological features: leukocyte subsets, fibroblast activation, cellular proliferation, apoptosis, and fibrin deposition in the glomerulus and the tubulointerstitium in human post-mortem kidney biopsy tissue. Biopsy tissue samples from 27 patients with sepsis-AKI were collected 33 min (range 24-150) after death in the ICU. The unaffected part of the kidneys from 12 patients undergoing total nephrectomy as a result of renal carcinoma served as controls. RESULTS: Immunohistochemical analysis revealed the presence of more neutrophils and macrophages in the glomeruli and more neutrophils in the tubulointerstitium of renal tissue from patients with sepsis compared to control renal tissue. Type II macrophages were predominant, with some macrophages expressing both type I and type II markers. In contrast, there were almost no macrophages found in control kidneys. The number of activated (myo)fibroblasts was low in the glomeruli of sepsis-AKI kidneys, yet this was not observed in the tubulointerstitium. Cell proliferation and fibrin deposition were more pronounced in the glomeruli and tubulointerstitium of sepsis-AKI than in control kidneys. CONCLUSIONS: The extensive heterogeneity of observations among and within patients emphasizes the need to thoroughly characterize patients with sepsis-AKI in a large sample of renal biopsy tissue from patients with sepsis.

Microsphere-Based Rapamycin Delivery, Systemic Versus Local Administration in a Rat Model of Renal Ischemia/Reperfusion Injury.

PURPOSE: The increasing prevalence and treatment costs of kidney diseases call for innovative therapeutic strategies that prevent disease progression at an early stage. We studied a novel method of subcapsular injection of monodisperse microspheres, to use as a local delivery system of drugs to the kidney. METHODS: We generated placebo- and rapamycin monodisperse microspheres to investigate subcapsular delivery of drugs. Using a rat model of acute kidney injury, subcapsular injection of placebo and rapamycin monodisperse microspheres (monospheres) was compared to subcutaneous injection, mimicking systemic administration. RESULTS: We did not find any adverse effects related to the delivery method. Irrespective of the injection site, a similar low dose of rapamycin was present in the circulation. However, only local intrarenal delivery of rapamycin from monospheres led to decreased macrophage infiltration and a significantly lower amount of myofibroblasts in the kidney, where systemic administration did not. Local delivery of rapamycin did cause a transient increase in the deposition of collagen I, but not of collagen III. CONCLUSIONS: We conclude that therapeutic effects can be increased when rapamycin is delivered subcapsularly by monospheres, which, combined with low systemic concentrations, may lead to an effective intrarenal delivery method.

Epithelial-to-mesenchymal transition in fibrosis: collagen type I expression is highly upregulated after EMT, but does not contribute to collagen deposition.

The hallmark of fibrosis is an accumulation of fibrillar collagens, especially of collagen type I. There is considerable debate whether in vivo type II epithelial-to-mesenchymal transition (EMT) is involved in organ fibrosis. Lineage tracing experiments by various groups show opposing data concerning the relative contribution of epithelial cells to the pool of myofibroblasts. We hypothesized that EMT-derived cells might directly contribute to collagen deposition. To study this, EMT was induced in human epithelial lung and renal cell lines in vitro by means of TGF-ß1 stimulation, and we compared the collagen type I (COL1A1) expression levels of transdifferentiated cells with that of myofibroblasts obtained by TGF-ß1 stimulation of human dermal and lung fibroblasts. COL1A1 expression levels of transdifferentiated epithelial cells appeared to be at least one to two orders of magnitude lower than that of myofibroblasts. This was confirmed at immunohistochemical level: in contrast to myofibroblasts, collagen type I deposition by EMT-derived cells was not or hardly detectable. We postulate that, even when type II EMT occurs in vivo, the direct contribution of EMT-derived cells to collagen accumulation is rather limited.

Reduced Tie2 in Microvascular Endothelial Cells Is Associated with Organ-Specific Adhesion Molecule Expression in Murine Health and Endotoxemia.

Endothelial cells (ECs) in the microvasculature in organs are active participants in the pathophysiology of sepsis. Tyrosine protein kinase receptor Tie2 (Tek; Tunica interna Endothelial cell Kinase) is thought to play a role in their inflammatory response, yet data are inconclusive. We investigated acute endotoxemia-induced changes in the expression of Tie2 and inflammation-associated endothelial adhesion molecules E-selectin and VCAM-1 (vascular cell adhesion molecule-1) in kidneys and lungs in inducible, EC-specific Tie2 knockout mice. The extent of Tie2 knockout in healthy mice differed between microvascular beds, with low to absent expression in arterioles in kidneys and in capillaries in lungs. In kidneys, Tie2 mRNA dropped more than 70% upon challenge with lipopolysaccharide (LPS) in both genotypes, with no change in protein. In renal arterioles, tamoxifen-induced Tie2 knockout was associated with higher VCAM-1 protein expression in healthy conditions. This did not increase further upon challenge of mice with LPS, in contrast to the increased expression occurring in control mice. Also, in lungs, Tie2 mRNA levels dropped within 4 h after LPS challenge in both genotypes, while Tie2 protein levels did not change. In alveolar capillaries, where tamoxifen-induced Tie2 knockout did not affect the basal expression of either adhesion molecule, a 4-fold higher E-selectin protein expression was observed after exposure to LPS compared to controls. The here-revealed heterogeneous effects of absence of Tie2 in ECs in kidney and lung microvasculature in health and in response to acute inflammatory activation calls for further in vivo investigations into the role of Tie2 in EC behavior.

Pattern of tamoxifen-induced Tie2 deletion in endothelial cells in mature blood vessels using endo SCL-Cre-ERT transgenic mice.

Tyrosine-protein kinase receptor Tie2, also known as Tunica interna Endothelial cell Kinase or TEK plays a prominent role in endothelial responses to angiogenic and inflammatory stimuli. Here we generated a novel inducible Tie2 knockout mouse model, which targets mature (micro)vascular endothelium, enabling the study of the organ-specific contribution of Tie2 to these responses. Mice with floxed Tie2 exon 9 alleles (Tie2floxed/floxed) were crossed with end-SCL-Cre-ERT transgenic mice, generating offspring in which Tie2 exon 9 is deleted in the endothelial compartment upon tamoxifen-induced activation of Cre-recombinase (Tie2ΔE9). Successful deletion of Tie2 exon 9 in kidney, lung, heart, aorta, and liver, was accompanied by a heterogeneous, organ-dependent reduction in Tie2 mRNA and protein expression. Microvascular compartment-specific reduction in Tie2 mRNA and protein occurred in arterioles of all studied organs, in renal glomeruli, and in lung capillaries. In kidney, lung, and heart, reduced Tie2 expression was accompanied by a reduction in Tie1 mRNA expression. The heterogeneous, organ- and microvascular compartment-dependent knockout pattern of Tie2 in the Tie2floxed/floxed;end-SCL-Cre-ERT mouse model suggests that future studies using similar knockout strategies should include a meticulous analysis of the knockout extent of the gene of interest, prior to studying its role in pathological conditions, so that proper conclusions can be drawn.

TOF-secondary ion mass spectrometry imaging of polymeric scaffolds with surrounding tissue after in vivo implantation.

Supramolecular polymeric materials are of increasing interest for the use as drug delivery carriers. A thorough insight in the biocompatibility and the degradation of these materials in vivo are of fundamental importance to further their development and application in medical practice. Molecular imaging techniques are powerful tools that enable the elucidation of molecular distributions in and around such polymer implants. A supramolecular polymeric hydrogel was implanted under the renal capsule to study its biocompatibility with TOF-SIMS. This results in a molecular cartography of the polymer implant combined with the cellular signature of the implantation environment. In this experiment, molecular signals are observed from cells that are involved in the biological response to the implant, e.g., macrophages. These molecular signatures are compared with macrophage standards cultured in different polarization environments. On the basis of this comparison, information can be acquired on the various macrophage differentiations that are connected to different stages in the foreign body response. Mass spectrometric imaging techniques offer the opportunity to visualize different histological phenomena in a single experiment without the need for specific immunohistochemical markers. Cellular infiltration into the polymer is visualized, offering a clear view on both biological and polymer features in a single imaging experiment.

Ciclosporin does not influence bone marrow-derived cell differentiation to myofibroblasts early after renal ischemia/reperfusion.

BACKGROUND: Ischemia/reperfusion injury (IRI) is a risk factor for the development of interstitial fibrosis. Previously we had shown that after renal IRI, bone marrow-derived cells (BMDC) can differentiate to interstitial myofibroblasts. Here we hypothesized that the immunosuppressant ciclosporin A (CsA), known for its profibrotic side effect, promotes myofibroblast differentiation of BMDC in the postischemic kidney. METHODS: Using a model of unilateral renal IRI in rats reconstituted with R26-human placental alkaline phosphatase transgenic bone marrow, CsA was administered in a previously defined critical window for differentiation of BMDC to myofibroblasts. We evaluated fibrotic changes in the kidney and myofibroblast differentiation of BMDC on day 14 after CsA treatment. RESULTS: CsA treatment for 14 days led to increased transforming growth factor-beta transcript levels and collagen III deposition in the postischemic kidney. However, neither the total number of alpha-smooth-muscle-actin-positive interstitial myofibroblasts, nor the bone marrow-derived fraction thereof was affected by CsA administration, irrespective of dosage and duration of treatment. CONCLUSIONS: In the critical postischemic window of BMDC differentiation to myofibroblasts, CsA did not promote BMDC differentiation to myofibroblasts, suggesting that, in the clinical setting, CsA is not involved in myofibroblastic differentiation of BMDC.

Early Heterogenic Response of Renal Microvasculature to Hemorrhagic Shock/Resuscitation and the Influence of NF-κB Pathway Blockade.

Hemorrhagic shock (HS) is associated with low blood pressure due to excessive loss of circulating blood and causes both macrocirculatory and microcirculatory dysfunction. Fluid resuscitation after HS is used in the clinic to restore tissue perfusion. The persistent microcirculatory damage caused by HS and/or resuscitation can result in multiple organ damage, with the kidney being one of the involved organs. The kidney microvasculature consists of different segments that possess a remarkable heterogeneity in functional properties. The aim of this study was to investigate the inflammatory responses of these different renal microvascular segments, i.e., arterioles, glomeruli, and postcapillary venules, to HS and resuscitation (HS/R) in mice and to explore the effects of intervention with a nuclear factor-kappa B (NF-κB) inhibitor on these responses. We found that HS/R disturbed the balance of the angiopoietin-Tie2 ligand-receptor system, especially in the glomeruli. Furthermore, endothelial adhesion molecules, proinflammatory cytokines, and chemokines were markedly upregulated by HS/R, with the strongest responses occurring in the glomerular and postcapillary venous segments. Blockade of NF-κB signaling during the resuscitation period only slightly inhibited HS/R-induced inflammatory activation, possibly because NF-κB p65 nuclear translocation already occurred during the HS period. In summary, although all three renal microvascular segments were activated upon HS/R, responses of endothelial cells in glomeruli and postcapillary venules to HS/R, as well as to NF-κB inhibition were stronger than those in arterioles. NF-κB inhibition during the resuscitation phase does not effectively counteract NF-κB p65 nuclear translocation initiating inflammatory gene transcription.

Heterogenous Renal Injury Biomarker Production Reveals Human Sepsis-Associated Acute Kidney Injury Subtypes.

To identify mechanisms associated with sepsis-acute kidney injury based on the expression levels of renal injury biomarkers, neutrophil gelatinase-associated lipocalin, and kidney injury molecule-1 in renal biopsies which may allow the identification of sepsis-acute kidney injury patient subtypes. DESIGN: Prospective, clinical laboratory study using "warm" human postmortem sepsis-acute kidney injury kidney biopsies. SETTING: Research laboratory at university teaching hospital. SUBJECTS: Adult patients who died of sepsis in the ICU and control patients undergoing tumor nephrectomy. MEASUREMENTS AND MAIN RESULTS: Reverse transcription quantitative polymerase chain reaction and immunohistochemical staining were used to quantify messenger RNA and protein expression levels of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in the kidney of sepsis-acute kidney injury patients and control subjects. Morphometric analysis was used to quantify renal and glomerular neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 protein levels. Neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 messenger RNA and protein levels were increased in kidneys of sepsis-acute kidney injury patients compared with control kidney tissue. Neutrophil gelatinase-associated lipocalin was localized in the distal tubules, collecting ducts, the adventitia of the renal arterioles, and in the glomerular tufts of renal biopsies from sepsis-acute kidney injury patients. In contrast, kidney injury molecule-1 was localized at the brush border of the proximal tubules. There was no correlation between neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 levels. Furthermore, renal neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 levels were not associated with the extent of renal injury, the severity of critical illness, or serum creatinine levels at either ICU admission or day of expiration. By laser microdissecting glomeruli, followed by reverse transcription quantitative polymerase chain reaction, we identified heterogenous glomerular neutrophil gelatinase-associated lipocalin production in the kidney of sepsis-acute kidney injury patients. CONCLUSION: We found differences in the expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in patients with the same syndrome "sepsis-acute kidney injury" meaning there is no single pathway leading to sepsis-acute kidney injury. This underscores the beliefs that there are many/different pathophysiological pathways that can cause sepsis-acute kidney injury. Hence, patients with criteria that meet the definitions of both acute kidney injury and sepsis can be divided into subtypes based on pathophysiological features.

Partial Deletion of Tie2 Affects Microvascular Endothelial Responses to Critical Illness in A Vascular Bed and Organ-Specific Way.

Tyrosine kinase receptor (Tie2) is mainly expressed by endothelial cells. In animal models mimicking critical illness, Tie2 levels in organs are temporarily reduced. Functional consequences of these reduced Tie2 levels on microvascular endothelial behavior are unknown. We investigated the effect of partial deletion of Tie2 on the inflammatory status of endothelial cells in different organs. Newly generated heterozygous Tie2 knockout mice (exon 9 deletion, ΔE9/Tie2) exhibiting 50% reduction in Tie2 mRNA and protein, and wild-type littermate controls (Tie2), were subjected to hemorrhagic shock and resuscitation (HS + R), or challenged with i.p. lipopolysaccharide (LPS). Kidney, liver, lung, heart, brain, and intestine were analyzed for mRNA levels of adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular cell adhesion molecule 1 (ICAM-1), and CD45. Exposure to HS + R did not result in different expression responses of these molecules between organs from Tie2 or Tie2 mice and sham-operated mice. In contrast, the LPS-induced mRNA expression levels of E-selectin, VCAM-1, and ICAM-1, and CD45 in organs were attenuated in Tie2 mice when compared with Tie2 mice in kidney and liver, but not in the other organs studied. Furthermore, reduced expression of E-selectin and VCAM-1 protein, and reduced influx of CD45 cells upon LPS exposure, was visible in a microvascular bed-specific pattern in kidney and liver of Tie2 mice compared with controls. In contrast to the hypothesis that a disbalance in the Ang/Tie2 system leads to increased microvascular inflammation, heterozygous deletion of Tie2 is associated with an organ-restricted, microvascular bed-specific attenuation of endothelial inflammatory response to LPS.

Cryoinjury: a model of myocardial regeneration.

INTRODUCTION: Although traditionally adult cardiomyocytes are thought to be unable to divide, recent observations provide evidence for cardiomyocyte proliferation after myocardial injury. Myocardial cryoinjury has been shown to be followed by neovascularization. We hypothesize that, in addition to neovascularization, cardiomyocyte proliferation after myocardial cryoinjury contributes to regeneration. METHOD: Cryolesions were applied to the left ventricle of mouse hearts. Inflammatory cell infiltration (F4/80, neutrophils), neovascularization (CD31), and cardiomyocyte proliferation (5-bromo-2-deoxyuridine, Ki-67, mitotic spindle) were determined at different time points (2-70 days) after cryoinjury. RESULTS: Between Days 7 and 14 after injury, a 150- and 280-fold increase in number of proliferating cardiomyocytes was observed, as compared to controls. At the same time, numerous proliferating capillaries were found in between the proliferating cardiomyocytes. Presence of high numbers of macrophages in the cryolesion preceded and coincided with this proliferation. The area of cryolesion decreased significantly between Days 7 (23+/-5%) and 14 (8+/-2%) after cryoinjury. Moreover, regeneration of viable, nonhypertrophied myocardium was observed. After 14 days, cardiomyocyte proliferation decreased to numbers observed in controls, and concomitantly, the number of macrophages strongly decreased. CONCLUSION: Our data show that adult cardiomyocytes proliferate in sufficiently high numbers to effectuate myocardial regeneration after left ventricular cryoinjury in mice.

Tubular engraftment and myofibroblast differentiation of recipient-derived cells after experimental kidney transplantation.

BACKGROUND: In human renal allografts, recipient-derived cells engrafted in various kidney substructures, have been detected in the long term after transplantation. Here we investigated tubular engraftment and myofibroblast differentiation of recipient-derived cells at short term after experimental kidney transplantation, during a previously described window of regeneration and possible onset of renal interstitial fibrosis. METHODS: Fisher (F344, syngeneic) and Dark Agouti (DA, allogeneic) kidneys were transplanted into F344-hPAP transgenic recipient rats, which allowed tracing of recipient-derived cells in nontransgenic donor kidneys. We evaluated tubular engraftment and myofibroblast differentiation of recipient-derived cells on day 14 after kidney transplantation. RESULTS: Kidney transplantation resulted in tubular engraftment of recipient-derived cells. After allogeneic kidney transplantation, 9.7% of tubular cross-sections contained at least one recipient-derived cell, which represented a significant increase in comparison to syngeneic transplantation (4.0%, P<0.05). Moreover, recipient-derived myofibroblasts were present in the renal interstitium of the transplanted kidney. These cells contributed 39% of the total interstitial myofibroblast population in allografts, which was comparable to the syngeneic situation (28%, P=0.25). CONCLUSIONS: In a defined early window of regeneration and possible onset of renal interstitial fibrosis after kidney transplantation, rejection-associated injury, superimposed on ischemic damage, increases tubular engraftment of recipient-derived cells, although it does not affect their relative contribution to the renal interstitial myofibroblast population.

Dependence of neovascularization mechanisms on the molecular microenvironment.

In vivo vascularization of implanted (bio)artificial constructs is essential for their proper function. Vascularization may rely on sprouting angiogenesis, vascular incorporation of bone marrow-derived endothelial cells (BMDECs), or both. Here we investigated the relative contribution of these 2 mechanisms to neovascularization in a mouse model of a foreign body reaction (FBR) to subcutaneously implanted Dacron and in hind limb ischemia (HLI) in relation to the molecular microenvironment at these neovascularization sites. Neovascularization was studied in C57Bl/6 mice reconstituted with enhanced green fluorescent protein (EGFP) transgenic bone marrow. Sprouting angiogenesis, detected using nuclear incorporation of bromodeoxyuridine in endothelial cells was present in both models, whereas vascular incorporation of EGFP(+) BMDECs was restricted to HLI. In HLI, the presence of a pro-angiogenic molecular microenvironment comprising vascular endothelial growth factor, fibroblast growth factor 2, and granulocyte colony-stimulating factor corroborated the importance of these factors for vascular BMDEC incorporation, whereas this microenvironment was absent in FBR. Enhanced mobilization of BMDECs by granulocyte-macrophage colony-stimulating factor administration or by combining HLI and FBR with Dacron did not induce incorporation of BMDECs in FBR neovessels. We conclude that the efficacy of BMDEC-based therapy is not generally warranted, but it depends on the molecular microenvironment in the targeted tissue.

Spatial and temporal expression patterns of the epithelial cell adhesion molecule (EpCAM/EGP-2) in developing and adult kidneys.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is expressed by most epithelia and is involved in processes fundamental for morphogenesis, including cell-cell adhesion, proliferation, differentiation, and migration. Previously, a role for EpCAM in pancreatic morphogenesis was confirmed in vitro. Furthermore, changes in the EpCAM expression pattern were found in developing lung and thymus and in the regenerating liver. Therefore, EpCAM was proposed to be a morphoregulatory molecule. METHODS: Using immunohistochemistry, the expression pattern of human and murine homologues of EpCAM was characterized in adult and embryonic kidneys from humans and human-EpCAM (hEpCAM)-transgenic mice. RESULTS: EpCAM expression was found in the ureteric bud throughout nephrogenesis. EpCAM was not expressed in the metanephric mesenchyme. In comma- and S-shaped bodies, both metanephric mesenchyme derived structures, EpCAM expression appeared by E13.5. In adult kidneys, most epithelia expressed varying levels of EpCAM, as confirmed by double staining for human EpCAM and segment-specific nephron markers. Podocytes were EpCAM negative. At the cellular level, the EpCAM expression shifted from apical in embryonic to basolateral in adult kidneys. CONCLUSIONS: The spatiotemporal expression pattern of EpCAM changes during nephrogenesis. In the adult kidney, the expression varies markedly along the nephron. These data provide a basis for further studies on EpCAM in developing and adult kidneys.

Organ-Specific Differences in Endothelial Permeability-Regulating Molecular Responses in Mouse and Human Sepsis.

In patients with sepsis-induced multi-organ dysfunction syndrome, diverging patterns of oedema formation and loss of function in organs such as lung and kidney suggest that endothelial permeability-regulating molecular responses are differentially regulated. This potential differential regulation has been insufficiently studied at the level of components of adherens and tight junctions. We hypothesized that such a regulation by endothelial cells in sepsis takes place in an organ-specific manner. We addressed our hypothesis by studying by quantitative real time polymerase chain reaction the expression of a predefined subset of EC permeability-related molecules (occludin, claudin-5, PV-1, CD-31, endomucin, Angiopoietin-1, Angiopoietin-2, Tie2, VEGFA, VEGFR1, VEGFR2, and VE-cadherin) in kidney and lung after systemic lipopolysacharide injection in mice, and in kidneys of patients who died of sepsis. We showed that baseline endothelial expression of permeability-related molecules differs in mouse kidney and lung. Moreover, we showed differential regulation of these molecules after lipopolysacharide injection in the two mouse organs. In lung we found a decrease in expression levels of molecules of the adherence and tight junctions complex and related signaling systems, compatible with increased permeability. In contrast, in kidney we found expression patterns of these molecules compatible with decreased permeability. Finally, we partially corroborated our findings in mouse kidney in human kidneys from septic patients. These findings may help to understand the clinical difference in the extent of oedema formation in kidney and lung in sepsis-associated organ failure.

Endothelial Interferon Regulatory Factor 1 Regulates Lipopolysaccharide-Induced VCAM-1 Expression Independent of NFκB.

Sepsis is a severe systemic inflammatory response to infection. Endothelial activation and dysfunction play a critical role in the pathophysiology of sepsis and represent an important therapeutic target to reduce sepsis mortality. Interferon regulatory factor 1 (IRF-1) was recently identified as a downstream target of TNF-α-mediated signal transduction in endothelial cells. The aim of this study was to explore the importance of IRF-1 as a regulator of lipopolysaccharide (LPS)-induced endothelial proinflammatory activation. We found that renal IRF-1 was upregulated by LPS in vivo as well as in LPS-stimulated endothelial cells in vitro. Furthermore, we identified intracellular retinoic acid inducible gene-I (RIG-I) as a regulator of LPS-mediated IRF-1 induction. IRF-1 depletion specifically resulted in diminished induction of VCAM-1 in response to LPS, but not of E-selectin or ICAM-1, which was independent of NFκB signaling. When both IRF-1 and the RIG-I adapter protein mitochondrial antiviral signaling (MAVS) were absent, VCAM-1 induction was not additionally inhibited, suggesting that MAVS and IRF-1 reside in the same signaling pathway. Surprisingly, E-selectin and IL-6 induction were no longer inhibited by MAVS knockdown when IRF-1 was also absent, revealing a redundant endothelial activation pathway. In summary, we report an IRF-1-mediated proinflammatory signaling pathway that specifically regulates LPS-mediated VCAM-1 expression, independent of NFκB.

Local therapeutic efficacy with reduced systemic side effects by rapamycin-loaded subcapsular microspheres.

Kidney injury triggers fibrosis, the final common pathway of chronic kidney disease (CKD). The increase of CKD prevalence worldwide urgently calls for new therapies. Available systemic treatment such as rapamycin are associated with serious side effects. To study the potential of local antifibrotic therapy, we administered rapamycin-loaded microspheres under the kidney capsule of ureter-obstructed rats and assessed the local antifibrotic effects and systemic side effects of rapamycin. After 7 days, microsphere depots were easily identifiable under the kidney capsule. Both systemic and local rapamycin treatment reduced intrarenal mTOR activity, myofibroblast accumulation, expression of fibrotic genes, and T-lymphocyte infiltration. Upon local treatment, inhibition of mTOR activity and reduction of myofibroblast accumulation were limited to the immediate vicinity of the subcapsular pocket, while reduction of T-cell infiltration was widespread. In contrast to systemically administered rapamycin, local treatment did not induce off target effects such as weight loss. Thus subcapsular delivery of rapamycin-loaded microspheres successfully inhibited local fibrotic response in UUO with less systemic effects. Therapeutic effect of released rapamycin was most prominent in close vicinity to the implanted microspheres.

Donor-derived circulating endothelial cells after kidney transplantation.

BACKGROUND: In solid-organ transplantation, the allograft vasculature, in particular the endothelium, is prone to injury inflicted by peritransplantational and posttransplantational factors. Previously, we have shown that circulating endothelial cells (cEC) can be detected in the peripheral blood of kidney allograft recipients and are often associated with acute rejection and active infections with human cytomegalovirus. In the present study we hypothesized that cEC after kidney transplantation are of donor origin, thus reflecting transplantation-related damage to the allograft endothelium. METHODS: Using hydraulic micromanipulation equipment, we isolated single cEC (n=153) from the peripheral blood of nine kidney allograft recipients at various time points after transplantation. We demonstrated the origin of these cells (donor or recipient) by typing their HLA-DRB alleles by single-cell, genomic, nested polymerase chain reaction. RESULTS: The majority (71.8%) of cEC were of donor origin and could be detected up to 141 days after onset of acute rejection episodes. Although less frequent (28.2%), recipient-type cEC were detected in the same time course as donor-type cEC. CONCLUSION: We conclude that posttransplantational injury to the allograft endothelium is reflected by the presence of donor-derived cEC in the blood.

Distribution patterns of the membrane glycoprotein CD44 during the foreign-body reaction to a degradable biomaterial in rats and mice.

Although biomaterials have been used in the clinical setting for a long time, little is known of the molecular mechanisms underlying the foreign-body reaction (FBR). A good understanding of these mechanisms is requisite for the controlled regulation of the FBR needed to prevent adverse tissue reactions and thus to improve the function of the biomaterial. Macrophages are essential in the inflammatory reaction in, as well as around, the implants, and they also are believed to initiate most of the adverse responses. Typically, during the FBR macrophages become activated and fuse into multinucleated giant cells (MnGCs). CD44, an integral membrane glycoprotein expressed on a broad spectrum of cell types, is involved in MnGC formation in vitro and in inflammation processes in general. In vivo it is not known whether CD44 is part of a specific protein machinery that enables macrophage fusion or whether it has additional functions in the FBR. In the present in vivo study, CD44 expression patterns were followed in rats and mice during the FBR to a degradable collagen type I biomaterial. We found that CD44 is upregulated on all migrating cells and on newly formed blood vessels at the onset of the FBR and that MnGCs, up to week 15 postimplantation, expressed CD44. Although no evidence was found that CD44 participates in macrophage fusion leading to multinucleation, it nevertheless may be an interesting target molecule for modulating the FBR in vivo, possibly by affecting cell activation, cell migration towards the biomaterial, vascularization, and MnGC formation.

Ongoing foreign body reaction to subcutaneous implanted (heparin) modified Dacron in rats.

Dacron-containing heart valve repair devices trigger chronic inflammation characterized by the presence of activated macrophages, foreign body giant cells, and capsule formation. Upon blood contact, proinflammatory proteins adsorb to the material and provide a substrate for monocyte binding and differentiation. Various heparin-coated polymers have been shown to reduce adsorption of proinflammatory proteins in vitro and in vivo. In this study, the effect of knitted, heparin-coated Dacron on the foreign body reaction was tested subcutaneously in rats. We hypothesized that the anti-inflammatory effect of heparin would reduce monocyte recruitment and differentiation and therefore limit the inflammatory reaction. An ongoing foreign body reaction, characterized by the presence of foreign body giant cells and high vascularization, was observed in uncoated as well as (heparin-)coated Dacron at up to 180 days of implantation. Also, a thin capsule was formed around each material up to this time. In conclusion, although heparin coatings might have an effect on the acute inflammatory response, we were not able to show a difference between heparin-coated and uncoated Dacron after 180 days' implantation in rats. Further research needs to be conducted to assess the difference in proinflammatory protein adsorption between the tested materials and the effect this has on the long-term foreign body reaction.