Bone marrow-derived cells in kidney repair: A double-edged sword?

From NIER

by Dr. Martine Broekema

University Medical Center Groningen, Groningen, The Netherlands.

Originally published as general introduction to the PhD thesis of Dr. Martine Broekema which she published in november 2007 under supervision of her promotores Prof. dr. Gerjan Navis and Prof. dr. Marja J.A. van Luyn and co-promotores Dr. Eliane R. Popa and Dr. Marco C. Harmsen at the University Medical Center Groningen in The Netherlands.

Contents 1 Abstract 2 Introduction 3 Acute renal failure 4 Renal repair mechanisms after acute injury 5 Chronic renal failure 6 Bone marrow-derived cells (BMDC) in kidney repair 7 References

[edit] Abstract

Bone marrow-derived cells (BMDC) possibly play a therapeutic role in organ repair after injury. Here, an overview is provided on some general aspects of renal damage, repair, and the possible therapeutic potential of BMDC therein.

[edit] Introduction

The kidney can recover after acute renal injury due to its highly effective endogenous regenerative capacity. However, under certain conditions the balance between injury and repair can get disturbed. This can ultimately lead to chronic renal failure, which is an increasing problem in the clinical setting. Therapy for renal failure has greatly improved over the years, especially by the introduction of kidney transplantation. Nevertheless, due to the shortage of donor organs and the relatively low long-term success rate after kidney transplantation, new therapeutic strategies are highly desirable. In this thesis we explored the differentiation choices of BMDC after acute renal injury. In this introductory chapter we will introduce some general aspects of renal damage, repair, and the possible therapeutic potential of BMDC therein and we will discuss the research questions addressed in this thesis.

[edit] Acute renal failure

Acute renal failure (ARF), which affects up to 5% of all long-term hospital patients (1), can be triggered by various insults, among which ischemia/reperfusion holds a forefront role. The pathophysiology of ischemia/reperfusion injury (IRI) involves renal endothelial and tubular epithelial injury, oxidative stress, inflammation and wound healing. Post-ischemic endothelial damage results, due to an imbalance in the production of endothelin (2) and NO (3), in intra-renal vasoconstriction, which is assumed to contribute to loss of renal function. However, the use of renal vasodilators, which return renal blood flow to normal in experimental IRI, do not improve renal function, therefore the vasomotor component may not be the culprit. Studies on the mechanisms mediating IRI have been primarily focused on the renal tubule. The straight segment of the proximal tubule (S3 segment, pars recta) is most susceptible to ischemic injury (4). Both the deprivation of oxygen during ischemia and the restoration of oxygen supply during reperfusion cause tubular damage (Figure 1). The damage caused by renal ischemia depends largely on the duration of oxygen deprivation. Upon ischemia, oxygen deprivation of the tubular cell leads to depletion of cellular ATP, which initiates a cascade of biochemical events that lead to mitochondrial damage, impaired solute and ion transport, loss of cell polarity, and cytoskeletal disruption (5). In case of severe ischemia these events lead to cell death, shedding and excretion of proximal tubule brush border membranes and tubular epithelial cells into the urine (6). Histologically, ischemic damage is represented by patchy and focal loss of tubular epithelial cells, resulting in areas of denuded basement membrane, and the presence of intraluminal casts (7).

Although restoration of blood flow during reperfusion is essential for survival of the kidney, it leads to further cellular damage. Reperfusion is therefore known as the “reflow paradox”. The mechanism of reperfusion injury includes the generation of reactive oxygen species (ROS) (8). ROS have numerous deleterious effects on cells, such as lipid peroxidation, oxidation of cell proteins, and damage to DNA (9). Moreover, generation of ROS causes endothelial activation and recruitment of leukocytes through the upregulation of chemokines (10) and the proinflammatory cytokines interleukin-1 (IL-1), IL-2, IL-8, tumor necrosis factor-alpha (TNF-) and interferon-γ (IFN-γ) by affected cells (11-13). Adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM) and P-selectin are upregulated during IRI and promote endothelial-leukocyte interactions. These adhesion molecules facilitate inflammation by increasing the infiltration of mainly neutrophils and macrophages in the renal interstitium early after ischemic injury (14), while later phases are characterized by infiltration of macrophages and T lymphocytes (15;16). The infiltration of leukocytes leads to a vicious circle of inflammation by secondary activation of leukocytes which release mediators, such as free oxygen radicals, proteases, and cytokines that further aggravate tissue damage.

Kidney transplantation can also lead to acute renal injury and consequently, delayed graft function. Renal transplantation is the treatment of choice for end stage renal failure (17). Due to the introduction of immunosuppressive therapy (especially of cyclosporin A (CsA) in the 1980’s) and improvements in HLA matching (18), ischemic times and organ preservation (19), graft survival after kidney transplantation has improved to about 90% at 1 year post-transplantation (20). Despite major short-term graft survival, 50% of all renal transplant recipients experience acute rejection, which is the strongest risk factor for development of chronic renal transplant failure. Beside acute rejection, transplant injury is caused by cold and warm ischemia and nephrotoxicity from immunosuppressive medication. Ischemia/reperfusion injury (IRI) augments the specific immune response to the allograft through the upregulation of MHC class II and adhesion molecule expression in the allografted kidney, thereby increasing the risk of acute rejection (21). In principle, IRI of the donor kidney is unavoidable, especially when a kidney of a deceased donor is transplanted. However, when ischemic times are short, for example during transplantation of a kidney of a living donor, IRI and the risk for delayed graft function is substantially reduced.

Acute rejection is primarily a cell-mediated immune-response of the recipient against the alloantigens present in the renal graft. Histologically, acute rejection is characterized by interstitial inflammatory infiltrates, tubulitis and various degrees of arteritis. For diagnostic purposes, the abnormalities are classified in renal biopsies according to the Banff classification (22). Acute rejection is initiated when recipient CD8+ T-cells recognize allogeneic MHC class I-molecules on the tubular epithelium, endothelium or mesangial cells of the donor kidney. Upon costimulation by professional antigen-presenting cells (APCs) or cytokines provided by CD4+ helper T-cells, CD8+ T-cells differentiate to cytotoxic T-cells (CTLs). These alloreactive CTLs directly lyse target cells of the donor kidney. Activation of CD4+ helper T-cells, by recognition of MHC class II molecules on APCs, results in secretion of cytokines and activation of macrophages, B cells and CD8+ T-cells, thereby maintaining inflammation and further aggravating graft injury. Not only cell-mediated, but also a humoral immune response against allo-antibodies contributes to rejection. These allo-antibodies bind to endothelium, activate the complement system and injure graft blood vessels.

[edit] Renal repair mechanisms after acute injury

After acute renal injury innate mechanisms are activated that result in replacement of damaged tubular epithelial cells and deposition of extracellular matrix to restore renal morphology and function. Better understanding of these innate mechanisms of repair will uncover new therapeutic targets.

Acute tubular injury is usually reversible. The prevailing theory is that the innate repair mechanisms of the kidney are dependent on surviving proximal tubular epithelial cells (Figure 1). After injury the surviving tubular cells have the ability to repair themselves by re-polarization and restoration of their cytoskeleton, mitochondria and solute and ion transport system. In addition, surviving tubular cells serve to replace lost tubular cells. In the scenario of replacement, sublethally injured tubular cells dedifferentiate to mesenchymal cells (23;24). Subsequently, the dedifferentiated tubular epithelial cells proliferate rapidly and extensively to restore original epithelial cell number (25-27). The dedifferentiated cells spread and migrate over the basement membrane to denuded areas (28;29) where they differentiate to tubular epithelial cells, leading to restoration of morphology and function (30). Several growth factors have been identified that may facilitate these regenerative responses, such as epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) (31).

Repair of the kidney after acute injury depends not only on epithelialization of the denuded basement membrane, but also on wound healing. Wound healing is the process of structural remodeling in order to maintain tissue integrity. Fibroblasts and myofibroblasts participate in wound healing by producing extracellular matrix (ECM) components and by responding to and synthesizing cytokines, chemokines and other mediators of inflammation. When wound healing is completed, fibroblasts and myofibroblasts generally disappear by apoptosis (32). However, when apoptosis is lacking and proliferation and/or activation of fibroblasts and myofibroblasts is out of control, this can result in the excessive deposition ECM components. Long-term consequence of the disturbed balance between ECM production and degradation is interstitial fibrosis and chronic renal failure.

[edit] Chronic renal failure

If innate renal repair mechanisms are inadequately controlled, this can eventually lead to progressive loss of renal function. Chronic renal failure (CRF) is characterized by the progressive loss of renal function and can result in end-stage renal disease and the need for replacement therapy, i.e. dialysis or transplantation. Renal interstitial fibrosis is the hallmark of CRF because the extent of renal interstitial fibrosis correlates with loss of renal function (33). As discussed above, renal interstitial fibrosis is the result of inadequately controlled wound healing. Control over wound healing is lost when transforming growth factor-β (TGF-β), generally regarded as the inducer of fibrosis, is overexpressed in the kidney. TGF-β increases expression of connective tissue growth factor (CTGF) (34), which induces proliferation of (myo)fibroblasts (35). Similar to CTGF, TGF-β causes upregulation of basic fibroblast growth factor-2 (FGF-2) in renal interstitial (myo)fibroblasts, inducing proliferation of these cells (36). The downstream effect of TGF-β upregulation and subsequent (myo)fibroblast proliferation is excessive interstitial accumulation of ECM components, such as fibronectin, collagen I, III and proteoglycans. Formation of peptide bonds between these ECM components is catalyzed by transglutaminases, resulting in extensively cross linked protein polymers, which are resistant to degradation. Together with excessive production of ECM, this will lead to a disturbance in the balance between production and degradation of ECM components, resulting in interstitial scarring and suppression of tubular epithelial cells (Figure 1).

Myofibroblasts, are considered the major producers of ECM components during renal interstitial fibrosis (37). These cells possess phenotypical characteristics of both fibroblasts and smooth muscle cells. The origin of the myofibroblast remains subject of discussion. Several options for their origin have been reported, i.e. that myofibroblasts may represent an activated population of resident fibroblasts (38), or that myofibroblasts originate from injured tubular epithelial cells by epithelial-to-mesenchymal transition (EMT) (39), from perivascular smooth muscle cells (40) or from bone marrow-derived cells (BMDC) (41) (Figure 1). Chronic renal injury can also occur in the renal transplant and thus lead to graft loss. Despite the excellent clinical perspective at 1 year post-transplantation, half of the transplanted kidneys are lost within 10 years after engraftment (42). The most important cause of graft loss is chronic allograft nephropathy (CAN); progressive deterioration of renal function ultimately leading to graft loss. Histopathologically, CAN is characterized by mononuclear cell infiltration, glomerulosclerosis, interstitial fibrosis, tubular atrophy, perivascular inflammation, vascular obliteration and vascular wall thickening. In contrast to the allo-antibody-driven event of acute rejection, CAN is the consequence of both immune factors, such as acute rejection episodes, and non-immune factors, such as donor-related factors (e.g. age, brain death), IRI during transplantation and immunosuppressive drug-induced toxicity (43). To date, there is no effective way to prevent or treat CAN.

Currently, preventing or treating renal failure by simulating or promoting the innate capacity of the kidney to reverse acute tubular injury, has attracted interest as a new therapeutic strategy.

[edit] Bone marrow-derived cells (BMDC) in kidney repair

The bone marrow-derived cell (BMDC) population is heterogeneous, consisting mainly of inflammatory cells, i.e. neutrophils, macrophages and T lymphocytes that infiltrate the kidney upon injury. The BMDC population contains, beside inflammatory cells, small numbers of stem cells and/or progenitor cells. A large body of evidence supports the idea that stem- and progenitor cells can infiltrate multiple organs and engraft tissue structures upon injury. BMD stem- and progenitor cells were reported to show the plasticity to differentiate towards cells from any of the three germ layers. The potential of BMDC subpopulations to replace damaged tissue cells by engraftment and differentiation, favors therapeutic cell therapy with BMDC.

The bone marrow contains two stem cell populations that were believed to serve only as the blood-forming compartment of the body. However, multiple investigators have suggested that these stem cells mediate tissue repair by repopulation and differentiation in injured tissues. Firstly, hematopoietic stem cells (HSC), appeared capable to differentiate to multiple cell types (44). HSC are lineage-uncommitted (Lin-) bone marrow cells that are characterized in human and mice by the expression of the cell surface markers CD34, Sca-1 and c-kit (45;46). Secondly, mesenchymal stem cells or marrow stromal cells (MSC) that are typically characterized as plastic adherent, non-hematopoietic bone marrow cells that can be cultured in vitro and maintained as fibroblast-like cells. MSC have the potential to differentiate into mesenchymal lineages such as chondrocytes, osteocytes and adipocytes, but also into other, non-mesenchymal cell lineages such as endothelial and muscle cells (47). Possibly, these bone marrow-derived (BMD) stem cell populations have the ability to mediate kidney repair by differentiation towards tubular epithelial cells.

Beside stem cells, progenitor cells can be part of the BMDC population that infiltrates the kidney upon injury. While progenitor cells share many common features with stem cells, they are more restricted in terms of plasticity and self renewal. Endothelial progenitor cells (EPC) are BMD progenitor cells that, based on their expression of a restricted set of surface markers (CD34, VEGF-R2 and/or CD133), can be isolated from bone marrow, umbilical vein blood and peripheral blood. In vitro, EPC differentiate into endothelial cells and in animal models of ischemia, EPC were shown to incorporate into sites of active angiogenesis and mediate neovascularization (48;49). This potential renders the EPC an attractive candidate for therapeutic application to achieve endothelial repair in the injured kidney.

In several organs BMDC or BMD stem- or progenitor cells were reported to engraft and differentiate towards tissue specific cell types. Since in most studies bone marrow transplantation was performed with whole bone marrow, the investigators were unable to define which BMDC subpopulation differentiated. In the infarcted heart, injection of lineage-depleted, c-kit+ BMDC have been shown to differentiate into cardiomyocytes and vascular structures, thereby ameliorating the function of the infarcted heart (50;51). In the damaged liver an undefined BMDC subpopulation was shown to infiltrate and differentiate to hepatocytes, thereby improving liver function (52;53). Furthermore, undefined BMDC subpopulations have been reported to differentiate into endocrine β cells of the pancreas (54;55), cells of the central nervous system, such as neurons, oligodendrocytes and astrocytes (56-59), skeletal muscle cells (60) and intestinal epithelial cells (44;61). The recurrent hypothesis in all mentioned organ system is that upon injury, inflammation leads to the recruitment of BMDC subpopulations to the site of injury, where some BMDC subpopulations can repopulate the damaged structure by engraftment and differentiation into tissue specific cell-types.

The hypothesis that BMDC can infiltrate and repopulate the damaged organ upon injury was also tested in the kidney. In experimental studies, BMDC are usually detected by bone marrow transplantation with labeled bone marrow cells to allow tracing of BMDC in an injured organ. In humans, bone marrow transplantation with sex- or MHC-mismatched bone marrow followed by renal injury would allow tracing BMDC in the injured kidney. Since this sequence of events is not likely to occur, the presence of recipient-derived cells after sex- or MHC- mismatched kidney transplantation was investigated instead. In these studies, the presence of recipient-derived cells was reported in vessels and tubuli of the renal transplant (62-68). Recipient-derived cells that engrafted the tubular epithelium adopted a tubular epithelial phenotype (63;65;66). Since the extent of engraftment of recipient-derived cells in donor epithelium or endothelium correlated with the severity of sustained graft damage (64;67), it was suggested that recipient-derived cells mediate graft repair by replacing damaged graft cells. Moreover, since the recipient-derived cells are extra-renal and showed plasticity to differentiate in vivo, these cells were suggested to represent a BMD population of stem- or progenitor cells.

[edit] References

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