Acute Kidney Injury: Clinical and Experimental Issues 316
Clinical Acute Kidney Injury 316
Treatment Resistance of Clinical Acute Kidney Injury 317
Experimental Models of Acute Kidney Injury 317
Design of Clinical Trials 318
Novel Diagnostic Biomarkers for Acute Kidney Injury 318
Pathophysiology-based Therapy 318
Treatment of Acute Kidney Injury with Stem Cells: Preclinical Studies 319
Pathophysiology and Repair of Acute Kidney Injury 319
Hematopoietic Stem Cell Mobilization and Administration 320
Administration of Endothelial Precursor Cells 320
Administration of Multipotent Marrow Stromal Cells 321
Administration of Multipotent Marrow Stromal Cells in Experimental Acute Kidney Injury 323
Treatment of Cisplatinum and Glycerol-induced Acute Kidney Injury with Mesenchymal Stem Cells 324
Treatment of Ischemia–Reperfusion Acute Kidney Injury in Rats with Autologous and Allogeneic Mesenchymal Stem Cells 324
Mediator Mechanism of Mesenchymal Stem Cells in Ischemia–Reperfusion Acute Kidney Injury 326
Detachment and Fate of Mesenchymal Stem Cells 327
Hemodynamic Actions 327
Anti-inflammatory Actions 328
Trophic Actions 329
Role of Vascular Endothelial Growth Factor Expression by Mesenchymal Stem Cells 329
Vascular Actions 330
Fusion and Diapedesis 330
Late Consequences of Ischemia–Reperfusion Acute Kidney Injury Therapy with Allogeneic Mesenchymal Stem Cells 331
Phase I Clinical Trial 332
Prevention and Treatment of Postoperative Acute Kidney Injury with Allogeneic Mesenchymal Stem Cells in Patients who Require On-pump Cardiac Surgery 332
Study Design 333
Study Objectives 333
Patient Selection 333
Preoperative Data Collection 333
Allogeneic Mesenchymal Cell Stem Therapy 333
Postoperative Data Collection 333
Summary and Conclusion 334
Competing interests 337
Acute kidney injury (AKI) is a common complication with poor early outcome and significant later development of chronic kidney disease. Current therapies remain only supportive, demonstrating that adequate recovery depends on the kidney’s ability to repair itself. Extensive preclinical data show that the administration of mesenchymal stem cells (MSCs) is highly effective in protecting renal function and stimulating repair, principally mediated by paracrine trophic and anti-inflammatory actions. Translating these data, a phase I clinical trial was conducted in which allogeneic MSCs were administered to open-heart surgery patients who are at high risk for postoperative AKI. The intervention was safe, and none of the study subjects, compared with well-matched controls, developed postoperative AKI or subsequent chronic kidney disease. A phase II clinical trial will be conducted next, and the generated data, together with the use of early diagnostic biomarkers for AKI, are expected to translate into a significant improvement in the outcomes of patients with AKI.
Acute Kidney Injury: Clinical and Experimental Issues
Clinical Acute Kidney Injury
In 1951 Homer Smith introduced the term “acute renal failure” in his textbook The Kidney: Structure and Function in Health and Disease , describing the abrupt decrease in renal function, caused by various renal insults, resulting in nitrogenous waste product accumulation. Currently, acute renal failure (ARF) due to acute kidney injury (AKI) is defined as the abrupt partial or complete loss of kidney functions that results in disturbed volume, electrolyte and acid–base balance, and uremic, multisystem complications due to retention of toxic waste products, a heightened inflammatory state and generation of reactive oxygen species (ROS), together resulting in life-threatening multiorgan complications with high mortality rates, particularly in elderly patients and those with significant other comorbidities .
Because of widely divergent definitions of ARF, in 2004 the Acute Dialysis Quality Initiative (ADQI) ( www.ccm.upmc.edu/adqi/ADQI2/ADQI2g1.pdf ) published a consensus definition for this syndrome, the Risk–Injury–Failure–Loss–Endstage renal disease (RIFLE) classification . The definition is based on the change in serum creatinine and/or urine output and combines severity grades based on these variables as well as on clinical outcomes, together combined into the RIFLE criteria (risk, injury and failure, with the outcome classes loss and end-stage kidney disease). The term acute kidney injury (AKI) was introduced to define the syndrome broadly, identifying both early and mild forms as well as severe forms, requiring renal replacement therapy. The ADQI group subsequently published a simplified version of the RIFLE classification, known as the AKI Network classification . The categories risk, injury, and failure were replaced by stages 1, 2 and 3, respectively. An absolute increase in creatinine of at least 0.3 mg/dl was added to stage 1; patients needing renal replacement therapy are automatically classified as stage 3. The RIFLE outcome categories loss and endstage renal disease were deleted.
Clinical settings that result most commonly in AKI include ischemia–reperfusion insults of the kidney due to major surgery, shock, sepsis, trauma and nephrotoxins such radiocontrast medium, aminoglycosides, cisplatin and others . Comorbidities that greatly enhance the susceptibility of patients to develop severe AKI include underlying renal disease , a recently challenged risk factor , older age, diabetes mellitus, nephrotic syndrome, congestive heart failure, chronic obstructive pulmonary disease and prolonged cardiopulmonary bypass times in patients undergoing coronary artery bypass graft (CABG) or valve surgery .
Severity and extension of injury are related to the nature and duration of the causative agent or insult. Pathological data were initially limited to autopsy findings, based on which AKI has long been termed acute tubular cell necrosis (ATN). The subsequent utilization of renal biopsies demonstrated that in patients with significant AKI only very limited tubular cell necrosis is seen, and only discrete cell loss, affecting mainly the proximal tubule and occasionally the medullary thick ascending limb (mTAL) and rarely the collecting duct. This pattern has recently been described as “physiologic and pathologic dissociation” .
The morphological manifestations of AKI develop in several overlapping phases. Initially, ultrastructural changes manifest as cytoskeletal alterations leading to changes in the polarization of proximal tubular cells and loss of their brush border . Cell detachment is a consequence of these cytoskeletal alterations and eventually some cells undergo apoptosis or may become necrotic if an insult is severe and rapid. This is followed by repair and regeneration. Cell loss and detachment make the tubular epithelium appear flattened and expose areas of denuded basement membrane. Detached tubular cells are seen in the tubular lumen and can be cultured from the urine. Debris as well as dead and viable cells cause tubular obstruction downstream. Vascular changes in AKI affect mainly the peritubular capillaries and result in vascular congestion mainly in the corticomedullary region. The acute vascular insult results in leaks and subsequent rarefaction of peritubular capillaries with distension of surviving capillaries .
Data on the renal pathology of human AKI remain limited because the lack of systematic studies describing early, intermediate and late changes. In contrast, the renal pathology in animal models of AKI has been extensively described. Yet, the morphological changes in animal kidneys cannot be directly compared to the human situation since the majority of animal studies are conducted in otherwise healthy animals, i.e. animals that lack comorbidities that are regularly present in patients with significant AKI. It holds, therefore, that effective therapies can only be developed if cellular, molecular, pathophysiological and morphological manifestations of AKI are fully understood and thus comprehensively targeted.
The incidence of AKI is becoming more common in the hospital, mainly caused by an aging population subjected to high-risk procedures while being affected by high rates of cardiovascular, diabetic, hepatic and pulmonary comorbidities, rendering these patients particularly susceptible to AKI . AKI is also becoming an increasingly important cause of end-stage renal disease (ESRD) since a significant percentage of patients with AKI progresses to ESRD within 2 years after diagnosis (United States Renal Disease Survey, 2006) .
It is well recognized that, despite the provision of intensive, continuous renal replacement and all other needed supportive therapies, morbidity and mortality, exceeding 50%, and associated treatment costs in patients with dialysis requiring degrees of AKI have remained disturbingly high . This dismal situation, together with the late development of ESRD post-AKI and high cost burdens, continue to make the development of effective interventions a therapeutic urgency. Despite the fact that extensive preclinical studies with novel agents showed and continue to show renoprotective efficacy in experimental AKI, the data from a subsequent clinical trial that evaluated insulin-like growth factor-1 (IGF-1) were either inconclusive or failed to demonstrate efficacy . The use of the highly original Renal Assist Device in a group of patients with severe AKI was promising, as its use improved both renal outcomes and patient survival . Currently, however, this complex system is not available in the clinic.
In conclusion, the frequency of AKI in hospitalized patients and its impact on the outcome of underlying comorbidities, as well as the high mortality and therapeutic costs associated with AKI, urgently warrant the development of effective therapeutic interventions. The reality, however, is that there is no specific pathophysiology-based treatment approach, and care remains limited to supportive measures such as fluid, electrolyte and blood pressure management and, as needed, provision of various renal replacement modalities.
Treatment Resistance of Clinical Acute Kidney Injury
There are numerous reasons why significant clinical AKI has essentially remained and/or appears to be unresponsive to renal replacement therapy (RRT) and resistant to numerous novel interventions that have been tested in clinical trials . All utilized modalities of hemodialysis provide only partial replacement of renal functions and, in addition, are invasive and thereby affected by their associated complications.
It is well documented that more elderly and severely ill patients with significant comorbidities are being subjected to high-risk surgical procedures, resulting in an increased incidence of AKI. Recent studies have confirmed that different dialysis modalities and higher doses of dialysis in patients with AKI do not improve outcomes . The ideal treatment for AKI targets its complex pathophysiology directly by interfering with all major cellular and molecular processes causing AKI, and thereby improving adverse multisystem complications. At the time of writing, essentially all novel therapeutic approaches in AKI lack a solid, broad-based pathophysiological focus.
Experimental Models of Acute Kidney Injury
While it is well recognized that major comorbidities increase the susceptibility of patients to developing severe AKI, practically all animal studies that show therapeutic benefit in AKI have ignored this critical fact by having been conducted in otherwise healthy animals. This approach is in frank contrast to AKI in the hospital setting, where patients with comorbidities such as diabetes or other multisystem diseases are at highest risk for severe AKI. These limitations of commonly used models are in part responsible for the slow and sometimes unsuccessful translation of experimental therapies into clinical practice. The present authors recently began to address this issue, showing that AKI induced in rats with underlying chronic kidney disease (CKD) (5/6th nephrectomy), i.e. acute on chronic renal failure, can be readily treated with mesenchymal stem cells (MSCs) (unpublished data).
For the purpose of simplification, clinical and experimental AKI have been divided into categories based on ischemic and toxic insults. For each of these, a number of animal models is available in which their respective pathophysiology and experimental therapies can be studied. These models are widely used; however, they are associated with significant limitations and their pathophysiology only reflects certain aspects of human injury .
Ischemic models (primarily warm ischemia with reperfusion) most commonly use clamping of the renal arteries with complete cessation of blood flow for varying times, causing different degrees of injury. The principal determinant of injury in this setting is tissue hypoxia due to blood flow cessation with changes in outer medullary microcirculation, subsequent induction of inflammation and tubular cell injury, mainly affecting the S3 segment of the proximal tubule. Reperfusion induces formation of ROS with resulting cellular injury. While hypoxemia and hypotension are commonly seen clinically, complete cessation of blood flow with massive cellular injury and frank cortical necrosis, as seen in animal models, is rare.
Nephrotoxic models include exposure to cisplatin, heavy metals, aminoglycoside antibiotics and glycerol-induced rhabdomyolysis with heme pigment release. These agents cause multifactorial tissue damage, including apoptosis and hypoxia, mainly in tubular cells, but some are not nephrotoxic in humans or toxicity can be prevented, e.g. by vigorous hydration before and after the administration of cisplatinum .
Several models have been developed that claim to be similar in their pathophysiology to the decline in renal function that is associated with sepsis. These sepsis models include endotoxin administration, bacterial infusion, cecal ligature and perforation, and intraperitoneal infusion of bacteria. While sepsis-associated AKI is an important independent contributor to morbidity and mortality, these models only satisfy some aspects of the complicated pathophysiology of AKI in critically ill patients . The cecal ligation model is primarily a model of cytokine storm and to some extent neglects the fluid imbalance and vascular aspects of human sepsis. The response to endotoxin is very variable across species and rather inconsistent in rodents. There is also considerable variability in morbidity and mortality of animals among laboratories using these models.
Design of Clinical Trials
The design of a successful clinical trial requires that a suitable clinical setting is chosen in which the efficacy of a novel intervention can be reliably tested. For example, lack of a proper study design probably contributed to the apparent failure of a trial with IGF-1 in AKI patients . In these studies, the enrolled subjects represented an inhomogeneous cohort of patients with ARF in whom a tested intervention was given at widely different time-points after the initial insult, and the study drugs were harmful because they caused hypotension.
These examples strongly argue for study designs that test novel therapies in a clinical setting where the time-point of a potential renal insult and the subsequent intervention are clearly defined . Mindful of this, the present authors chose to test the safety and preliminary efficacy of allogeneic MSC therapy in patients undergoing on-pump CABG and/or valve surgery, the potential renal insult, and precisely defined the postoperative time-point at which allogeneic MSCs, i.e. the therapy, were given (see below). In addition, realistic endpoints were chosen, changes in novel biomarkers for AKI were assessed and the RIFLE criteria were used to define AKI (see below). Based on the same rationale, a clinical trial will be conducted in recipients of cadaveric kidney transplants who, being at high risk of delayed graft function (DGF), will be treated with allogeneic MSCs. In this setting, both the renal insult (cold ischemia time, surgery) and time of cell administration are well defined, facilitating the optimal assessment of safety and preliminary efficacy of such an intervention.
Novel Diagnostic Biomarkers for Acute Kidney Injury
Under most steady-state conditions serum creatinine levels are a reasonable indicator of glomerular filtration rate (GFR) but are not specific for an acute renal injury per se. There can be marked injury with relative preservation in GFR and vice versa. An ideal marker for AKI would identify risk, provide prognostic information for the possible development of AKI, specifically diagnose both renal injury and loss of function, and indicate successful recovery with resolution of the causative derangements. In recent years several novel biomarkers for kidney injury have been identified. The most promising include kidney injury molecule-1 (KIM-1), interleukin-18 (IL-18), neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C . Since a rise in serum creatinine in patients with AKI may not be the result of an actual renal injury, and even if caused by an actual injury, it is often delayed by 24–48 h, its levels may be affected by its generation rate or by changes in extracellular volume. From this it follows that sole reliance on serum creatinine levels is inadequate for the proper management of this complication. In addition, a decrease in urine output, which can also be the result of prerenal or postrenal complications and thus not represent actual renal injury, may further prevent the prompt institution of potentially effective therapies. The introduction, therefore, of such novel diagnostic biomarkers for AKI is essential for its early and specific diagnosis, permitting the administration of therapies within a few hours after a given insult. This approach is expected to significantly improve overall outcomes in patients with AKI. However, whether this is seen with the currently available treatment options remains to be shown.
The authors believe that single pharmacological agents that focus only temporarily on some aspects of the highly complex pathogenesis and pathophysiology of AKI (summarized in Fig. 21.1 ) are inferior to stem cell-based interventions. The reason for this may lie in the fact that infused MSCs, as described below, home to the site of injury, adhere to the microvascular endothelium in a regulated fashion, as has been shown in the heart , interpret and respond to cues from the damaged microenvironment by changing their own gene expression profile and that of responsive renal cells to a cytoprotective, anti-inflammatory and repair-stimulating pattern. After effectively carrying out these actions, which requires generally less than 72 h in the kidney, MSCs detach in a regulated fashion, leave the kidney and undergo anoikis/apoptosis in the circulation. As further discussed in detail below, these complex organ protective and regeneration-stimulating effects of MSCs are mediated by paracrine and endocrine mechanisms, and not by their differentiation into and replacement of lost target cells or significant fusion with target cells.
Injury to the kidney or other solid organs may result in the transient expression or export of the biology of the stem cell niche, i.e. the creation of a facultative niche, in which small numbers of administered MSCs function as they do in the bone marrow or in a pericyte location, i.e. they respond to insults at these sites by carrying out anti-inflammatory, cytoprotective and repair-supporting programs. These nursing effects of MSCs are well recognized in clinical bone marrow transplantation, where these cells are known to facilitate hematopoietic stem cell (HSC) engraftment and afford their protection through identical paracrine actions .
Treatment of Acute Kidney Injury with Stem Cells: Preclinical Studies
Pathophysiology and Repair of Acute Kidney Injury
Ischemia–reperfusion and toxic insults to the kidney result in a rapid succession of pathophysiological events or stages of AKI that lead to loss of function ( Fig. 21.1 ). Molitoris and Sutton recently described these successive pathophysiological stages as an initiation phase, injury phase, vascular extension phase and recovery phase . These phases are characterized by decreases in renal blood flow, decline in GFR, vascular congestion, tubular and vascular damage, epithelial cell depolarization, transepithelial and vascular leaks, cell loss due to apoptosis and cell necrosis, tubular obstruction and accumulation of inflammatory cells in the microvasculature. At the molecular level, the generation of ROS and upregulation of proinflammatory cytokines and other maladaptive responses are recognized as major mediators of the evolving renal injury .
The kidney, particularly in younger individuals or those with less severe degrees of kidney injury, is readily capable of repairing itself, achieving within days return of normal or adequate kidney function. Organ repair, as is now well established, is primarily carried out by intrinsic renal cells . At the gene level, re-expression of developmental programs that are active in organogenesis participate in the overall repair process . It appears likely that additional contributions to the repair process are carried out by circulating stem and progenitor cells, such as MSCs, HSCs and endothelial progenitor cells (EPCs) . In addition, a contribution of kidney intrinsic stem cells to the repair process has been suggested by Oliver et al. . These investigators identified cells with stem cell characteristics in the rodent papilla, a location that may represent bona fide stem cell niches. Post-AKI, these cells appear to migrate towards the cortical areas of tissue damage, where they may support the repair process. MSCs that are thought to reside in a pericyte location within the kidney may similarly participate in local repair, a possibility that awaits investigation.
It is apparent, however, that the collective capacity of these kidney intrinsic and extrinsic organ-protective and repair-stimulating mechanisms is overwhelmed when the insult is too severe. This is the reason why several investigations have tested whether the therapeutic boosting of stem or progenitor cell delivery to the injured kidney would improve outcomes. The efficacy of these cell-based interventions was reproducibly demonstrated in cisplatin-, glycerol- and ischemia–reperfusion injury (IRI)-induced models of rodent AKI (see below).
Hematopoietic Stem Cell Mobilization and Administration
The initial rationale for the subsequent administration of multipotent, bone marrow-derived stem cells was based on the hypothesis that the acutely injured kidney would generate signals for stem cell mobilization, from the bone marrow, and their homing to the kidney. There, they would adhere to endothelial cells at sites of injury and contribute to repair by differentiation into and replacement of lost renal cells. This response, it was hypothesized further, may represent another recapitulation of a developmental program, according to which stem cell delivery, via the primitive circulation of the embryo, is boosted during organogenesis. Data were generated that were partly in support of this generally held paradigm. Bilateral or unilateral IRI AKI in mice resulted in the prompt mobilization, from the bone marrow, of CD34 + cells (HSCs, endothelial progenitor and other cells) into the circulation and recruitment to the injured kidney only. The mobilization and homing signal that was strikingly upregulated in the injured kidney was stromal cell-derived factor-1 (SDF-1) or CXC chemokine receptor-12 (CXCR12), a chemokine that has a central function in hematopoietic bone marrow niches, where it is produced by reticular and mesenchymal precursor cells, i.e. MSCs . It facilitates recruitment and engraftment of intravenously administered HSCs to the bone marrow niches of a bone marrow transplant recipient. Both HSCs and MSCs express CXCR4, the cognate receptor for SDF-1, albeit only in low numbers in MSCs . Physiologically, and even after bone marrow ablation, the highest SDF-1 levels are found in the bone marrow. However, this gradient was reversed following AKI, resulting in higher renal than bone marrow levels of SDF-1. Both in vivo and in vitro, inhibition of CXCR4 activation reversed the recruitment of CD34 + CXCR4 + cells to the injured kidney or their migration across a transwell, identifying SDF-1 as the principal mobilization and homing signal in this experimental setting. Similar roles of the SDF-1–CXCR4 axis have been identified in the injured brain, liver, heart and other organs . Of note was the fact that the renal expression of CXCR4 was also upregulated following AKI, a response whose ultimate significance remains to be elucidated .
Since AKI resulted in a significant increase in CD34 + cell numbers both in the circulation and in the kidney, it was hypothesized that these multipotent cells must have the capacity to contribute to organ protection and repair. Accordingly, it was tested whether G-CSF and cytoxan-induced augmentation of circulating CD34 + cell numbers at the time of AKI induction would exert a robust kidney protective effect, i.e. in analogy to the beneficial effects that stem cell mobilization had in a mouse myocardial infarct model . In the latter, mobilized HSCs were reported to differentiate and engraft in the myocardium, resulting in overall better outcomes. Unexpectedly, outcomes (survival, renal function, histopathology) in animals that were pretreated with an HSC mobilization protocol were extremely poor compared with vehicle-treated controls ( Fig. 21.2 ). This was the result of concurrent and marked increases in peripheral neutrophil numbers, which abrogated any potential beneficial effects of mobilized HSCs by causing frank renal cortical necrosis. Animals rendered neutropenic before the induction of AKI had significantly improved outcomes, confirming that the adverse actions of stem cell mobilization were exerted by the renal delivery of high numbers of neutrophils. However, several studies with HSCs alone or following bone marrow transplantation and mobilization in experimental AKI suggested their therapeutic potency, mediated by their engraftment following differentiation , while administration of HSCs to animals with AKI by other investigators were without benefit . From this, it follows that stem cell/progenitor cell mobilization protocols that are associated with a simultaneous rise in circulating leukocytes must be avoided. A recent elegant study by Park et al. demonstrated that spontaneous mobilization and recruitment to the kidney of HSCs, MSCs and EPCs play an important role in the postobstructive repair of the kidney that is injured by unilateral ureteral obstruction (UUO). Additional evidence for the role of the SDF-1–CXCR4 axis in MSC recruitment to the obstructed kidney was also provided.
In analogy of what has been shown for HSCs, the SDF-1–CXCR4 axis is also involved in the recruitment of CXCR4-expressing MSCs that are administered to animals with AKI . This also applies, at least in theory, to the homing of EPCs given to rodents with AKI. Herrera et al. identified CD44, hyaloadherin, the receptor for hyaluronic acid that is expressed on MSCs, as an important mediator of MSC recruitment by the kidney that is injured by glycerol-induced rhabdomyolysis . In this model, interstitial hyaluronic acid is robustly upregulated, facilitating intrarenal binding of administered MSCs and subsequent improvement of kidney function.
Administration of Endothelial Precursor Cells
Patschan et al. demonstrated that interventions that use EPC administration were highly effective in improving renal function by repairing microvascular injury and hemodynamics in animals with AKI . Details of this work are described in Chapter 18 . In analogy to these observations, MSCs elicit a similar vasculoprotective effect early after AKI in rats and such treatment of AKI protected microvascular density later after the renal insult .
Administration of Multipotent Marrow Stromal Cells
This second type of adult stem cell in the bone marrow cannot be readily mobilized into the circulation, in part owing to its extravascular localization both in the bone marrow niches and in their pericyte location on blood vessels . Bone marrow niches are low oxygen pressure compartments in which MSCs and HSCs are localized both adjacent to the endostium (endosteal or osteoblastic niche) and next to sinusoids (perivascular niche) . At these sites, MSCs secrete, together with reticular cells and megakaryocytes, soluble factors in a paracrine fashion, which importantly include SDF-1. These collectively regulate the maintenance and survival of HSCs in their respective niches . Unlike relatively large MSCs, comparably small HCSs can readily enter the peripheral circulation via characteristic fenestrations in the walls of sinusoidal vessels.
In 1970, Friedenstein et al. first described fibroblast-like cells growing out in culture of bone explantants and bone marrow suspensions , cells that were subsequently named MSCs . Friedenstein recognized the bone-forming potential of these cells and distinguished this new cell population from cells undergoing hematopoietic differentiation. These cells were initially named colony-forming units of fibroblasts (CFU-F) that could be readily obtained from the bone marrow, spleen and thymus of mice. Being clonogenic, these cells could additionally be induced to differentiate into osteocyte precursors, chondrocytes, adipocytes and bone marrow stroma cells in vitro. Subsequent studies reported that MSCs can also differentiate into cells from unrelated germ-line lineages, but some of these reports have been questioned and the full nature of the differentiation potential or plasticity of MSCs is a matter of debate that will have to await full elucidation of their in vitro and in vivo biology .
In an effort to standardize the nomenclature, a consortium of experts suggested the name “multipotent mesenchymal stromal cells” to refer to this population of fibroblast-like cells that are plastic adherent and can be differentiated into mesenchymal lineages . The defining property of MSCs is their ability to differentiate into various mesenchymal lineages, including bone, cartilage and adipose tissue, as well as tendon, muscle and marrow stroma . In the laboratory, trilineage differentiation into bone, adipose tissue and cartilage is taken as a major defining criterion for MSCs.
The research community is still debating whether these cells have properties of true stem cells and can therefore be called stem cells. This depends largely on the criteria by which a stem cell is defined, and whether that includes the necessity to differentiate into cell types of unrelated germ-line layers. Potential artifacts, induced during in vitro culture and by in vivo tracking methods, and their fusion with target cells, have raised questions as to their true differentiation into cells of non-mesodermal germ layers. Further complicating the issue of their stemness is the fact that true stem cells must be tightly interacting with niche cells to exist as such within a niche system . Since these aspects of the MSC biology remain incompletely understood, the question as to their definitive stemness (self-renewal, differentiation into a mesodermal cell type) remains unsettled, a point that may or may not be important.
The nature of resident MSCs in vivo is only poorly described, and many of their in vivo characteristics are unknown. It has been shown that MSCs can be derived from many tissues, including the bone marrow, adipose tissue, vasculature, cord blood and umbilical cord matrix . MSCs in the microvasculature are found in a pericyte location, from which, it has been suggested, they exert their organ-protective and vasculoprotective activity when a local injury occurs . They locally elicit potent anti-inflammatory effects that cooperate with their antiapoptotic and repair-inducing trophic actions. Despite defined functional differences between tissues of origin, MSCs express a number of common surface markers (CD29, CD44, CD49a–f, CD51, CD73, CD90, CD166, CD271, Stro-1) and lack expression of markers typical of hematopoietic lineages including CD11b, CD14 and CD45 .
A number of bone marrow-derived pluripotent cells that have the ability to differentiate into cells of mesodermal, endodermal and neuroectodermal lineages have been described.
The yield of MSCs from various tissues is very low. Their frequency has been estimated to be in the order of 0.001–0.01% of total nucleated cells, a percentage that is also dependent on harvesting methods and separation processes . Adipose tissue contains the highest numbers of MSC at approximately 400 × 10 6 /ml . MSCs can be expanded in vitro to hundreds of millions of cells from a 10–20 ml bone marrow aspirate. Most clinical studies have used fetal calf serum to expand MSCs in vitro, adding a potential biohazard to the culture system and potentially alloimmunizing the recipient . Effective culture expansion in animal serum-free media, using platelet lysate or other additives, is now well established . Because of their ability to proliferate extensively in vitro, MSCs seem ideal candidates for large-scale industrial expansion under good manufacturing practice (GMP) conditions. Development of culture systems that include closed bioreactors with larger overall surface areas for effective cell expansion is currently ongoing, and is expected to improve the economy and efficiency of the cell culture process, and thus provide for more efficient large-scale production in commercial applications .
MSCs do not induce significant alloreactivity in vivo because they express low levels of major histocompatibility complex (MHC) class I antigens and do not express MHC class II as well as costimulatory molecules including CD40, CD80 and CD86 on their surface . These features protect MSCs from natural killer (NK) cell-mediated lysis and make them attractive for large-scale applications in clinical practice . Figure 21.3 summarizes the currently identified immune-modulating activities that are exerted by MSCs . Additional details are provided in Chapter 9 .
Treatment with allogeneic human MSCs in the clinical setting did not induce MSC antibody production or T-cell priming, while antibodies against xenogenic tissue culture medium components such as fetal bovine serum were found . Because of this immune privilege, MSCs have become the premier cell type for clinical applications, representing an off-the-shelf allogeneic cell product.
Allogeneic MSCs have already been successfully used clinically in a number of different diseases. Horwitz et al. showed that allogeneic MSCs derived from the same donor who provided the initial bone marrow transplant for pediatric patients with osteogenesis imperfecta were able to engraft in the majority of recipients, resulting in improved growth velocity and bone mineralization. However, their overall contribution to bone cells was very low (in the range of 1%) and declined over time . Lazarus et al. reported that the combined administration of hematopoietic stem cells and MSCs from the same marrow donor in patients undergoing a bone marrow transplant for hematological malignancies was safe, feasible and resulted in improved bone marrow engraftment . It was reported that steroid-refractory acute graft-versus-host disease was resolved in six out of eight transplant recipients after MSC treatment .
To date, MSCs, also termed multipotent marrow stromal cells, have been the mainstay of non-hematological cell therapy in the clinical setting. This is facilitated by the fact that they can be safely administered in an allogeneic fashion, and their use in most conditions has been safe so far. Intravenous infusions of MSCs has been shown to improve repair of multiple organs such as bone , ischemic brain , heart and pancreas , and found to favorably modulate the immune system (see above). Importantly, these beneficial effects were shown to be independent of long-term engraftment but instead were mediated by local effects of these cells after transient vascular adhesion or engraftment at the site of injury. Figure 21.4 summarizes the spectrum of trophic actions that have been identified in endogenous and administered MSCs. Importantly, in humans, no long-term adverse effects have been reported so far.
Administration of Multipotent Marrow Stromal Cells in Experimental Acute Kidney Injury
In recognition of the biology of MSCs in the bone marrow niches and as pericyte-like cells in practically all blood vessels, as discussed above and in Chapter 9 , together with their effective use in a number of experimental and clinical disorders, their therapeutic potential and mechanisms of action were tested in rats with IRI AKI.
As stated above, the ideal treatment of AKI would address its complex pathogenesis and pathophysiology directly, comprehensively and throughout its successive phases, thereby interfering with harmful processes that lead to the full expression of this syndrome, while initiating and carrying out regenerative programs that will result in return of function ( Fig. 21.1 ). Cell therapy appears to possess the unique capacity to address virtually all major aspects of the pathophysiology of AKI, thereby providing a fundamentally novel therapeutic approach that appears vastly superior to single-agent pharmacotherapy. Cell therapy has, in principle, the potential to replace cells that are lost through injury, to enhance the survival of sublethally injured cells that are about to undergo apoptosis, to stimulate regeneration by boosting mitogenesis, to decrease inflammation at the site of injury, to modify cytokine secretion patterns of damaged cells, to enhance recovery of damaged endothelial cells and thereby improve microcirculation, and to activate tissue-resident stem cells at the site of injury .
Since the pathophysiology of AKI involves mainly tubular and vascular damage, an optimally effective agent to prevent or attenuate AKI should have vascular, hemodynamic and tubular effects. Cells are capable of interpreting and reacting to microenvironmental changes, such as hypoxia, oxidant stress or altered cytokine and growth factor levels, in real time and can thereby specifically react to pathological derangements at sites of injury. Such actions have the capacity to change the microenvironment at the site of injury from one that is deleterious to one that results in a favorable outcome.
Despite the substantial theoretical advantages of cell therapy in clinical applications, the main safety concern with cell therapy is their long-term engraftment with potential adverse effects such as fibrosis, scar formation, ectopic differentiation and tumorigenesis, issues that are further addressed in Chapter 23 . Exogenously administered MSCs have been shown to protect kidney function and improve kidney repair in a number of animal models of AKI, and it was shown in humans that bone marrow cells contribute to renal cell types including tubular cells .
Treatment of Cisplatinum and Glycerol-induced Acute Kidney Injury with Mesenchymal Stem Cells
Injection of MSCs protected against cisplatin-induced AKI , while infusion of purified HSCs had no protective effect in the same setting. Fluorescence in situ hybridization (FISH) for Y-chromosomes in a model in which male MSCs were injected into female hosts showed that donor-derived MSCs contributed and integrated into the tubular epithelium. The exact quantity of the contribution was not determined, but proved to be very small in later studies. Herrera et al. provided data showing a relatively large contribution of 20% of donor derived MSCs to tubular epithelium in a glycerol model of AKI . Other studies from different groups did not observe significant engraftment of donor-derived cells and thus argue against long-term engraftment or significant fusion with target cells, and significant restoration of epithelia by donor-derived cells .
The process of MSC differentiation and incorporation into tubular epithelia is rare and inconsistent, and occurs after renal function is essentially normalized, and therefore cannot explain the improvement in outcome compared to controls. Subsequent studies using more rigorous methodologies showed that mechanisms independent of differentiation are responsible for the overall improvement of AKI in animal models .
Most of the diverging data regarding renal engraftment of differentiated MSCs can be explained by differences in the models and, most importantly, by the cell labeling and tracking methods that were used . Methodically more rigorous studies, cited above, established the absence of significant and long-term engraftment of MSCs that have assumed an epithelial cell phenotype, and thus further argue for paracrine mediator mechanisms . Timing of cell delivery relative to injury and recovery from injury, seen as early as within 24–48 h, does not correlate with the very rare and late engraftment of differentiated MSCs. This discrepancy represents another powerful argument in favor of an overall paracrine mode of action of these cells. Rigorous lineage analysis provided conclusive evidence that most epithelial regeneration is undertaken by endogenous tubular cells.
Based on these observations in the kidney and other organs it is currently generally accepted that paracrine mechanisms underlie the organ-protective activity of administered MSCs in most tested injury models. These mechanisms involve immunomodulation ( Fig. 21.3 ), secretion of growth factors and antiapoptotic actions ( Fig. 21.4 ). Immunomodulation involves all of the MSC properties described above, including T-cell modulation, effects on NK cells, and suppression of B-lymphocyte proliferation and antibody production . MSCs secrete a broad range of growth factors that have been shown to be renoprotective and data have been published that link some of these factors, namely vascular endothelial growth factor (VEGF) and IGF-1, directly to renoprotection . Data showing remote endocrine effects of MSCs injected intraperitoneally provide further evidence of the paracrine/endocrine hypothesis of action . Recent data show that MSC-derived microvesicles enhanced proliferation in vitro and conferred resistance of tubular epithelial cells, via horizontal transfer of mRNA, to apoptosis in vivo .
Homing mechanisms of administered cells are largely unknown but a number of studies provide evidence of the involvement of cytokines such as SDF-1 in this process . Hypoxic preincubation of MSCs appears to increase engraftment in vivo and the mechanism involves upregulation of the SDF-1–CXCR4 axis . Another potential homing receptor is CD44, which is expressed on MSC and binds to upregulated hyaluronic acid in the interstitial space of the kidney in glycerol-induced AKI . The migration response of MSCs is induced by growth factors and chemokines, the latter augmented by preincubation of MSC with tumor necrosis factor-α (TNF-α) . Understanding the processes involved in homing and migration of stem cells after injury is vital in designing effective therapies that optimize the targeting efficiency of therapeutic cells to injured organs.
Treatment of Ischemia–Reperfusion Acute Kidney Injury in Rats with Autologous and Allogeneic Mesenchymal Stem Cells
Renal Function and Histopathology
As others in the field had primarily focused on toxic models of AKI (see above), the present authors chose to investigate the therapeutic utility of syngeneic and later allogeneic MSCs in the rat with IRI AKI. It was established that the immediate or delayed administration, at 24 h after reflow, of syngeneic as well as allogeneic MSCs promptly protected renal function and hastened subsequent recovery of function, associated with decreased injury and apoptotic scores, and increased proliferative indices ( Fig. 21.5 ) . Instead of using an intravenous, intraperitoneal or intraparenchymal route of administration, as used by others, these cells were injected into the suprarenal aorta. This was done to avoid the induction of potential respiratory distress in animals (and in future study subjects), an adverse effect that has been shown to result from trapping of these large cells in the microcirculation of the lungs . In addition, the reasoning was that with this administration route a smaller cell dose for the effective treatment of AKI may be sufficient. Extensive dose–response studies in rats with IRI AKI were performed, which found that this therapy is safe and effective when either syngeneic or allogeneic MSCs are infused . Infusion of identical doses of syngeneic fibroblasts to animals with AKI failed to improve outcomes, demonstrating that the organ-protective effects are specific to MSCs.