Outline
Introduction 341
Clinical Trials of Regenerative Medicine in Other Disciplines 342
Clinical Trials on Renal Regeneration 346
Future Perspectives in Clinical Regenerative Nephrology 347
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.
Introduction
Kidney disease is a growing global public health issue. Chronic kidney disease (CKD) has a prevalence of over 10% in the general population and this number is increasing by almost 3% per year . Advanced CKD is associated with a more than three-fold increased risk for cardiovascular events and an almost six times increased mortality . Acute kidney injury (AKI) is also an increasing clinical problem, with an incidence of 15 patients per 1000 patient years (Medicare database). Mortality rates of AKI are up to 80% and more than 10% of the survivors eventually develop end-stage renal disease (ESRD). More than 350 new cases of ESRD per 1,000,000 population are seen every year, according to the United States Renal Data System (USRDS) 2009 annual data report. Thus far, therapeutic strategies for kidney disease have focused on the reduction of tissue damage and supportive care.
However, renal disease and renal disease progression can be envisioned as the result of the kidney injury in combination with an insufficient regenerative response. Both glomeruli and tubuli are known to have regenerative capacity in humans . In the past decade, the recognition that growth factors and progenitor cells are involved in the regeneration of tissues has heightened interest in enhancing tissue regeneration ( Table 22.1 ). Preclinical trials have shown that therapeutic strategies can enhance renal regeneration. Therapeutic regeneration may be an interesting therapeutic strategy in renal disease.
| Therapy | Subtype | Administration |
|---|---|---|
| Growth factor therapy | Recombinant human | Subcutaneous |
| Gene therapy | Systemic intravenous | |
| Plasmid | Local intra-arterial | |
| Adenoviral | Direct tissue injection | |
| Cell therapy | Unselected BMDCs | Systemic mobilization |
| Selected BMDCs | Systemic intravenous | |
| MSCs | Local intra-arterial | |
| CD133 | Direct tissue injection | |
| CD34 | ||
| Cultured MNCs | ||
| Mature differentiated cells |
Clinical trials investigate the relation between a certain input variable (e.g. a therapeutic intervention or prognostic test) and a certain output variable (e.g. organ function, disease outcome, patient survival) in a well-defined patient population, and aim to estimate phenomena in a population, by making measurements on samples from the target population . Clinical trials form the cornerstone for the introduction of new prognostic and therapeutic procedures in the clinic. The number of clinical trials in nephrology is quite low compared with other internal medicine disciplines . There appears to be a barrier in the translation of basic science to human studies, which may relate to regulatory burden, high research costs and lack of funding. However, despite similar hurdles, strategies to enhance regeneration in cardiovascular medicine and hepatology have been translated into clinical trials with positive results. These results, combined with promising preclinical studies on therapeutic renal regeneration, suggest that clinical introduction of therapeutic renal regeneration is at hand. This chapter will discuss regenerative strategies in other organ systems, (pre)clinical studies on renal regenerative therapies and the translation of successful preclinical strategies into clinical trials on therapeutic renal regeneration.
The first section of this chapter will review strategies and outcome parameters applied in prognostic and therapeutic clinical trials on regeneration in other disciplines. The focus will be on cardiac neovascularization because of the crucial role of the vasculature in renal function, maintenance and repair, and on hepatic regeneration because kidney and liver are both highly vascularized parenchymatous organs. In the second section the available clinical data on renal regeneration will be discussed. The third section of this chapter will discuss translation of animal experiments from cage to clinic. As the majority of preclinical studies so far have investigated therapeutically enhanced regeneration in models of acute tubular damage, the primary focus will be on the translation of therapeutic strategies for acute tubular damage.
Clinical Trials of Regenerative Medicine in Other Disciplines
Therapeutic Neovascularization in Cardiovascular Medicine
Blood vessels are of crucial importance for renal function, maintenance of renal integrity and renal repair . Cardiovascular studies aiming at the generation of new blood vessels (neovascularization) may have great relevance for regenerative nephrology. During neovascularization, proliferating mature endothelial cells are one source of new endothelial cells. Growth factors play an important role in the regulation of endothelial cell proliferation. In addition, circulating endothelial progenitor cells stimulate neovascularization, both by structural participation in the vasculature and by paracrine effects ( Table 22.1 ) . Both prognostic studies to assess regenerative capacity and therapeutic trials to enhance regeneration, using growth factors or stem cells, have been performed in patients with cardiovascular disease (CVD). The different diagnostic and therapeutic strategies, as well as the outcome parameters used in these studies, will be discussed here.
Prognostic clinical trials have shown that parameters of regeneration relate to clinical outcome in CVD. Elevated plasma levels of hepatocyte growth factor (HGF) were associated with improved collateralization and a favorable prognosis in patients with myocardial ischemia . A decreased number of circulating endothelial progenitor cells (EPCs), suggesting low regenerative capacity, correlated with increased cardiovascular mortality in patients with coronary artery disease . However, others report data that seem conflicting. Serum growth factor levels in patients with angina correlated with the development of myocardial infarction and several studies have shown positive correlations between concentrations of circulating growth factors and myocardial damage and mortality . The increase in circulating growth factors or progenitor cells depends on both the amount of tissue injury and the quality of the regenerative response and therefore may reflect not only the process of regeneration but also the amount of tissue damage.
Therapeutic trials of growth factor administration to enhance neovascularization in patients with cardiac ischemia showed variable results . Growth factors regulate the proliferation and migration of endothelial (progenitor) cells, and form a promising strategy to enhance neovascularization. Therapeutic use of recombinant growth factors allows for the precise control of the administered dose. Recombinant growth factors can be administered systemically (e.g. intravenous injection) or locally (e.g. intracoronary or intramyocardial administration). Local delivery can specifically increase the local concentration while reducing possible systemic adverse events, but requires invasive procedures such as catheterization or the surgical implantation of minipumps.
In the Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis (VIVA) trial recombinant human vascular endothelial growth factor (rhVEGF) in patients with stable angina was shown to improve clinical parameters . Fibroblast growth factor (FGF) has been shown to increase the formation of capillaries, reduce myocardial ischemia, increase left ventricular ejection fraction and lower the rate of angina recurrence . However, these results were not reproduced in the large, multicenter, randomized, double-blind, placebo-controlled FGF-2 Initiating RevaScularization Trial (FIRST) . The disadvantage of recombinant protein therapy is the short half-life in vivo due to rapid degradation by circulating proteases and hence the need for a relatively high dose and/or repeated administration of the recombinant protein to induce a therapeutic effect. This may increase the occurrence and intensity of adverse events. Therefore, administration of plasmids has been used to deliver proangiogenic factors locally and continuously. All clinical studies that used gene therapy to enhance neovascularization used local administration by either intracoronary infusion or intramyocardial injection. Plasmids have a good safety profile because they are not integrated into the host genome and plasmid levels become undetectable within weeks of administration because of breakdown by nucleases. Plasmids of several isoforms of FGF and VEGF enhanced myocardial perfusion and myocardial wall motion, and reduced angina in patients with chronic myocardial ischemia . However, results were not very consistent: some found increased perfusion without functional improvement, while others found functional improvement without increased perfusion. In addition, although some studies demonstrated increased plasma growth factor levels after transfection and improved cardiac function, others demonstrated improved cardiac function without a significant increase in plasma growth factors . Moreover, the GENASIS trial, designed to enhance exercise tolerance using VEGF-C plasmid in “no-option” coronary artery disease patients, was stopped prematurely because of the high likelihood of lack of effect . The limited effect of plasmids may be the flipside of their good safety profile: low transfection efficacy and rapid breakdown.
Viral vectors can be used to obtain a more efficient transfection and sustained gene expression. However, the use of viral vectors can be complicated by an inflammatory response . In addition, the integration into the host genome raises important safety concerns, specifically the possibility of insertional mutagenesis with subsequent malignant transformation . In the REVASC study, “no-option” patients with coronary artery disease were treated with replication-deficient adenovirus containing VEGF . This study showed improvement in exercise-induced ischemia and decreased angina symptoms but no increase in myocardial perfusion. The Angiogenic GENe Therapy (AGENT) trials used replication-deficient adenovirus containing the human FGF gene to enhance neovascularization in patients with angina . Although the safety profile was good and initial results were promising, the AGENT-3 and 4 trials were stopped prematurely because the interim analysis indicated that a significant difference in the primary efficacy endpoint was unlikely. In these trials adenoviral transfection did not increase the plasma levels of FGF. In summary, gene therapy, using either plasmids or replication-deficient adenoviruses, has not been shown to give consistently positive clinical results.
Progenitor cells have also been used in clinical trials to enhance neovascularization in patients with ischemic heart disease. Recent meta-analyses demonstrate that intracoronary and intramyocardial infusion of bone marrow-derived cells (BMDCs) in patients after myocardial infarction appears to be safe and shows a significant, consistent, clinically relevant but moderate improvement in left ventricular ejection fraction, left ventricular end-systolic volume and myocardial lesion area . Initially, it was speculated that BMDCs would enhance regeneration by reducing apoptosis of cardiomyocytes and stimulating proliferation of myocardial (progenitor) cells. However, more recent data suggest that the beneficial actions of BMDCs on myocardial function are mainly due to enhanced neovascularization and paracrine effects . BMDC infusion 5–7 days after myocardial infarction appeared to be superior to early administration, suggesting that postinfarction inflammation may attenuate BMDC-enhanced regeneration.
Infusion of subpopulations of BMDCs or cultured progenitor cell populations has also been used to enhance regeneration in patients with myocardial infarction. In the Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI) trial both intracoronary infusion of cultured autologous peripheral blood-derived progenitor cells and bone marrow mononuclear cells improved coronary blood flow reserve, coronary conductance capacity, myocardial viability, regional wall motion, end-systolic left ventricular volume and left ventricular ejection fraction. The CD34 + subpopulation of BMDCs reduced angina frequency and increased exercise time, whereas the CD133 + subpopulation enhanced myocardial perfusion and left ventricular function . Another interesting subpopulation is the mesenchymal stromal cell, also known as the mesenchymal stem cell (MSC). MSCs have been shown to have therapeutic properties in several preclinical studies . They may offer advantages over BMDCs. MSCs are immunologically privileged cells that are not rejected after allogeneic transplantation . However, it should be noted that under certain conditions MSCs are subject to immune rejection . MSCs home to areas of injury and can be infused intravenously . Preclinical studies have shown that MSCs are able to enhance neovascularization . Hare et al. infused in vitro expanded allogeneic MSCs intravenously in patients after myocardial infarction and showed a reduction in ventricular arrhythmias and global symptom score without significant adverse events . Furthermore, ejection fraction was increased and reverse remodeling was seen using magnetic resonance imaging (MRI). The mechanism by which MSCs enhanced cardiac function was not evaluated. Because of the clinical effect combined with the possibility of allogenic use and a good safety profile, MSCs are a promising new therapeutic strategy to enhance regeneration. Infusion of (subpopulations of) BMDCs appears to enhance myocardial perfusion and, subsequently, myocardial function in patients after myocardial infarction.
The outcomes of clinical trials on cardiac neovascularization were evaluated at different levels ( Table 22.2 ). Several authors investigated whether infused BMDCs reach the infarcted region in human myocardium, by isotopically labeling BMDCs and tracing them using positron emission tomography (PET). They demonstrated that some of the injected BMDCs home to infarcted areas of the myocardium . This property also makes them ideal vehicles for the delivery of genes, e.g. for paracrine stimulation of regeneration . Homing differed between the various subpopulations of BMDCs. The direct effect of the intervention on tissue vascularization was assessed by quantitative coronary angiography, including coronary flow reserve. The effect on myocardial perfusion was assessed by contrast-enhanced MRI and myocardial perfusion scintigraphy [single-photon emission computed tomography (SPECT)]. In addition, the functional consequences of the intervention were evaluated by parameters such as left ventricular ejection fraction, end systolic volume and end diastolic volume, with the aid of echocardiography, left ventricular angiography and MRI, respectively. Heart failure was further quantified using N-terminal pro-brain natriuretic peptide (NT-proBNP) and N-terminal pro-atrial natriuretic peptide (NT-proANP) serum levels . Besides these functional parameters, clinical parameters such as exercise duration, angina score, recurrence of myocardial infarction, any revascularization procedure, death, clinical heart failure and quality of life were evaluated. Finally, possible side-effects of treatment were evaluated such as neointima accumulation, edema formation, inflammatory response, restenosis, elevated liver enzyme concentrations, increased tumor/teratoma formation, neovascularization in non-target organs, vascular malformations, increased atherogenesis or plaque destabilization and arrhythmias .
| Intervention | Intervention evaluation | Cell/tissue outcome | Clinical outcome | |
|---|---|---|---|---|
| Heart |
|
|
|
|
| Liver |
|
|
|
|
| Kidney |
|
|
|
|
Hepatic Regeneration
The liver shares several characteristics with the kidney. It is a highly vascularized parenchymatous organ that under normal conditions has a relatively low cell turnover, but is also able to recover from acute damage by an enormous capacity to proliferate. Under physiological conditions, as few as one out of 2000–3000 hepatocytes divides to maintain the physiological liver mass. However, liver damage or loss of liver mass can stimulate the regenerative capacity until the tissue mass has been restored by the proliferation of mature parenchymal liver cells . In rodents, up to 75% of surgically removed liver mass can be regenerated within 1 week . This model reflects what happens in human liver after partial hepatectomy (e.g. in case of tumor resection). Compensatory hyperplasia is also seen after liver transplantation when the recipient is larger than the donor .
Hepatic cell renewal is thought to be derived from three different sources. Mature hepatocytes and cholangiocytes are the earliest and most important source of tissue repair . In times of overwhelming cell loss, with longstanding iterative injury, or when hepatocyte replication is impeded, regeneration occurs via a second, poorly defined, cell compartment. This compartment seems to arise from a less differentiated cell population within the terminal branches of the intralobular biliary tree. Finally, bone marrow-derived hepatocytes and cholangiocytes have been reported in human bone marrow transplant recipients, although the relevance of this source for hepatocyte renewal is debated . BMDCs may enhance hepatic regeneration by other mechanisms than transdifferentiation. BMDCs have been shown to enhance liver regeneration by fusing with mature, differentiated liver cells . Furthermore, BMDCs can stimulate mature hepatocytes by paracrine mechanisms or by stimulation of neovascularization, which subsequently stimulates liver regeneration .
No trials have been performed that investigated the relation between regenerative parameters and liver disease prognosis. However, assessment of regeneration in liver biopsies may be used to evaluate the severity of disease . Human hepatic progenitor cells can be identified immunohistochemically in liver biopsies using markers such as OV6, CK7 and CK19. Livers of patients with massive necrosis showed increased numbers of hepatic progenitor cells over time. In addition, these cells migrated from their ductal progenitor cell niche into the liver lobule. The location and number of hepatic progenitor cells correlate to the severity of hepatic disease . Here, regenerative parameters may be viewed as an indirect parameter of organ damage, similar to the relation between serum levels of growth factors and CVD risk, because increased damage can also initiate increased regeneration.
Therapeutic trials to enhance liver regeneration, in patients with advanced stages of cirrhosis and in patients with a partial hepatectomy because of a hepatic malignancy, used several techniques . First, portal vein embolization or ligation has been shown to cause hypertrophy of the contralateral hepatic lobe . Makuuchi et al. first reported a beneficial effect of this novel approach for routinely inducing contralateral hypertrophy in patients with cholestatic liver disease, chronic hepatitis or cirrhosis . However, this strategy will not be applicable for the kidney because a reduction in nephron mass causes detrimental hemodynamic and structural changes in the remaining nephrons . A second strategy to enhance liver regeneration in patients with cirrhosis or patients with partial hepatectomy is mobilization of progenitor cells from the bone marrow using granulocyte colony-stimulating factor (G-CSF). Mobilization of CD34 + bone marrow-derived stem cells using G-CSF in patients with alcoholic steatohepatitis increased hepatic progenitor cell proliferation in liver biopsies, increased serum levels of regenerative growth factors (e.g. HGF) and improved liver function as measured by the Model for End-Stage Liver Disease (MELD) score . Third, peripheral infusion of undifferentiated BMDCs, obtained by bone marrow aspiration, has been shown to increase the number of proliferating cells in hepatic biopsies and subsequently the amount of viable hepatic tissue in patients with liver cirrhosis . Similar results have been shown for portal infusion of BMDC subpopulations. Autologous CD133 + BMDCs infused in patients after partial hepatectomy enhanced liver volume assessed by computer tomography (CT) . As a fourth strategy, a combination of G-CSF-induced mobilization, CD34 + cell isolation using apheresis, in vitro expansion and local infusion of CD34 + cells was used in patients with (alcoholic) liver disease. This strategy decreased serum bilirubin and transaminases, and improved ascites and Child–Pugh score, in uncontrolled trials .
Finally, intraperitoneal infusion of allogeneic fetal hepatocytes was used to treat fulminant hepatic failure . Ethical considerations may hamper the routine clinical use of such a strategy. Taken together, mobilization as well as local and systemic infusion of selected and unselected BMDCs can enhance liver mass and improve liver function in patients with cirrhosis and alcoholic steatohepatitis.
The outcome of these therapeutic strategies for regeneration of hepatic tissue was quantified at different levels ( Table 22.2 ). Progenitor cell mobilization from the bone marrow into the circulation using colony-stimulating factors was quantified by fluorescence-activated cell sorting (FACS) analysis of peripheral blood. The direct effect of therapeutic intervention at a cellular level has been evaluated by quantifying proliferation of hepatic (progenitor) cells in liver biopsies. Liver function was assessed using serum parameters such as albumin, prothrombin time, transaminases and bilirubin. Liver volume was serially measured using CT, and patient survival was evaluated. Finally, possible adverse events were assessed. Bone marrow infusion was complicated by a short period of fever, and leukapheresis by a reversible thrombocytopenia. In addition, infection rate, bleeding complications, alterations in liver perfusion and the appearance of focal liver lesions were assessed in liver regeneration trials; however, no increased incidence of these adverse effects was found in the intervention groups.
Clinical Trials on Renal Regeneration
Thus far, no clinical trials have specifically addressed therapeutic renal regeneration. However, some trials that used growth factors in patients with AKI or CKD have reported possible regenerative effects of these factors. In addition, prognostic studies have evaluated the relation between several parameters and renal disease prognosis. Although most of these parameters reflect inflammation or tubular damage, some can be considered parameters of regeneration.
Prognostic clinical trials in nephrology have mainly used markers of tubular inflammation and damage in blood and urine to predict renal prognosis ( Table 22.3 ). Increases in protein biomarkers such as urinary kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) or interleukin-18 (IL-18) have been shown to correlate with a worse renal prognosis . A relation between tissue and urinary mRNA levels of genes encoding for molecules involved in the pathogenesis of renal disorders was found to be related to the outcome of various renal diseases 1–12 months after biopsy . One recent prognostic clinical trial demonstrated a relation between increased VEGF mRNA level and improved clinical outcome in patients with lupus nephritis class III or IV. Levels of VEGF mRNA, assessed in renal biopsies, correlated negatively with crescent formation, histological activity index and endocapillary proliferation. Furthermore, renal VEGF mRNA levels accurately predicted deterioration of renal function in the next 12 months, defined as doubling of serum creatinine or the development of ESRD . To the authors’ knowledge, this is the only prognostic clinical trial that correlates the level of a regenerative parameter with improved clinical outcome. Other studies reported that higher plasma and urine levels of VEGF and HGF were associated with worse renal survival. This discrepancy is similar to that observed in cardiovascular studies and again may be explained by the fact that levels of growth factors are determined by both the extent of tissue injury and the quality of the regenerative response and therefore reflect both injury and regeneration.
| Serum | Urine |
|---|---|
| Neutrophil gelatinase-associated lipocalin (NGAL) | Neutrophil gelatinase-associated lipocalin (NGAL) |
| Cystatin C | Cystatin-C |
| Pro-atrial natriuretic peptide (pro-ANP) | γ-Glutamyltransferase (GGT) |
| Neutrophil CD 11b | IL-18 |
| Interleukin-6 (IL-6) | Glutathione- S -transferase (GST) |
| IL-8 | γ-GST |
| IL-10 | π-GST |
| A-GST | |
| Alkaline phosphatase (AP) | |
| Lactate dehydrogenase (LDH) | |
| β 2 -Microglobulin | |
| N -Acetyl-β- d -glucosamidase (NAG) | |
| α 1 -Microglobulin | |
| Kidney injury molecule-1 (KIM-1) | |
| Sodium–hydrogen exchanger isoform-3 (NHE-3) | |
| Matrix metalloproteinase-9 (MMP-9) | |
| Liver fatty acid-binding protein (FABP) |
Although no therapeutic clinical trials have primarily aimed to enhance renal regeneration, insulin-like growth factor-1 (IGF-1) administration was found to improve renal function in clinical trials that may, at least in part, be through regenerative mechanisms. IGF-1 in the circulation is bound to binding proteins (IGFBPs), of which seven subtypes have been identified. These carrier proteins are responsible for the bioavailability of the hormone, but mechanisms are ill-defined . IGF-1 was used to enhance glomerular filtration rate (GFR) and renal plasma flow (RPF) . However, IGF-1 is also abundantly expressed during tubular regeneration and may act as a renotropic agent, which can induce hypertrophy of tubules and glomeruli . Furthermore, IGF-1 reduces protein breakdown and exerts a generalized anabolic action . In animal models of ischemia–reperfusion, continuous subcutaneous administration of IGF-1, for 4 days starting 30 min after reperfusion, not only reduced serum creatinine and blood urea nitrogen and enhanced inulin clearance post-ischemia–reperfusion, but also reduced histological damage and mortality . The latter suggests that the beneficial effects of IGF-1 are mediated not only by temporary hemodynamic effects but also by structural changes in the kidney. In 1993, the first uncontrolled clinical trial, in four patients with CKD and a creatinine clearance between 25 and 60 ml/min, showed that administration of recombinant human (rh) IGF-1 (s.c. twice daily for 4 days) increased inulin and para-aminohippurate (PAH) clearance in all patients during treatment . After cessation of rhIGF-1, PAH clearance remained elevated in two out of four patients whereas inulin clearance returned to baseline values in all patients. In addition, IGF-1 treatment caused a temporary increase in renal volume, which returned to baseline after cessation of therapy. The acute and partially reversible effect on renal function suggests that IGF-1 stimulates the functional reserve of the damaged kidney. The persistently elevated PAH clearance in two patients may suggest some structural improvement in the kidney.
In a subsequent uncontrolled trial in nine patients with more severe renal failure (inulin clearance below 21 ml/min/1.73 m 2 ) treatment with rhIGF-1 (s.c. twice daily for 4–27 days) caused an increase in GFR and RPF and a decrease in blood urea nitrogen and plasma phosphate . No difference in renal volume was observed by CT. However, the effects of IGF-1 did not persist over time, possibly owing to increased removal of IGF-1 by the increase in GFR, downregulation of IGF-1 receptors, and reduced availability of the administered IGF-1 due to altered IGF-binding proteins during treatment. Furthermore, side-effects were reported: jaw pain, nasal congestion, Bell’s palsy, pericarditis and gingival hyperplasia .
Franklin et al. were the first to perform a randomized, placebo-controlled, double-blind trial aimed at preventing decline in renal function after major vascular surgery by administration of IGF-1 (twice daily for 3 days) starting immediately after surgery . They showed that 3 days after surgery the IGF-1-treated group had a significantly higher creatinine clearance than the control group. However, there was no improvement in secondary endpoints: length of intensive care unit stay, length of hospital stay, length of intubation and creatinine at discharge. No side-effects were reported. In a subsequent double-blind, randomized, placebo-controlled clinical trial in 72 patients with AKI due to surgery, trauma, hypotension or sepsis, rhIGF-1 (s.c. twice daily for 14 days) caused no improvement in clinical recovery or renal function, but even a tendency towards delayed recovery of GFR and urine flow rate, despite a significant increase in serum IGF-1 concentrations . These results were in contrast to the promising results of the previous animal experiments and clinical trials. The authors speculated that the role of IGF might differ between rodents and humans as well as between different disease state or etiology . Furthermore, the first dose of IGF-1 was given as late as 6 days after onset of AKI and only continued for 2 weeks. Finally, the bioavailability of IGF-1 might have been affected by the IGFBPs.
The variable results on the potential role of IGF to enhance renal function illustrate that promising strategies in animal models do not guarantee successful clinical results. Furthermore, these studies underline the fact that evaluation of bioavailability of the therapy (e.g. by measuring growth factor binding proteins) is necessary to evaluate whether the therapy reaches its target. In addition, these trials stress the difference between improvement of renal function by hemodynamic alterations and structural renal regeneration. The acute improvement in renal function probably represents the functional reserve of the kidney, whereas a persistent increase in renal function after cessation of growth factor therapy may reflect structural improvement, and therefore renal regeneration. The latter, however, should be confirmed by renal biopsy, because a lowered serum creatinine as a result of hyperfiltration may be harmful in the long term . Finally, timing of the initiation of therapy may influence its efficacy, as was also demonstrated in cardiovascular trials.
Future Perspectives in Clinical Regenerative Nephrology
Prognostic Clinical Trials
Prognostic assessment of the outcome of renal disease is important to guide clinical decisions. To adequately predict renal outcome, which can be envisioned as the balance between renal damage and renal repair, both detrimental processes and (potential) regenerative capacity should be quantified. Nowadays, prognostic information about renal diseases is obtained by combining the damage inflicted and the intensity of the current disease process . Quantification of potential regenerative capacity will improve prognostic evaluation, which is crucial to guide clinical decisions, especially when toxic therapies are considered, for example in patients with glomerulonephritis or with rejection after kidney transplantation.
Several new biomarkers have been introduced to evaluate the severity of renal damage, as reviewed by Coca et al. ( Table 22.3 ) . Considerably fewer data are available on evaluation of renal regenerative capacity . The regenerative capacity of the kidney may be assessed using blood or urine samples and renal biopsies. Trials in cardiovascular medicine have demonstrated a relation between circulating growth factors, such as HGF, and angiogenic capacity . Growth factors such as IGF-1, HGF and epidermal growth factor (EGF) have been shown to be upregulated in renal regeneration and to enhance renal recovery in animal models . These regenerative growth factors can be related to outcome parameters. Renal (progenitor) cells can also be evaluated to assess regenerative capacity. Proliferating tubular cells are the most important source of new tubular cells after AKI . Progenitor cells, both local and circulating, are thought to enhance this process, predominantly by paracrine stimulation . The number of progenitor cells may correlate with regenerative capacity. In addition, functional parameters such as migration and proliferative capacity of both progenitor and mature tubular cells may correlate with renal prognosis, comparable to the relation between EPC function and cardiovascular prognosis . Furthermore, regenerative potential might be evaluated at the protein and mRNA level in renal biopsies or urine. Similar to previous studies that correlated increased tissue and urinary mRNA levels of genes encoding for molecules involved in the pathogenesis of renal disorders with worse outcome, an increase in mRNA levels of regenerative factors, such as HGF, may be an independent predictor of improved disease outcome . Assessment of mRNA and protein levels of factors involved in renal regeneration such as HGF, IGF-1 and EGF in renal biopsy, urine samples or blood may be able to provide prognostic information.
Clinical trials correlate a certain prognostic or therapeutic intervention with an outcome parameter, which can cover the complete spectrum from histology to mortality. In animal studies the damage to the kidney is often standardized, and the intervention group is compared with a control group with the same amount of damage. In human trials the research population as well as the cause and degree of kidney injury will be more heterogeneous, necessitating not only the inclusion of a large number of patients but also multifactorial analysis of the correlation between parameters of renal damage, disease progression, regeneration and outcome. In prognostic clinical trials, regenerative parameters should therefore be considered in the context of initial renal damage and possibly a continuing and damaging process.
The continuing process of injury to the kidney can be evaluated using specific disease parameters such as antineutrophil cytoplasmic antibodies (ANCAs) in the case of Wegener’s disease or anti-double stranded DNA antibodies in the case of systemic lupus erythematosus. More specific parameters of renal damage can also be used ( Table 22.3 ). Immunohistochemistry is an important source of information on both the disease and regenerative process in animal experiments. In clinical trials protocol biopsies yield small amount of tissue and are performed only at a limited number of time-points. Using standardized scoring systems, histological improvement and quantification of (progenitor) cell proliferation can be used as a parameter of damage, disease and possibly regeneration . However, with only a few small sequential biopsies, the risk of sample bias is great. Another outcome parameter could be kidney volume assessed by ultrasound or CT. Furthermore, commonly used renal function tests can be used, such as creatinine clearance and proteinuria (see also Table 22.4 ) . Finally, clinical outcome parameters, such as quality of life, hospitalization, need for renal replacement therapy and mortality, should be used. Information about regenerative potential will cover an important blind spot in disease prognostication in renal medicine.
| Parameter | Test |
|---|---|
| GFR | Inulin clearance |
| 125 I-Iothalamate clearance | |
| 51 Cr-Ethylenediaminetetra-acetic acid (EDTA) clearance | |
| 99m Tc-Mercaptoacetyltriglycine (MAG 3 ) clearance | |
| Iothalamate sodium clearance | |
| Iohexol clearance | |
| Diatrizoate meglumine clearance | |
| Creatinine clearance | |
| Urea clearance | |
| Cystatin-C clearance | |
| RPF | P-aminohippurate (PAH) clearance |
| 131 I-Hippuran clearance | |
| 99m Tc-Mercaptoacetyltriglycine (MAG 3 ) clearance | |
| Renal concentrating ability | Water deprivation test |
| Renal diluting capacity | Water loading test |
| Urinary acidification | Urinary anion gap |
| Urine osmolal gap | |
| Alkali loading test | |
| Urine P co 2 − Blood P co 2 | |
| Sodium sulfate infusion test | |
| Response to loop diuretic | |
| Tubular function in AKI | Fractional excretion of sodium |
| Fractional excretion of urea | |
| Fractional excretion of uric acid | |
| Urinary β 2 -microglobulin | |
| Urinary amylase | |
| Urinary lysozyme | |
| Urinary retinol-binding protein | |
| Glomerular damage | Non-selective proteinuria |
Intervention Trials
Clinical trials will have to determine in which patient population a new therapeutic intervention will be investigated. Diabetic nephropathy is the leading cause of CKD that ultimately progresses to ESRD. The number of patients with diabetic nephropathy is steadily increasing worldwide. Diabetic nephropathy has been shown to be a reversible glomerular disease . Therefore, patients with diabetic nephropathy form a relevant population for therapeutic enhanced regeneration. However, the number of preclinical data on therapeutically enhanced regeneration in models of diabetes-induced renal failure is limited ( Table 22.5 ). In contrast, numerous preclinical studies have investigated therapeutically enhanced regeneration in acute tubular damage models, both toxic and ischemic. Although generally considered a reversible renal disease, AKI is associated with a 28-fold increase in the risk of developing stage 4 or 5 CKD . Moreover, AKI is independently associated with a two-fold increased risk of death. Based on the extensive preclinical experience on therapeutically enhanced regeneration in models of acute tubular damage, clinical trials to enhance renal regeneration in patients with acute tubular damage will soon be initiated. A phase I clinical trial using allogeneic MSC infusion in patients with AKI after on-pump coronary artery bypass grafting has been initiated by Westenfelder et al. (see www.clinicaltrials.gov ) .
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