Tissue Protection and Regeneration Aided by Erythropoietin and Erythropoietin-derived Peptides

Outline
Introduction 93
Tissue Expression of Erythropoietin and the Erythropoietin Receptor 93
Erythropoietin in the Vascular System 94
- Erythropoietin and the Endothelium 94
- Erythropoietin and Endothelial Progenitor Cells 95
Erythropoietin and the Kidney 95
- Erythropoietin and Acute Kidney Injury 95
- Renoprotection by rHuEPO in Chronic Kidney Disease 97
Conclusion 98

In the hematopoietic system, the principal function of erythropoietin (EPO) is the regulation of red blood cell production. Consequently, following the cloning of the EPO gene, recombinant human EPO has been widely used for treatment of anemia in various clinical conditions such as chronic kidney diseases. However, a growing body of evidence indicates that EPO as well as recombinant human EPO and its analogs have tissue-protective effects and prevent ischemia-induced and hypoxic tissue damage. It is therefore conceivable that therapy with recombinant human EPO, whether acute high dose or (early) chronic low dose, may be a feasible way to prevent acute kidney injury and/or retard progressive chronic kidney disease. Furthermore, EPO and its recombinant forms also stimulate endothelial progenitor cells which promote endothelial regeneration. Available data support the hypothesis that EPO is a key molecule in the process of endothelial (vascular) repair and regeneration.

Introduction

In the hematopoietic system, the principal function of erythropoietin (EPO) is the regulation of red blood cell production in order to maintain tissue oxygenation and thus prevent tissue hypoxia. Consequently, following the cloning of the EPO gene , different recombinant human EPO (rHuEPO) forms (e.g. EPOetin-α and EPOetin-β), and their long-acting analogs darbepoetin-α and the continuous erythropoietin receptor activator (CERA) have been mainly used for treatment of anemia in patients with chronic kidney disease (CKD) and chemotherapy-induced anemia in cancer patients. However, recent experimental studies investigated the ability of exogenous EPO to modulate organ function and cellular responses to diverse types of injury. Thus, in addition to its essential role in the regulation of mammalian erythropoiesis, EPO signaling, activated either by exogenous EPO or by endogenous EPO in an autocrine or paracrine fashion, has emerged as a major tissue-protective survival factor in various non-hematopoietic organs. Indeed, numerous articles have been published on the effects of rHuEPO to prevent ischemia-induced tissue damage in several organs, e.g. brain, heart, liver, blood vessels and kidney (reviewed in ). In this chapter the focus will be on the effects of EPO and rHuEPO related to the field of nephrology, i.e. the effects on vascular and renal tissue.

Tissue Expression of Erythropoietin and the Erythropoietin Receptor

Characterization of the non-erythropoietic biological effects of EPO and understanding the mechanisms of EPO–EPO-receptor signaling activation in non-hematopoietic organs and cell types are critical to the future development of novel applications for rHuEPO and its derivatives. The primary sites of EPO production reside in the fetal liver and adult kidney where EPO gene expression occurs under the tight control of an oxygen-sensing, hypoxia-inducible factor (HIF)-dependent mechanism . In addition, recent studies could identify EPO expression in several extrarenal tissues and cell types, including astrocytes, neurons, the female genital tract, male reproductive organs, mammary glands, placental trophoblasts, bone marrow macrophages and erythroid progenitors . The expression of EPO receptors in non-erythroid tissues such as the brain, retina, heart, kidney, smooth muscle cells, myoblasts and vascular endothelium has been associated with the discovery of novel biological functions of endogenous EPO signaling in non-hematopoietic tissues. For example, targeted disruption of either EPO or EPO receptor in mice leads to in utero death because of a lack of definitive erythropoiesis in the fetal liver and defects in the onset of the circulatory system, indicating the vital role for EPO and EPO-receptor signaling during vascular development .

The EPO molecule is a glycoprotein with a molecular weight of 30.4 kDa, and its tertiary structure is defined by four antiparallel α-helices. Binding of a single molecule to two adjacent EPO receptors on the membrane of target cells leads to homodimerization of the EPO receptor and the triggering of different intracellular signaling cascades. The major mechanism for degradation of EPO in the body occurs in cells expressing the EPO receptor, through receptor-mediated endocytosis of EPO followed by degradation in lysosomes . However, these signaling cascades are highly complex and their relevance for the cellular effects of EPO is not completely understood so far. For example, by using EPO-receptor fusion proteins, it has been shown that distinct conformations of the EPO receptor exist which may activate different intracellular pathways. Thus, which receptor conformation is achieved and which signaling pathway is subsequently activated may depend on the extracellular binding site of EPO . Moreover, a physical association between the common β-receptor chain subunit (CD131) and the EPO receptor has been demonstrated by coimmunoprecipitation of the proteins in neuron-like cells. The tissue-protective effects of EPO appear to require the expression of CD131 and a low-affinity, heterodimeric EPO-receptor–CD131 receptor, which may exert a different signaling behavior than the “classical” EPO receptor . In addition, different classes of EPO receptor with respect to receptor affinity for EPO binding have been described: high (KD90–900 pM) and low (KD20–9000 pM)-affinity EPO receptor . The former mediate the well-known hematopoietic effects, whereas the latter seem to be involved in tissue protection by EPO. Collectively, these data indicate that the interaction of EPO with its receptor may be far more complicated than previously believed.

The activated EPO receptor exhibits more than 40 binding sites, and a pivotal molecule that induces intracellular signaling is JAK2 tyrosine kinase. Activation of this kinase leads to tyrosine phosphorylation and dimerization of signal transducers and activators of transcription (STATs). The JAK2–STAT5 signaling pathway not only is responsible for the effects of EPO on red blood cell differentiation, proliferation and survival, but also can mediate protection against programmed cell death (apoptosis) . It can therefore be anticipated that the JAK–STAT5 pathway plays an important role in the tissue-protective properties of rHuEPO not related to anemia correction. Another important signaling pathway triggered by EPO is phosphatidylinositol 3-kinase (PI3K), which activates Akt (i.e. serine/threonine protein kinase B). EPO-induced activation of the PI3K–Akt pathway and subsequent inhibition of apoptosis seems to be imperative for tissue protection by rHuEPO, because prevention of Akt phosphorylation abolished the beneficial action of rHuEPO in settings of experimental cardiovascular and neuronal injury. Although all of the above pathways seem to be activated via the classical EPO receptor it is currently unknown whether different ligands of the EPO receptor may induce different signaling pathways or whether the single EPO receptor chain associates and forms dimers with other membrane proteins. Theoretically, these novel receptors may be the target of newly designed rHuEPO compounds such as the carbamylated form of the hormone .

Erythropoietin in the Vascular System

Erythropoietin and the Endothelium

There is evidence that EPO is a major regulator of vascular formation (i.e. angiogenesis) and organ growth in the embryo, and EPO receptors have been found in almost every embryonic tissue . Kertesz et al. found both EPO and EPO receptors expressed in the vasculature during embryogenesis, and deletion of either in knockout animals leads to severe angiogenic defects resulting in an embryonic lethal phenotype. These angiogenesis defects can be partially rescued by expressing human EPO during embryogenesis. Furthermore, EPO receptors have been found also on mature endothelial cells, where direct biological effects of EPO have been described, e.g. stabilization of endothelial structures and vascular integrity such as cell–cell and cell–matrix contacts. Addition of rHuEPO to the culture medium increases endothelial cell proliferation and protects cells against ischemia and apoptosis . The latter effect is thought to be mediated via the PI3K/Akt pathway . Since mature endothelial cells obviously do not lose their EPO receptors, antiapoptotic signaling could persist much longer than in erythrocytes thus rendering endothelial cells more resistant to ischemia-induced cell death. Moreover, mature human endothelial cell lines also respond to rHuEPO by differentiating into primitive vascular structures . It is therefore tempting to speculate that EPO preserves its role as a key regulator of vascular protection and even vascular formation in the adult organism.

Recently, activation of endothelial nitric oxide synthase (eNOS) by EPO has been identified as an important mechanism in how EPO affects endothelial cells . d’Uscio et al. used a murine model of wire-induced injury of the carotid artery to examine the effect of rHuEPO (1000 IU/kg body weight s.c.) on endothelial repair in wild-type and eNOS-deficient mice. They studied the reactivity and vascular structure of isolated carotid arteries in vitro. The injured arteries exhibited impairment of endothelium-dependent relaxation to acetylcholine, and this was associated with an increase in medial cross-sectional area. In wild-type mice treatment with rHuEPO upregulated expression of eNOS, normalized the vasodilator response to acetylcholine and prevented the injury-induced increase in medial cross-sectional area. These protective effects were not only abolished in eNOS-deficient mice; the authors even observed a significant increase in systolic blood pressure and enhanced medial thickening of injured carotid arteries in these animals. These results show that the vasculoprotective effects of EPO may critically depend on the activation of eNOS, and point to the fact that a functionally active eNOS is crucial for the action of EPO at the vascular level.

Erythropoietin and Endothelial Progenitor Cells

Another aspect of EPO’s action on the endothelium is its stimulating effect on endothelial progenitor cells (EPCs). These cells promote endothelial regeneration and orchestrate vascular reparative processes with secretion of proangiogenic cytokines . Findings in patients with CKD together with results from animal and cell culture experiments permit the conclusion that EPO is a potent regulator of EPC proliferation and differentiation ( Fig. 5.1 ), and this effect is mediated, at least in part, via Akt activation . In addition, activation of eNOS in EPCs may contribute to EPO’s effects in endothelial regeneration, since recent work demonstrated that nitric oxide (NO) production is a key feature of EPCs that is required for endothelial repair . For example, Urao et al. investigated the role of rHuEPO in EPC mobilization and repair of injured endothelium in a wire injury model of the femoral artery in mice. Administration of rHuEPO inhibited neointimal formation and significantly increased the re-endothelialized area. The authors also showed that rHuEPO induced AKT phosphorylation and stimulation of eNOS in EPCs. These results were recently confirmed in a model of postmyocardial infarction heart failure, where rHuEPO-induced neovascularization was also mediated through a combination of EPC recruitment from the bone marrow and improved EPC engrafting . Collectively, these data support the hypothesis that EPO is a key molecule in the process of endothelial (vascular) repair. This is also of importance for physicians caring for patients with CKD, in whom reduced numbers and/or function of EPCs was documented in numerous studies, and possibly contributes to their high cardiovascular morbidity . Moreover, it is plausible that deficient EPC function may hamper vascular and thus tissue repair and regeneration in the kidney as a result of acute or chronic damage such as diabetes or hypertension.

Erythropoietin and the Kidney

Erythropoietin and Acute Kidney Injury

EPO receptors have been found in vascular as well as non-vascular kidney tissue, and it has been shown that rHuEPO activates different survival intracellular pathways in kidney tissue, such as the PI3K/Akt pathway . Moreover, numerous studies have revealed that administration of rHuEPO protected tissue and whole-organ function in various experimental settings of acute kidney injury (AKI), such as ischemia–reperfusion or toxic injury . These investigations unequivocally documented that treatment with even a single (high) dose of rHuEPO ameliorates kidney dysfunction by reducing apoptotic cell death in different renal tissue compartments ( Table 5.1 ). In addition, rHuEPO could theoretically increase the local NO bioavailability through stimulation of eNOS and prevent renal vessel injury . However, as already discussed, the EPO-mediated increase in vascular NO bioavailability was only seen in intact vessels, whereas administration of rHuEPO provoked vasoconstriction in injured arteries .

Table 5.1

In Vivo Studies on Acute Protective Effects of Recombinant Human Erythropoietin (rHuEPO) on the Kidney

Species (ref.)	Experimental model of acute kidney injury	Dose, route and time of application	Mechanism(s) of tissue protection
Rat	Cisplatin nephrotoxicity	100 U/kg rHuEPO i.p. daily for 9 days	Increased regeneration of renal tubular cells
Rat	Ischemia–reperfusion	3000 U/kg rHuEPO i.v. 24 h before ischemia	Heat shock protein 70 activation, reduced caspase activity (reduced apoptosis)
Rat	Hemorrhagic shock	300 U/kg rHuEPO i.v. before resuscitation	Reduced caspase activity (reduced apoptosis)
Rat	Ischemia–reperfusion	200 U/kg rHuEPO i.p. before ischemia, 6 and 24 h after reperfusion, thereafter daily	Reduced downregulation of renal aquaporins (water channels) and sodium transporters
Rat	Ischemia–reperfusion	300 U/kg rHuEPO i.v. 30 min before ischemia, 5 min before reperfusion or 30 min after ischemia	Reduced caspase activity (reduced apoptosis)
Rat	Ischemia–reperfusion	5000 U/kg rHuEPO i.p. 30 min before ischemia	Reduced apoptosis, increased regeneration of renal tubular cells
Mouse	Ischemia–reperfusion	1000 U/kg rHuEPO s.c. for 3 days before ischemia or 5 min before reperfusion	Reduced oxidative stress and lipid peroxidation
Rat	Ischemia–reperfusion	5000 U/kg rHuEPO or 25 μg/kg darbepoetin-α i.p. immediately after ischemia or 6 h after ischemia	Reduced apoptosis, increased regeneration of renal tubular cells
Rat	Ischemia–reperfusion	500 U/kg rHuEPO i.p. 20 min prior to ischemia–reperfusion	Reduced apoptosis and inflammation
Rat	Radiocontrast injury + nitric oxide inhibition	3000 U/kg and 600 U/kg rHuEPO i.v. 24 and 2 h before administration of iothalamate + indomethacin	Not reported
Rat	Cisplatin nephrotoxicity	5000 U/kg rHuEPO i.v. before injection of cisplatin	Reduced apoptosis, preserved renal perfusion

i.v.: intravenously; i.p.: intraperitoneally; s.c.: subcutaneously.

The encouraging experimental results have certainly prepared the ground for studies exploring the therapeutic potential of rHuEPO in humans with AKI, but before that some safety issues must be resolved. In particular, the dose-dependent effects on the number and activation state of thrombocytes and the stimulation of platelet adherence to (injured) endothelium could mitigate the beneficial effects of rHuEPO on damaged renal (vascular) tissue . Recent experimental work revealed that darbepoetin-induced erythropoiesis with increased hematocrit levels is not associated with an increased risk for thrombosis as long as endothelial NO production in intact vessels serves as a compensatory mechanism . In other words, with preserved (local) NO generation, the beneficial vascular actions of EPO prevail over potential adverse effects. Unfortunately, this is almost never the case in the clinical situation, particularly in renal patients in whom NO bioavailability is known to be significantly reduced .

It is conceivable, however, that administration of a single high dose or a few repetitive doses of rHuEPO (to avoid the increase in hematocrit levels) to prevent acute ischemic injury may be a promising approach to limit loss of kidney tissue in patients with AKI. In this respect, a small study in patients after coronary bypass grafting has provided encouraging preliminary results . However, data from a prospective multicentre study from France in patients receiving a cadaveric kidney transplant have not revealed a significant effect of high-dose rHuEPO treatment on the ischemia–reperfusion injury after kidney transplantation . In comparison with placebo treatment, administration of EPOetin-β 4 × 30,000 IU i.v. immediately after, 12 h, 7 and 14 days after transplantation did not reduce the incidence of primary kidney graft non-function or need for dialysis. Thus, further studies are needed to explore the potential of rHuEPO to prevent or ameliorate AKI in humans.

Renoprotection by rHuEPO in Chronic Kidney Disease

Even more important than prevention of AKI may be renoprotection by rHuEPO in settings of CKD, that is, for prevention of progression of primary as well as secondary kidney diseases such as diabetic nephropathy. So far, therapeutic efforts in CKD patients have been made only to correct anemia and the putative hypoxic renal tissue damage as a result of anemia . Whereas results of smaller randomized studies suggested that anemia correction with rHuEPO could slow progression , data from recently published large trials in patients with CKD revealed no beneficial effect on progression . However, in later studies, patients with advanced CKD have been studied, and (almost) complete correction of anemia was accomplished within weeks using relatively high doses of rHuEPO; the initial weekly starting dose in the CHOIR study was 10,000 IU of rHuEPO-α . Such an abrupt increase in the hematocrit level in patients with serious vascular problems might have mitigated putative beneficial effects of rHuEPO on the kidney, particularly if one assumes that much lower doses could be adequate for tissue protection.

A hematologically non-effective dose of the long-acting rHuEPO analog darbepoetin (i.e. a dose that did not affect hematocrit levels in treated animals) was used in the established 5/6 nephrectomy remnant kidney model in the rat . This model features progressive injury to the renal microvascular endothelium leading to glomerular sclerosis and ischemia-induced tubulointerstitial damage. It was demonstrated that chronic treatment with darbepoetin conferred renal vascular and tissue protection and preserved renal function in the 5/6 nephrectomized animals. This culminated in significant better survival compared with saline-treated animals ( Fig. 5.2 ). In this experimental setting of CKD, persistent activation of the Akt pathway in endothelial and glomerular epithelial cells was found, along with reduced apoptotic cell death in renal tissue. Further, it was shown that escalating doses of darbepoetin mitigate the protective effects on the remnant kidney tissue and even worsen microvascular renal injury, that is glomerulosclerosis . These findings were expanded in experimental models of diabetic (db/db mouse), toxic and immunological kidney injury . Chronic administration of CERA had beneficial dose-dependent effects on molecular pathways of diabetic kidney damage . However, only the non-hematologically effective (low) dose was also clinically renoprotective, whereas high-dose CERA aggravated albuminuria in this experimental setting despite clear-cut beneficial molecular effects. Moreover, phlebotomy in high-dose CERA-treated mice preserved its tissue-protective effect. These experimental observations could be of considerable clinical relevance, because low-dose rHuEPO treatment may be a safe strategy to avoid potential adverse effects of high-dose therapy, i.e. doses that cause an increase in hematocrit with accompanying changes in rheology and activate thrombocytes. Thus, earlier administration of rather low doses of rHuEPO or analogs may be a feasible way to limit renal tissue damage in patients with CKD.