4Hemotologic Aspects of Kidney Disease IntroductionBrenner & Rector’sThe Kidney 9th EditionChapter 56Hemotologic Aspects of Kidney Disease
5Introduction Definition of anemia in CKD： Hgb <13.5 g/dl for male; Hgb <12.0 g/dl for femaleErythropoietin-stimulating agents：Recombinant human erythropoietin was introduced as a treatment for the anemia associated with chronic kidney disease (CKD) since 1989.Erythropoietin therapy rendered many patients free of blood transfusions with dramatic benefits on quality of life (particularly physical capacity) and other physiologic effects of increasing hemoglobin levels from ~6 g/dL up to approximately g/dL.
6IntroductionLimitations of EPO：inconveniences of the need for regular administration 3 times a week and the need to inject this protein parenterally.=> Longer acting erythropoietin analogue, darbepoetin alfa (2001) and pegylated epoetin beta (2007)
7IntroductionSeveral studies showed that partial correction of anemia (to hemoglobin levels in the range of g/dL) was a safer strategy, reducing the risk of increased arterial and venous thromboembolism and other possible harmful effects in CKD patients.Rajiv Agarwal, Clin J Am Soc Nephrol , 2010 (5): 1340–1346
8Large Randomized Controlled trials Trial to Reduce Cardiovascular Events with Aranesp Therapy (TREAT)-- the first large trial to compare ESA therapy with placebo in patients with CKD not on dialysisCREATE trial
9TREATObjectivesto determine whether treating the anemia associated with CKD with DPO to a target Hgb of 13g/DL would reduce the occurrence of primary composite endpoints –1. CV primary end point：All- cause death, MI, stroke, CHF requiring medical attention or hospitalization for MI2. Renal primary end point：All-cause death or progression to ESRD
10(“rescue” darb if Hgb < 9) TREAT: Study designDarbepoetin alfaTo target Hgb 13N=20264038 subjects withCKD(eGFR ≥ 20 and ≤ 60 ml/min/1.73 m2Type 2 DMAnemiaHgb ≤ 11 g/dLIron Repletetransferrin sat ≥ 15%Randomize1:1Control(“rescue” darb if Hgb < 9)N=2012Randomized, controled, double blind, international trialStratified by baseline proteinuria and cardiovascular diseaseControl subjects received darbepoetin if Hgb dropped below 9 g/dLAll subjects could receive RBC transfusion if neededEvent driven – to conclude when 1203 subjects experienced a primary event
14Vascular access thrombosis HbEPO doseerythropoiesisHb > 13 g/dLA recent meta-analysis of studies evaluating higher Hb target with the use of ESA in chronic kidney disease (CKD) suggests there is a greater risk for stroke, cardiovascular events, and mortality rate.However, it is not clear whether the adverse outcomes are related to EPO dosing, higher Hb mass, or some (hidden) variables.CV riskMortalityStrokeVascular access thrombosis
15Naturally Occurring Higher Hemoglobin Concentration Does Not Increase Mortality among Hemodialysis PatientsGoodkin D A et al. JASN 2011;22:Among 29,796HD patients in 12nations545 EndogenousEPO patientswere included ineach model.Adjusted risk ofmortality does notdiffer significantlyfor Endogenouscompared withOther patients(RR, 0.81; 95% CI, 0.66 to 0.98RR, 0.98; 95% CI, 0.80 to 1.19Adjusted risk of mortality does not differ significantly for Endogenous EPO patients compared with Other patients. 545 Endogenous EPO patients were included in each model. The models were stratified by region and study phase, accounted for facility clustering effects, and adjusted for age, sex, black versus other race, vintage, smoking, cause of ESRD, catheter hemoaccess, serum PTH and albumin concentrations, Kt/V, and 14 summary comorbid conditions.RR 0.94 (95% CI, 0.72 to 1.22
16Effect of erythropoietin levels on mortality in old age: the Leiden 85-plus study den Elzen W P et al. CMAJ 2010;182:Cumulative all-cause mortalityamong 428CKD 1~2 patientaged 86 atbaseline, bytertile oferythropoietinlevel atbaseline(lowest = 3.4–8.6 IU/L, middle= 8.7–12.3 IU/L,highest = 12.4–103.0 IU/L).Cumulative all-cause mortality among 428 participants aged 86 at baseline, by tertile of erythropoietin level at baseline (lowest = 3.4–8.6 IU/L, middle = 8.7–12.3 IU/L, highest = 12.4–103.0 IU/L).Higher concentration of endogenous EPO => fatal outcome ↑Excessive EPO synthesis => off-target biological consequences ↑ ??Limitations: undiagnosed hypoxemia? Impaired bone marrow response?EPO resistance?
17Association of Mean Weekly Epoetin Alfa Dose with Mortality Risk in a Retrospective Cohort Study of Medicare Hemodialysis PatientsAm J Nephrol 2011;34:298–308The cohort included 137,918HD patients. Mean age was63.2 yearsRelative hazards of deathover mean EPO dose perweek during 3-monthexposure period, in patient-months with meanhemoglobinThe distribution of mean EPOdose per week is displayed inthe shaded area, and thereferent dose is shown bythe solid vertical line.
18Oluwatoyin F. Bamgbola, Kidney international (2011) 80, 464-474 ReviewPattern of resistance to erythropoietin-stimulating agents in chronic kidney diseaseOluwatoyin F. Bamgbola, Kidney international (2011) 80,Uremic toxins/Oxidative stress/InflammationNutrition deficiencyHeavy metalHematologic disordersAngiotensin-modulating agentsEPO inhibitors
19Erythropoietin-stimulating agents (ESA) resistance： Hb < 11g/dL over 3 month despite of EPOdose > 400 IU/kg/wk or IU/wk (≥1.0μg/kg for darbepoietin)1. Age and gender： Greater need of EPO in adult females due to androgenic stimulation of erythropoiesis in males and blood loss from menstrual cycles in females.
20The risk factors, pathogeneses, and therapeutic modulation of erythropoietin resistance in chronic kidney disease
22Uremic toxins The mechanism is uncertain There is fair correlation of urea nitrogen clearance with improved cytokine profiles (IL-6, CRP) and lower requirement for ESA.Uremia may be related to：Poor bone marrow response to ESAAccelerated turn over rate of RBC from altering erythrocytemorphology by inducing outward expression of thephosphatidyl-serine content of its inner membraneUremia alters erythrocyte morphology by inducing outward expression of the phosphatidyl-serine content of its inner membrane, which is removed by circulating macrophages.19,20 In addi- tion, there is impaired cellular generation of ATP by uremic inhibition of pentose phosphate shunt and tricarboxylic acid cycle.21,22 These cells, which are depleted of ATP, manifest poor capacity for cytosolic calcium efflux and thereby inhibit mitochondrial oxidative phosphorylation, thus, setting up a vicious cycle.23,24and progressive depletion of adenosine triphosphate (ATP)Uremic toxins -- EPO synthesis / erythroid response -- Longer effective dialysis
23Oxidative stress Depletes the capacity of ATP generation Downregulates the generation of hypoxic-inducible factor (HIF) protein, thereby reducing EPO synthesis.1. Depletes the capacity of ATP generation
24✗ Oxidative stress *Oxidative stress downregulates the generation of hypoxicinducible factor(HIF) protein.Haase V H Am J Physiol Renal Physiol 2010;299:F1-F13
25InflammationCKD pro-inflammatory cytokines: IL-6* Pro-inflammatory cytokines such as IL-1, IL-6, INF-γ, TNF could disrupt iron recycling and absorptionOluwatoyin F. Bamgbola, Kidney international (2011) 80,DMT 1, divalent methyl transporter-1Process of physiological iron recycling involving macrophages and enterocytes
26Inflammation* Pro-inflammatory cytokines promote EPO resistence by activation of suppressor of cytokine signaling and inhibition of nuclear factor κB4.5% USA dialysis patient had at least one failed allograft. Transplant rejection, persistent elevation of inflammatory indices and intractable anemia despite of large doses of ESA => nephrectomy is a reasonable arrpoach.*Failed allograft – nephrectomy is a reasonable approach for transplant rejection with persistent elevation of inflammatory indices and intractable anemia.* Dialysis cathetersPro-inflammatory cytokines(+)SOCS, suppressor of cytokine signaling; STAT-5 signal transducer and activator of transcription-5Oluwatoyin F. Bamgbola, Kidney international (2011) 80,Erythropoietin receptor activation and intracellular signal transduction
27InflammationFailed allograft – nephrectomy is a reasonable approach for transplant rejection with persistent elevation of inflammatory indices and intractable anemia.Dialysis catheters – additional sources of oxidative inflammation are the use of per-cath and synthetic grafts as vascular access in HD subjects.
28Nutrition deficiency1. Iron2. Folate3. Vitamin C
29Nutrients deficiency Iron Principally due to poor diet, frequent blood sampling and high frequency of infectionAnnual loss of 2g of elemental Fe among HD patientDiagnosis: serum iron < 100 mg/dl, TSAT <20%, and serum Ferritin < 100mg/dl ~ at least 2 indicesTreatment:1. Iron therapy2. Vit-C – mobilize iron from RES(hold if serum ferritin > mg/dl)Vit-C – mobilize iron from RES
30Nutrients deficiency Folate Principally due to poor gastrointestinal absorption, poor diet, water-soluble nutrient loss, and high catabolic statusAn essential ingredient for nucleotide synthesis, DNA repair, and re-methylation of homocysteineFolic acid deficiency produces oxidative vascular injury by potentiating uremic inhibition of homocysteine catabolism血管壁中的上皮細胞(Epithelial cells)具有毒性，而且能夠促進凝血及膠原蛋白(Collagen)的製造，並減少一氧化氮的利用率，而導致動脈硬化的形成。改變了血液的凝結機制，增加血管的斑痕與傷害。
31Nutrients deficiency Vitamin C A cofactor for several enzymatic metabolismPromotes GI absorption of iron and enhances its mobilization from RES.Increases Hb synthesis by facilitating incorporation of Fe into protoporphyrinAn anti-oxidative free oxygen scavenger, downregulates hepatic synthesis of cytokinesGreater loss of Vit-C in HD compared with PDits deficiency must be considered in the event of persistent anemia that failed to respond to EPO and Fe supplements
33Heavy metalBone marrow function is often impaired in individuals with heavy metal poisoningAluminum toxicity – P-binder, dialysate, poor control of pharmaceutical standardsCadmium, lead, mercury – industrial pollution, some folk remedies of Indian and Middle Eastern originsIncreases gastrointestinal absorption of heavy metals
34Heavy metalLead toxicity is more likely to occur in patient with CKD due to：Iron and calcium deficiencies => GI absorption Uremia may increase mobilization of lead from bonetissue storesLead toxicity causes anemia by precluding incorporation of iron into a protoporphyrin ring for heme synthesisLead 95% bone, hair
36Hematological disorders The mechanism of EPO resistance includes bone marrow infarction, hemolysis, hypersplenism, and ineffective erythropoiesis.Common chronic hemolytic conditions are auto-immune diseases, sickle cell disease, thalassemia, hereditary spherocytosis, glucose 6-phosphate dehydrogenase deficiency.*comorbid hemato- logical disorders (B-thal, HbS, HbA2, HbH), is frequently associated with elevated endogenous EPO 。 The most common hemoglobinopathy is sickle cell disease.*Hemolytic diseases are more susceptible to the morbid impact of oxidative stress, nutrient deficiency, and dialysis blood loss.
37Hematological disorders Autoimmune hemolytic anemia occurs in 5-10% of patient with SLE. It is frequently associated with renal or neurological involvement.As reticulocytosis is common in patients on ESA therapy, early diagnosis of hemolytic events may be missed.It should be suspected when there is a progressive increase in EPO requirement while there is rapidly decreasing Hb concentration with megakaryocytic cell line and serum titer of anti-DS DNA.
39Angiotensin-modulating agents The influlence is controversial.A prototype study showed there is a higher EPO requirement in dialysis hypertensive patients who were treated with ACEI/ARB compared with CCB.ACEI suppresses the enzymatic degradation of N-acetyl-seryl-aspartyl- lysyl-proline (AcSDKP), a naturally occurring inhibitor of erythropoiesis.Stimulation of erythroid cellular proliferation by angiotensin binding of its type II surface receptor is inhibited by ARB=> therapeutic use of ARB in post-transplanterythrocytosis.
41Circulating EPO inhibitors Circulating EPO inhibitors may result in pure red cell aplasia.Pure red cell aplasia should be suspected：1. Received ESA therapy for 44 weeks2. Rapid decrease in Hb mass (> 0.5 g/dl per week),3. Reduction in absolute reticulocyte count <10, 000/ml and/or 1U of RBC transffusion per week4. Leucocytes and platelets are normal.5. The more common causes of EPO-resistantanemia should be excluded.Routine blood transfusion should be avoided except for life-threatening anemia.
42Circulating EPO inhibitors Diagnosis：1. Absence of erythroid precursors on bone marrowsample and low EPO content of the serum.2. The serum sample inhibits growth of erythroidcolonies in a bone marrow culture.3. Radioimmunoassay identifies circulating neutralizinganti-EPO IgG in serumTreatment：1. Discontinuation of rhEPO2. Steroid and/or calcineurin inhibitor3. Plasma exchange and/or allograft transplantationwill result in rapid fall of the neutralizing antibody.
43Secondary hyperparathyroidism and 1,25-vitamin D deficiency
44Secondary hyperparathyroidism There is a higher prevalence of anemia and greater EPO requirement HD subjects who are in the upper 50th percentile of intact parathyroid hormone.As a proof of causal relationship, surgical parathyroidectomy led to an improved control of anemia and a lower need for ESANevertheless, there is no evidence for a direct inhibition of erythropoiesis by excessive PTH.
451,25-vitamin D deficiencyVit-D may have synergistic effect on ESA control of anemiaCalcitriol treatment in subjects with uremic bone disease increases the proliferation of erythroid precursors.Lower EPO requirement among subjects with BB gene of vitamin D receptor genotype compared with those with the Bb/bb gene.
46Etiology of ESA resistance Risk factorMechanism of ESA resistanceTherapeutic interventionUremic toxinsEPO synthesis / erythroid responseLonger effective dialysisOxidative stressDownregulation of HIFVit E and Vit CInflammationCytokines: IL-1, IL-6, TNF-αAvoid sepsis and malnutritionIron deficiencyHemoglobin synthesisReplenish iron/ blood lossHyperparathyroidism/ Vit D deficiencyVitamin D synergism (erythropoiesis)Low P diet/ 1,25 (OH)2 vit DAluminum toxicityAluminum bone diseaseAvoid aluminum intakeHemolysisHbSS/G6PDD/AIHATreat underlying dieseaseDrug: angiotensin-modulating agentsErythroid ANG II receptors/ endogenous EPO inhibitor, AcSDKPDose of ACEI/ARB
47Potential Pharmacological intervention in EPO resistance Anti-inflammatory agentsNutritional supplementsEPO-mimetic peptideEndogenous induction of EPO
48Iain C. Macdougall, Am J Kidney Dis. 2012;59(3):444-451 Narrative ReviewNew anemia therapies: Translating Novel Strategies From Bench to BedsideIain C. Macdougall, Am J Kidney Dis. 2012;59(3):EPO-mimetic peptideHIF StabilizationHepcidin ModulationGATA-2 InhibitorsEPO gene therapy
50EPO-mimetic peptideThe concept that a peptide could activate the erythropoietin receptor and stimulate erythropoiesis was described first by Wrighton et al in 1996 in Science.EMP-1 (erythropoietin-mimetic peptide 1) was able to stimulate cellular proliferation of erythroid cells in a dose-dependent manner and also increase reticulocyte counts in animal models.However, EMP-1 had low affinity for the erythropoietin receptor and low biological activity.
51Iain C. Macdougall, Am J Kidney Dis. 2012;59(3):444-451 EPO-mimetic peptidePeginesatide is a dimeric peptide joined with a spacer linker to a pegylation chain to enhance its metabolic stability in vivo.No structural homology between peginesatide and erythropoietin. Antibodies against erythropoietin do not cross-react with peginesatide, and vice versa.=> potential therapeutic use for pure red cell aplasiaIain C. Macdougall, Am J Kidney Dis. 2012;59(3):=> pure red cell aplasia
52Peginesatide Phase 3 Clinical Trials Overview Iain C. Macdougall, Am J Kidney Dis. 2012;59(3):
54HIF StabilizationIt is now clear that the anemia associated with CKD is not due simply to deficient erythropoietin production, but rather to defective EPO gene regulation.Prolyl hydroxylase inhibitors results stabilization of HIF and consequently transcription of the EPO gene.(eg, the erythropoietin receptor, transferrin, transferrin receptor, ferroportin, and divalent metal transporter 1 )
55Regulation of hypoxia inducible factor (HIF) activity HIF stabilizationIt is now clear that the anemia associated with CKD is not due simply to deficient erythropoietin production, but rather to defective EPO gene regulation.Prolyl hydroxylase inhibitors results stabilization of HIF and consequently transcription of the EPO gene. (eg, the erythropoietin receptor, transferrin, transferrin receptor, ferroportin, and divalent metal transporter 1 )Iain C. Macdougall, Am J Kidney Dis. 2012;59(3):
56HIF Stabilization Prolyl hydroxylase inhibitors Advantages – 1. Orally active2. Modulate other genes involved in erythropoiesis inaddition to the EPO gene.Disadvantages –1. Upregulate several hundred other hypoxia-sensitivegenes, including those involved in glucose regulation,angiogenesis, such as VEGF (vascular endothelialgrowth factor)=> enhance tumor growth or proliferative diabeticretinopathy ??1. In phase 2 clinical trial
57HIF StabilizationThe first-generation HIF stabilizer molecule (prolyl hydroxylase inhibitors) -- FG 2216=> Patients developed abnormal liver enzyme test results,and one developed fatal hepatic necrosis in phase 2clinical trialThe second-generation HIF stabilizer molecule – FG 4592=> Significantly increase Hct and decrease serum Hepcidin levels=> This is now in phase 2 clinical trial.In addition to Fibrogen, several other companies are developing HIF stabilizers.
60Hepcidin ModulationHepcidin is a small defensin-like peptide produced largely by the liver, macrophage and adipocyte.Hepcidin has antimicrobial properties and it is the master regulator of iron metabolism., controlling the amount of dietary iron absorbed from the duodenum and also the release of iron from cells in the reticuloendothelial system (Kupffer cells, splenic macrophages)
61Hepcidin ModulationHepcidin is a small defensin-like peptide produced largely by the liver, macrophage and adipocyte.Hepcidin has antimicrobial properties and it is the master regulator of iron metabolism., controlling the amount of dietary iron absorbed from the duodenum and also the release of iron from cells in the reticuloendothelial system (Kupffer cells, splenic macrophages)
62Hepcidin Modulation * Hepcidin inhibits ferroportin, which controls iron efflux fromduodenalenterocytes,hepatocytes,and macrophages* Uremia is achronicinflammatorystate. Dialysispatients havemuch higherserum hepcidinlevels.The regulation of hepci- din is complex, but one of the major stimuli to its production is interleukin 6 (IL-6), produced as part of the inflammatory response. Other molecules, such as hemojuvelin and BMP-6 (bone morphogenetic protein 6), also have a role.39Babitt JL, Molecular mechansms of hepcidin regulation: implications for the anemia of CKD. Am J Kidney Dis (55):
63Hepcidin ModulationIt currently is believed that this has a part in the pathogenesis of anemia in CKD by limiting iron availability to the bone marrow.At a molecular level, hepcidin binds to the main iron exporter protein ferroportin, which controls iron efflux from duodenal enterocytes, hepatocytes, and macrophages.It currently is believed that this has a part in the pathogenesis of anemia in CKD by limiting iron availability to the bone marrow.
64Hepcidin Modulation Strategies： Monoclonal antibody against hepcidin (NOX-H94) has been shown the effect on inhibition of IL-6 induced anemia in mouse models.Inhibition of the hepcidin production by using antisense oligonucleotides or sliencing messenger RNA transcribed from the hepcidin gene(HAMP)None of the strategies have been subjected to clinical trials.=> Hepcidin has antimicrobial properties. Inhibition ofhepcidin might exacerbate the risk of infection ?=> Is there a “safe” levels not to suppress hepcidinactivity completely?
66Iain C. Macdougall, Am J Kidney Dis. 2012;59(3):444-451 GATA-2 InhibitorsGATA-2 inhibitors: K-7174 and KGATA-2inhibitorIain C. Macdougall, Am J Kidney Dis. 2012;59(3):Series of experiments studied the effects of K-7174 in both a human hepatoma cell line and an animal model of anemia and showed that this GATA-specific inhibitor potentiate erythropoietinprotein production and EPO promoter activitythat previously had been suppressed with IL-1β,TNF-α, or NG-monomethyl L-arginine (L-NMMA).Imagawa S. Negative regulation of the erythropoietin gene expression by the GATA transcription factors. Blood. 1997(89):
67GATA-2 InhibitorsK was found to evoke greater hypoxic induction compared with K-7174, possibly through stimulation of HIF-1 binding activity in addition to GATA inhibition.Potential role for an orally administered GATA inhibitor in the treatment of anemia.the HIF stabilizers, there is concern that GATA inhibition will promote activation of other genes in addition to erythropoietin.Imagawa S. Negative regulation of the erythropoietin gene expression by the GATA transcription factors. Blood. 1997(89):
69Erythropoietin Gene Therapy In 2005, a group of Israeli scientists developed a functional delivery system for the EPO gene using skin cells on SCID mice (using adenovirus vector).The mice responded by producing increased levels of erythropoietin, and this was associ- ated with an increase in hematocrit. No such effect was seen with the vector alone.* using an adenovirus vector in which the cytomegalovirus immediate early promoter drives EPO* One patient maintained hemoglobin levels in the normal range for more than 18 months without erythropoietin injections. Further evaluation of this novel therapeutic strategy is ongoing.
71Erythropietin Gene Therapy In 2010, a small group of patients with CKD in Israel have taken part in a proof-of-concept phase 1-2 clinical trial of this delivery system for the EPO gene.All patients showed increased erythropoietin production, with most showing sustained eleva- tion of hemoglobin levels (the primary end point) in the target range of g/dL for 6-12 months without receiving additional erythropoietin injections.
73ConclusionIt took us nearly 20 years to realize the limitations of ESAs and the potential for harm if used too aggressively.This review summarizes our current knowledge about a variety of new strategies for stimulating erythropoiesis.They will need to be subjected to the same degree of scientific investigation as the existing ESAs, and it may be many years before the true efficacy-safety balance of these novel scientific strategies is realized.None of the newer agents has outcomes data showing superiority to existing ESAs, and none has been tested in sufficient numbers of hyporesponsive patients to know whether the outcomes in these patients are different from those with conventional ESAs
74Currently Available Erythropoiesis-Stimulating Agents
76Oluwatoyin F. Bamgbola, Kidney international (2011) 80, 464-474 Clinical evaluation of resistance to erythropoietin stimulating agent in chronic kidney diseaseAl toxicityPb toxicityCu deficiencyHypothyroidismOluwatoyin F. Bamgbola, Kidney international (2011) 80,
77Proposed role of the bone morphogenetic protein(BMP) signaling pathway, HJV, HFE, and TFR 2 in iron sensing and hepcidin regulation in the liver