EPO

Question 1

What type of hormone is Erythropoietin (EPO)?

Answer 1

A glycoprotein hormone.

Question 2

How many amino acids make up the single chain of EPO?

Answer 2

165 amino acids.

Question 3

Describe the secondary structure elements of the EPO molecule.

Answer 3

Four alpha helices and two beta sheets.

Question 4

What is the function of the four glycans that make up 40% of the EPO molecule?

Answer 4

They protect EPO from proteases and modulate receptor binding affinity.

Question 5

How many disulfide bridges are found within the EPO chain?

Answer 5

Two intra-chain disulfide bridges.

Question 6

What is the structural configuration of the EPO receptor (EPOR)?

Answer 6

It is a homodimeric membranous receptor (two identical subunits).

Question 7

What are the two primary domains of the EPOR?

Answer 7

An extracellular domain and an intracellular domain.

Question 8

Which kinase is recruited to the EPOR upon EPO binding to catalyze transphosphorylation?

Answer 8

JAK-2.

Question 9

What molecule docks at the phosphotyrosines on the intracellular domain of the EPOR?

Answer 9

STAT5 (Signal Transducer and Activator of Transcription 5).

Question 10

Name the three main signaling pathways activated by EPOR phosphorylation.

Answer 10

1. STAT5 pathway
2. MAPK (ERK1 & 2)
3. PI3K / Akt

Question 11

Trace the pathway from Akt to NF-kB activation.

Answer 11

Akt stimulates IKK -> phosphorylates IkB -> IkB dissociates -> NF-kB is activated.

Question 12

What is the primary cellular effect of EPOR activation on erythroid progenitor cells?

Answer 12

It triggers an anti-apoptotic pathway, promoting proliferation and differentiation.

Question 13

What is the ultimate clinical result of successful EPO/EPOR signaling?

Answer 13

An increase in Red Blood Cells (RBCs).

Question 14

What is the specific regulating variable that triggers EPO synthesis?

Answer 14

Tissue pO2 (not just the RBC count).

Question 15

How does Anemia differ from Hypoxia in the context of EPO?

Answer 15

Anemia is a decrease in Hb concentration (a cause); Hypoxia is low tissue pO2 (the trigger).

Question 16

Name three causes of low tissue pO2 other than anemia.

Answer 16

1. High altitude
2. Lung/Heart disease
3. Airway obstruction.

Question 17

Trace the Kidney/Bone Marrow dialog starting with the stimulus.

Answer 17

Hypoxia -> Kidney (EPO) -> Bone Marrow (CFU-E) -> increase RBCs.

Question 18

What is the specific erythrocytic progenitor that EPO acts upon in the bone marrow?

Answer 18

The CFU-E (Colony-Forming Unit-Erythroid).

Question 19

What is the ultimate “feedback” result of increased RBC production?

Answer 19

Increased O2 carrying capacity, which restores tissue pO2 and shuts off EPO production.

Question 20

What is the definition of a Totipotent stem cell?

Answer 20

A cell that can differentiate into all cell types of the body PLUS extra-embryonic tissues (can form a complete organism).

Question 21

How does a Pluripotent cell differ from a Totipotent cell?

Answer 21

Pluripotent can develop into all cell types of the body except extra-embryonic tissues.

Question 22

What is a Multipotent stem cell?

Answer 22

A cell that can develop into multiple cell types within a specific lineage or tissue (e.g., hematopoietic stem cells).

Question 23

What is the correct sequence of erythroid precursors from BFU-E to Erythrocyte?

Answer 23

BFU-E -> CFU-E -> Proerythroblast -> Basophilic -> Polychromatophilic -> Orthochromatophilic -> Reticulocyte -> Erythrocyte.

Question 24

At which stages does EPO primarily bind to prevent apoptosis

Answer 24

CFU-E, Proerythroblasts, and early Basophilic erythroblasts.

Question 25

True or False: Hematopoietic Stem Cells (HSC) are pluripotent.

Answer 25

True.

Question 26

What is the typical ratio of hematopoietic cells to fat in a normal adult bone marrow?

Answer 26

50% hematopoietic cells and 50% fat.

Question 27

What is an erythroid island?

Answer 27

A specific organization where young, nucleated erythroid precursors surround a central macrophage.

Question 28

Which cell acts as the "nurse cell" in an erythroid island?

Answer 28

The Macrophage.

Question 29

What is the primary "nutrient" provided by the nurse cell to the developing erythroblasts?

Answer 29

Iron (essential for the production of hemoglobin).

Question 30

Which specific erythroid stages are found clustered around a nurse cell?

Answer 30

Proerythroblasts, basophilic, polychromatic, and orthochromatic erythroblasts.

Question 31

True or False: Hematopoietic precursors are randomly scattered throughout the bone marrow.

Answer 31

False. The tissue is highly organized into structures like erythroid islands.

Question 32

In normoxia, which enzyme hydroxylates HIF-2alpha using O2 as a co-substrate?

Answer 32

Prolyl Hydroxylase Domain (PHD) enzymes.

Question 33

What is the role of the von Hippel-Lindau (VHL) protein in normoxia?

Answer 33

It recognizes hydroxylated HIF-2alpha and targets it for ubiquitination and proteasomal degradation.

Question 34

Which HIF subunit is constitutively expressed (always present) regardless of oxygen levels?

Answer 34

HIF-1beta.

Question 35

Why does HIF-2alpha accumulate in the cytosol during hypoxia?

Answer 35

Because PHD lacks the O2 required to hydroxylate it, preventing its degradation.

Question 36

To what specific DNA sequence does the HIF heterodimer bind to trigger EPO transcription?

Answer 36

The Hypoxia Response Element (HRE) in the EPO gene enhancer.

Question 37

Which co-factor supports the HIF complex in triggering EPO gene transcription?

Answer 37

p300.

Question 38

What happens to the HIF-2alpha / HIF-1beta heterodimer once it forms in the cytosol?

Answer 38

It translocates to the nucleus to act as a transcription factor.

Question 39

Why does the renal vasculature facilitate shunt diffusion of O2?

Answer 39

Because the arterial and venous vessels (vasa recta) run countercurrent and in very close contact.

Question 40

Describe the O2 tension gradient from the renal cortex to the inner medulla.

Answer 40

It is high in the cortex (~50 mmHg) and very low in the inner medulla (~10–20 mmHg).

Question 41

Why is oxygen sensing in the kidney largely independent of blood flow rate?

Answer 41

The O2 gradient is generated by the anatomical arrangement of the network, so it is little affected by how fast blood moves through it.

Question 42

What specific cells are responsible for producing EPO in the kidney?

Answer 42

Renal Erythropoietin-Producing (REP) cells, which are primarily pericytes (PDGFR-beta interstitial cells).

Question 43

Where exactly are the REP cells located?

Answer 43

Along the post-glomerular blood vessels (peritubular capillaries).

Question 44

How does the kidney respond as hypoxia worsens?

Answer 44

The gradient of activated EPO-producing cells "goes deeper," activating more cells toward the outer medulla.

Question 45

How does the decrease in oxygen consumption by damaged tubules paradoxically reduce EPO?

Answer 45

It creates a "microenvironmental relative hyperoxia," which prevents HIF-2alpha from stabilizing, leading to less EPO production.

Question 46

What is the cellular transition that EPO-producing pericytes undergo during CKD?

Answer 46

They transdifferentiate into myofibroblasts.

Question 47

What is the consequence of the Extracellular Matrix (ECM) deposition and fibrosis in CKD?

Answer 47

It is associated with reduced HIF binding capacity, further lowering EPO synthesis.

Question 48

What epigenetic change occurs to the EPO gene in myofibroblasts?

Answer 48

DNA methylation of the 5' regulatory element (epigenetic silencing).

Question 49

How does DNA methylation affect the Hypoxia Response Element (HRE)?

Answer 49

It reduces the ability of the HIF complex to bind to the HRE, shutting down EPO transcription.

Question 50

What are the three primary "pillars" of EPO decrease in advancing CKD?

Answer 50

1. Relative hyperoxia (low O2 use) 2. Pericyte-Myofibroblast transition 3. Epigenetic silencing.

Question 51

How does the availability of EPO medications differ between human and veterinary medicine?

Answer 51

It is frequent and common in humans, but infrequent (if done at all) in veterinary medicine.

Question 52

Historically, how has EPO been measured in dogs and cats?

Answer 52

Using human immunoassays, though these were not originally validated for veterinary species.

Question 53

What modern method is used in research to quantify canine plasma EPO?

Answer 53

ELISA kits (e.g., from FUJIFILM VET Systems).

Question 54

What is a major hurdle for using human EPO agonists in veterinary patients?

Answer 54

They are human-sequence proteins, which can cause the animal to develop anti-EPO antibodies (potentially leading to pure red cell aplasia).

Question 55

What is true about erythropoiesis?

Answer 55

EPO stimulates precursors committed to the erythroid lineage EPO is triggered by lack of O2, not just the presence of anemia Hematopoietic precursors are not randomly distributed in the bone marrow

Question 56

What is the primary organ responsible for sensing low O2 tension and producing EPO?

Answer 56

The Kidney.

Question 57

What is the specific target tissue for circulating EPO?

Answer 57

The Bone Marrow.

Question 58

How does the body respond to EPO once it reaches the bone marrow?

Answer 58

By increasing Red Blood Cell (RBC) production (Erythropoiesis).

Question 59

What is the ultimate goal of increasing RBC production in this loop?

Answer 59

To increase O2 carrying capacity in the blood.

Question 60

Which specific molecule acts as the "oxygen sensor" because it is degraded in the presence of O2?

Answer 60

HIF-2alpha (Hypoxia-Inducible Factor 2-alpha).

Question 61

What does it mean for a molecule to be "O2-labile"?

Answer 61

It is unstable or degraded when oxygen is present.

Question 62

In normoxia, what is the first chemical modification that happens to HIF-alpha?

Answer 62

It is hydroxylated by PHD (Prolyl Hydroxylase Domain) enzymes.

Question 63

What is the specific role of VHL (von Hippel-Lindau) in the EPO pathway?

Answer 63

It recognizes hydroxylated HIF-alpha and targets it for proteasomal degradation.

Question 64

Regulation of EPO - Hypoxia

Answer 64

1. HIF-alpha accumulates in the cytosol 2. It forms a heterodimer with HIF-beta, the hypoxia-insensitive unit 3. The heterodimer translocates to the nucleus and acts as a transcriptional factor 4. This factor binds to Hypoxia-Responsive Elements (HREs), leading to EPO transcription

Question 65

Which statement is incorrect about the regulation of EPO by hypoxia?

Answer 65

HIF-1beta is consistently destructed in presence of O2

Question 66

Which statement is true about the regulation of EPO by hypoxia?

Answer 66

Hypoxia Responsive Element belongs to EPO's enhancer Hypoxia Inducible Factors bind to HRE in hypoxia Binding of HIF on HRE triggers transcription of EPO

Question 67

Which statement is incorrect about the sensing of hypoxia by the kidneys?

Answer 67

It is sensed by the juxtaglomerular apparatus JGA is the sensor for blood pressure and Na+//Cl- not hypoxia

Question 68

Hypoxia is sensed by ?

Answer 68

Renal Erythropoietin-Producing (REP) Cells, which are pericytes located along the post glomerular/peritubular capillaries.

Question 69

What anatomical arrangement allows for the oxygen shunt in the kidney?

Answer 69

The countercurrent arrangement of the vasa recta.

Question 70

Which statement is correct about the sensing of hypoxia by the kidneys?

Answer 70

1. It involves an O2 gradient throughout the parenchyma 2. It relies on the unusual renal vasculature 3. It does not depend on the rate of blood flow in kidney

Question 71

Why is there less HIF stabilization in a less functional (CKD) kidney?

Answer 71

Damaged tubules consume less O2, creating a relative "hyperoxia" that prevents HIF from stabilizing.

Question 72

What happens to pericytes during the progression of CKD?

Answer 72

They undergo a transition into myofibroblasts.

Question 73

How does ECM deposition (fibrosis) affect EPO production?

Answer 73

It is associated with reduced HIF binding capacity.

Question 74

What is the epigenetic reason for reduced EPO transcription in CKD?

Answer 74

Silencing of the HRE (Hypoxia Response Element) by DNA methylation.

Question 75

What is a "Total" summary of the result of these three mechanisms?

Answer 75

Less EPO production, leading to Non-regenerative Anemia.

Question 76

Is the buildup of metabolic waste (azotemia) responsible for the anemia seen in CKD?

Answer 76

No. The anemia is caused specifically by a lack of EPO.

Question 77

What is the "Take-home message" regarding the cause of anemia in CKD?

Answer 77

Loss of tubules and the pericyte-to-myofibroblast transition lead to decreased EPO production.

Question 78

Complete the Pathophysiology: CKD -> myofibroblast transition -> [?] -> Anemia.

Answer 78

Decreased EPO