Reevaluation of erythropoietin production by the nephron

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in β-intercalated or non α/non β-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.     

Renal carbonic anhydrase activity in DBA/2FG-pcy/pcy mice with inherited polycystic kidney disease.

DBA/2FG-pcy/pcy (D2-pcy) mice are a hereditary murine model of slowly progressive polycystic kidney disease (PKD) and characterized by the persistent excretion of acidic urine, in association with polyuria, after weaning. In this study, the activity of carbonic anhydrase (CA) and it histological distribution in the kidney of D2-pcy mice were investigated by immunohistochemistry. Significantly higher CA activity was detected in the cytosolic, but not membrane, fraction of kidney homogenates in 5-week-old D2-pcy mice than in age-matched, control DBA/2 (D2) mice, and a more rapid rate of urine acidification was noted in 11-week-old mice when acetazolamide, an inhibitor of the enzyme, was administered orally. By immunohistochemistry for the major renal CA isoenzyme (CA II), epithelial cells in the distal straight tubules and the cortical collecting ducts were stained intensely, whereas those of the proximal convoluted tubules had only weak and diffuse staining. The glomeruli, the proximal straight tubules and the ascending thin limb of Henle's loop were almost free from staining. In the cells lining cysts and/or dilated tubules, CA II activity was well preserved, although the staining intensity was considerably reduced in fully-flattened, lining cells of cysts, but no difference was found between D2-pcy and D2 mice in any segmental localization of renal CA II activity. From these results it seems that D2-pcy mice in the early stages of the cystic disease continue to secrete excess protons through the CA-mediated reaction that is stimulated for regulation of acid-base balance in the distal portion of the nephron and the collecting duct in kidney. It also suggests that monitoring urine pH may be useful in predicting the effects of early interventions on the progression of slowly developing renal cysts.     

Tissue-specific regulation of erythropoietin production in the murine kidney, brain, and uterus

Erythropoietin (Epo) produced by the kidney regulates erythropoiesis. Recent evidence suggests that Epo in the cerebrum prevents neuron death and Epo in the uterus induces estrogen (E(2))-dependent uterine angiogenesis. To elucidate how Epo expression is regulated in these tissues, ovariectomized mice were given E(2) and/or exposed to hypoxia, and the temporal patterns of Epo mRNA levels were examined. Epo mRNA levels in the kidney and cerebrum were elevated markedly within 4 h after exposure to hypoxia. Although the elevated level of Epo mRNA in the kidney decreased markedly within 8 h despite continuous hypoxia, the high level in the cerebrum was sustained for > or = 24 h, indicating that downregulation operates in the kidney but not in the brain. E(2) transiently induced Epo mRNA in the uterus but not in the kidney and cerebrum. Interestingly, the uterine Epo mRNA was hypoxia inducible only in the presence of E(2). Thus Epo expression appears to be regulated in a tissue-specific manner, endorsing the tissue-specific functions of Epo.     

Isolation and characterization of renal erythropoietin-producing cells from genetically produced anemia mice

Understanding the nature of renal erythropoietin-producing cells (REPs) remains a central challenge for elucidating the mechanisms involved in hypoxia and/or anemia-induced erythropoietin (Epo) production in adult mammals. Previous studies have shown that REPs are renal peritubular cells, but further details are lacking. Here, we describe an approach to isolate and characterize REPs. We bred mice bearing an Epo gene allele to which green fluorescent protein (GFP) reporter cDNA was knocked-in (Epo(GFP)) with mice bearing an Epo gene allele lacking the 3' enhancer (Epo(Δ3'E)). Mice harboring the mutant Epo(GFP/Δ3'E) gene exhibited anemia (average Hematocrit 18% at 4 to 6 days after birth), and this perinatal anemia enabled us to identify and purify REPs based on GFP expression from the kidney. Light and confocal microscopy revealed that GFP immunostaining was confined to fibroblastic cells that reside in the peritubular interstitial space, confirming our previous observation in Epo-GFP transgenic reporter assays. Flow cytometry analyses revealed that the GFP fraction constitutes approximately 0.2% of the whole kidney cells and 63% of GFP-positive cells co-express CD73 (a marker for cortical fibroblasts and Epo-expressing cells in the kidney). Quantitative RT-PCR analyses confirmed that Epo expression was increased by approximately 100-fold in the purified population of REPs compared with that of the unsorted cells or CD73-positive fraction. Gene expression analyses showed enrichment of Hif2α and Hif3α mRNA in the purified population of REPs. The genetic approach described here provides a means to isolate a pure population of REPs, allowing the analysis of gene expression of a defined population of cells essential for Epo production in the kidney. This has provided evidence that positive regulation by HIF2α and negative regulation by HIF3α might be necessary for correct renal Epo induction.     

DNA synthesis during larval development and compensatory growth in the mesonephros of Xenopus laevis.

Autoradiography following tritiated thymidine administration to Xenopus laevis tadpoles of stages 45–48 of larval development has revealed that, as the cells of the mesonephric kidney differentiate during organogenesis, there is a marked decrease in the percentage of cells synthesizing DNA (from 100% at stage 45 to less than 9% at stage 48). In the adult this figure is of the order of 0.1%. This reduced DNA synthetic activity was found to take place in the cells of both the proximal and distal tubules of the nephrons. Special mucous cells which serve as markers of distal tubules were not observed to synthesize DNA after the onset of their differentiation at stage 48 of larval development.Through the partial extirpation of tissue in one kidney of adult Xenopus laevis males, DNA synthesis was reactivated in differentiated cells. The increased DNA synthetic activity following partial unilateral nephrectomy was found to be maximal after 6 days of recovery when 19% of cells synthesize DNA. This increased DNA synthetic activity was found to occur only in the cells of the distal tubules, both mucous and nonmucous cells, while the cells of the proximal tubules did not respond to this reactivation.The apparent inability of proximal tubule cells to synthesize DNA following partial unilateral nephrectomy is discussed.     

Carbonic anhydrase activity in kidney and lower intestine of the European starling

A histochemical investigation of kidney and lower intestine of the European starling (Sturnus vulgaris) shows no carbonic anhydrase activity in proximal convoluted tubules, although activity is seen in similarly prepared sections of rat proximal tubules. Early distal tubule cells in the starling are stained throughout the cytoplasm and at the apical and highly infolded basolateral membranes. Late distal tubules lose apical activity and have reduced basolateral infolding, resulting in less intense staining. Darkly stained intercalated cells appear in the connecting tubules and cortical collecting ducts. Both of these segments also show intense basolateral staining. Medullary cones of the starling are highly organized, with central zones containing unstained thin descending limbs of loops of Henle, surrounded by both medullary collecting ducts with only scattered cells staining for enzyme, and by thick ascending limb segments. The latter contain many uniformly stained cells intermingled with occasional unstained cells. Scattered cells of the starling colonic villi demonstrate intense apical brush border membrane staining as well as cytoplasmic staining. Cells lining the cloaca stain less intensely. A biochemical assay for carbonic anhydrase was used to quantify enzyme activity in these tissues. Starling kidney contained 1.96 ± 0.33 (mean ± SEM) enzyme units/mg protein, less than half the activity seen in rat kidney. Stripped colonic epithelium contained 0.66 ± 0.15 enzyme units/mg protein. These quantitative results correlate well with the interpretations derived from the histochemical observations. The lack of proximal tubule carbonic anhydrase activity suggests that the avian kidney relies more on distal nephron segments to achieve net acidification of the urine.     

Role of TASK2 potassium channels regarding volume regulation in primary cultures of mouse proximal tubules

Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using beta-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 microM 293B, but blocked by 500 microM quinidine and 10 microM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.     

Beige granules as a cell marker for clonal analysis in kidney and liver of mouse aggregation chimaeras, and three-dimensional reconstruction from serial paraffin sections

Anomalous giant granules of beige (bg) mice have been used as a cell marker in the study of cell lineage of mast cells. Similar granules are known to exist in other tissues including kidney proximal tubules and liver parenchymal cells. In the present study, these granules were found to give yellow or orange autofluorescence when the tissue had been fixed with formaldehyde and embedded in paraffin. Thus, the granules can be used as a cell marker that can be visualized in serial paraffin sections without any specific histochemical staining. Chimaeric mice were produced by aggregation of 8-cell-stage embryos of beige (C57BL/6J-bgJ/bgJ) and A/J strains. The chimaeric liver showed beige cell patches with complicated shapes, although the patches frequently conformed to the shape of parenchymal cell cord or plate structures. In chimaeric kidney, beige cells formed coherent patches in the proximal tubules. The tubules were found to contain more than one clone. The patches frequently had long extended shapes suggesting growth of the clone along the tubule axis. Three-dimensional image reconstruction from the serial paraffin sections was carried out with the aid of a computer-assisted image analysis system, resulting in a clearer image of the patch shape.     

N-cadherin is depleted from proximal tubules in experimental and human acute kidney injury

Ischemia remains the most common cause of acute kidney injury (AKI). Decreased intercellular adhesion and alterations in adhesion molecules may contribute to the loss of renal function observed in AKI. In the present study, we evaluated the distribution of adhesion molecules in the human kidney and analyzed their expression in human and experimental AKI. Specimens of human kidneys obtained from patients with and without AKI were stained for the cell adhesion molecules E-cadherin, N-cadherin and β-catenin. Experimental AKI in rats was induced by renal artery clamping. Immunostaining and immunoblotting were carried out for E-cadherin, N-cadherin and β-catenin. Proximal tubule cells from opossum kidneys (OKs) were used to analyze the effect of chemical hypoxia (ATP depletion) in vitro. In the adult human kidney, N-cadherin was expressed in proximal tubules, while E-cadherin was expressed in other nephron segments. β-Catenin was expressed in both proximal and distal tubules. In human AKI and in ischemic rat kidneys, N-cadherin immunostaining was depleted from proximal tubules. There was no change in E-cadherin or β-catenin. In vitro, OK cells expressed N-cadherin only in the presence of collagen, and ATP depletion led to a depletion of N-cadherin. Collagen IV staining was reduced in ischemic rat kidneys compared to controls. The results of the study suggest that N-cadherin may play a significant role in human and experimental AKI.     

Expression of Somatostatin and Somatostatin Receptor Subtypes 1�C5 in Human Normal and Diseased Kidney

Somatostatin mediates inhibitory functions through five G protein–coupled somatostatin receptors (sst_1–5). We used immunohistochemistry, immunofluorescence, and RT-PCR to determine the presence of somatostatin receptors sst₁, sst_2A, sst_2B, sst₃, sst₄, and sst₅ in normal and IgA nephropathy human kidney. All somatostatin receptors were detected in the thin tubules (distal convoluted tubules and loops of Henle) and thick tubules (proximal convoluted tubules) in the tissue sections from nephrectomy and biopsy samples. Immunopositive sst₁ and sst₄ staining was more condensed in the cytoplasm of tubular epithelial cells. In normal kidney tissue sections, podocytes and mesangial cells in the glomeruli stained for sst₁, sst_2B, sst₄ and sst₅, and stained weakly for sst₃. In IgA kidney tissue, the expression of somatostatin receptors was significantly increased with particular immmunopositive staining for sst₁, sst_2B, sst₄, and sst₅ within glomeruli. In the epithelial cells, the staining for sst_2B and sst₄ in proximal tubules and sst₁, sst_2B, and sst₅ in distal tubules was increased. The mRNA expression of sst_1–5 was also detected by RT-PCR. Somatostatin and all five receptor subtypes were ubiquitously distributed in normal kidney and IgA nephropathy. The increased expression of somatostatin receptors in IgA nephropathy kidney might be the potential pathogenesis of inflammatory renal disease. (J Histochem Cytochem 56:733–743, 2008)     

Modifications of the genital kidney proximal and distal tubules for sperm transport in Notophthalmus viridescens (Amphibia,Urodela, Salamandridae)

Male salamanders use nephrons from the genital kidney to transport sperm from the testicular lobules to the Wolffian duct. The microstructure of the epithelia of the genital kidney proximal tubule and distal tubule was studied over 1 year in a population of Notophthalmus viridescens from Crawford and Pike counties in central Missouri. Through ultrastructural analysis, we were able to support the hypothesis that the genital kidney nephrons are modified to aid in the transportation of sperm. A lack of folding of the basal plasma membrane, in both the genital kidney proximal and distal tubules when compared to the pelvic kidney proximal and distal tubules, reduces the surface area and thus likely decreases the efficiency of reabsorption in these nephron regions of the genital kidney. Ciliated epithelial cells are also present along the entire length of the genital kidney proximal tubule, but are lacking in the epithelium of the pelvic kidney proximal tubule. The exact function of these cilia remains unknown, but they may aid in mixing of seminal fluids or the transportation of immature sperm through the genital kidney nephrons. Ultrastructural analysis of proximal and distal tubules of the genital kidney revealed no seasonal variation in cellular activity and no mass production of seminal fluids throughout the reproductive cycle. Thus, we failed to support the hypothesis that the cellular activity of the epithelia lining the genital kidney nephrons is correlated to specific events in the reproductive cycle. The cytoplasmic contents and overall structure of the genital and pelvic kidney epithelial cells were similar to recent observations in Ambystoma maculatum, with the absence of abundant dense bodies apically in the epithelial cells lining the genital kidney distal tubule. J. Morphol. 275:914–922, 2014. © 2014 Wiley Periodicals, Inc.     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

To determine if rat kidney Na+, K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na+, K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na+, K+-ATPase and rat kidney microsomes were treated with antiserum (1:200), followed by [125I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the alpha-subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the beta-subunit. When the Na+, K+-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na+, K+-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na+, K+-ATPase concentration in the rat kidney.     

Electron-microscopic and autoradiographic study of giant cells of foreign bodies in the focus of aseptic inflammation]

Ultrastructure of sinuous proximal and straight distal tubules, as well as collecting tubules of the cortical layer in the rat kidney fixed with perfusion has been studied with electron microscopic morphometry 7--8 weeks after contralateral nephrectomy. The volume of mitochondria, the area of their crists, the area of the membranes in intracellular labyrinth and other morphometrical parameters have been calculated. Relative volume of mitochondria in the nephron areas studied does not change, while in the collecting tubules it is elevated. The area of crists in mitochondria and "coefficient of morphological organization level" of these organells in the proximal tubules are increased, in the distal do not change, in the collecting tubules increase again. Subcellular changes described are discussed mainly in terms of enhancement of concentrating function of the compensatory-hypertrophic kidney.     

Carbonic anhydrase in the monkey kidney

The distribution of carbonic anhydrase in the kidney of the cynomolgus monkey was studied by the histochemical method of Hansson. Glomeruli and Bowman's capsule were inactive. Convoluted proximal tubules showed high enzyme activity at the brush border and the basolateral membranes and the cytoplasm. Straight proximal tubules were less intensely stained. In nephrons with long loops of Henle, the descending thin limb contained weak enzyme activity, whereas the ascending thin limb was inactive. The thick limb of Henle's loop displayed most enzyme activity at the luminal cell border. In distal convoluted tubules enzyme activity was restricted to the basal part of the cells. In the late distal tubule, intercalated cells appeared among the "ordinary" distal cells and contained abundant cytoplasmic enzyme. Many intensely stained intercalated cells were also found in the cortical and outer medullary segments of the collecting duct, intermingled with more weakly stained chief cells. In the inner medullary segment of the collecting duct, enzyme activity gradually disappeared. Many capillaries were clearly stained for enzyme activity. The capillary staining apparently varied with that of the kidney tubules; virtually all capillaries in the cortex, but very few in the inner medulla, were stained. The distribution of carbonic anhydrase in the kidney tubules of the monkey is very similar to that in man and in the rat, but the primate kidney differs from the rat kidney by the presence of capillary enzyme activity. The functional importance of this difference is not clear at present.     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.     

OCCURRENCE OF PHAGOSOMES AND PHAGO-LYSOSOMES IN DIFFERENT SEGMENTS OF THE NEPHRON IN RELATION TO THE REABSORPTION, TRANSPORT, DIGESTION, AND EXTRUSION OF INTRAVENOUSLY INJECTED HORSERADISH PEROXIDASE

The size, number, and location of lysosomes, phagosomes, and phago-lysosomes in different segments of the proximal and distal tubules, in the collecting tubules, and in invading macrophages of the kidneys of rats were compared by staining lysosomes (acid phosphatase) red, and phagosomes (injected horseradish peroxidase) blue in separate sections, and by staining phago-lysosomes purple by successive application of the reactions for the two enzymes in the same sections. It was concluded from these observations that the absorption of the foreign protein from the lumen and its gradual digestion in large phago-lysosomes took place mainly in the cells of the proximal convoluted tubules of the outer cortex. Several segments of the proximal convoluted tubules were distinguished on the basis of differences in the size and location of the phago-lysosomes and the amounts of peroxidase ingested. The distal tubules showed, in addition to moderate numbers of phago-lysosomes, many small phagosomes in the apical and basal zones of the cells. Moderate numbers of phagosomes and phago-lysosomes were observed in the cells of the collecting tubules. Macrophages showing very large phago-lysosomes were seen in the peritubular capillaries of the medulla, after injection of peroxidase. When high doses of peroxidase were administered, enlarged phago-lysosomes, parts of which seemed to be extruded into the lumen, were formed in the terminal segments of the proximal convoluted tubules.     

Immunoexpression of aquaporins 1, 2, 3 and 4 in kidney of yak (Bos grunniens) on the Qinghai-Tibetan Plateau

Aquaporins (AQP) 1, 2, 3 and 4 belong to the aquaporin water channel family and play an important role in urine concentration by reabsorption of water from renal tubule fluid. Renal AQPs have not been reported in the yak (Bos grunniens), which resides in the Qinghai Tibetan Plateau. We investigated AQPs 1?4 expressions in the kidneys of Yak using immunohistochemical staining. AQP1 was expressed mainly in the basolateral and apical membranes of the proximal tubules and descending thin limb of the loop of Henle. AQP2 was detected in the apical plasma membranes of collecting ducts and distal convoluted tubules. AQP3 was located in the proximal tubule, distal tubule and collecting ducts. AQP4 was located in the collecting ducts, distal straight tubule, glomerular capillaries and peritubular capillaries. The expression pattern of AQPs 1?4 in kidney of yak was different from other species, which possibly is related to kidney function in a high altitude environment.     

Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation

Erythropoietin (Epo) and its receptor (EpoR), critical for erythropoiesis, are expressed in the nervous system. Prior to death in utero because of severe anemia EpoR-null mice have fewer neural progenitor cells, and differentiated neurons are markedly sensitive to hypoxia, suggesting that during development Epo stimulates neural cell proliferation and prevents neuron apoptosis by promoting oxygen delivery to brain or by direct interaction with neural cells. Here we present evidence that neural progenitor cells express EpoR at higher levels compared with mature neurons; that Epo stimulates proliferation of embryonic neural progenitor cells; and that endogenous Epo contributes to neural progenitor cell proliferation and maintenance. EpoR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue but not in brain. Although these mice exhibited normal hematopoiesis and erythrocyte production and survived to adulthood, neural cell proliferation and viability were affected. Embryonic brain exhibited increased neural cell apoptosis, and neural cell proliferation was reduced in the adult hippocampus and subventricular zone. Neural cells from these animals were more sensitive to hypoxia/glutamate neurotoxicity than normal neurons in culture and in vivo. These observations demonstrate that endogenous Epo/EpoR signaling promotes cell survival in embryonic brain and contributes to neural cell proliferation in adult brain in regions associated with neurogenesis. Therefore, Epo exerts extra-hematopoietic function and contributes directly to brain development, maintenance, and repair by promoting cell survival and proliferation independent of insult, injury, or ischemia.