Localization of cells producing erythropoietin in murine liver by in situ hybridization

In situ hybridization using antisense RNA probes was used to localize cells that produce erythropoietin (EPO) in the livers of anemic transgenic mice expressing the human EPO gene and in livers of anemic nontransgenic mice. In transgenic mice bled from a hematocrit of 55% to one of 10%, hepatocytes surrounding central veins synthesized large amounts of human EPO mRNA. EPO-producing cells were very rare in the area of portal triads. In transgenic mice bled to a hematocrit of 20%, a similar number and distribution of cells contained human EPO mRNA as was found with a 10% hematocrit, but the cells were less heavily labeled, indicating increased EPO production per cell at 10% hematocrit as compared with 20% hematocrit. No human EPO mRNA was detected in the kidneys of anemic transgenic mice, although endogenous murine EPO mRNA was strongly expressed in cortical interstitial cells. In sections of livers from nontransgenic mice bled from a hematocrit of 45% to one of 10%, only isolated cells produced EPO. When the types of cells could clearly be identified, approximately 80% of these cells were hepatocytes, while 20% had a nonepithelial morphology and were located in or adjacent to the sinusoidal spaces. When the sense strand was used as the RNA probe for in situ hybridization, no labeled cells were seen in normal or anemic livers. These results demonstrate that hepatocytes are responsible for production of EPO in both transgenic and nontransgenic mice and that a second cell type that is similar in morphology to EPO-producing interstitial cells in the kidney also produces EPO in the livers of nontransgenic mice.     

Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration

In situ hybridization was used to quantitate the cells that produce erythropoietin (EP) in the renal cortices of mice with varying severities of acute anemia and of mice recovering from severe, acute anemia. The number of EP-producing cells in the renal cortex increased in an exponential manner as hematocrit was decreased. Individual EP- producing cells had very similar densities of silver grains in autoradiograms regardless of whether they were from normal mice or from slightly, moderately or severely anemic animals. With increasingly severe anemia, total renal EP mRNA levels and serum EP concentrations showed increases that correlated with the number of renal EP-producing cells. These results indicate that as mice become more anemic, additional cells are recruited to produce EP rather than the cells already producing EP being stimulated to increase their individual production. In mildly and moderately anemic animals, small clusters of EP-producing cells were found in the inner cortex with large areas of cortex containing no EP-producing cells. In severely anemic mice, EP- producing cells were found throughout the inner cortex with only a very few found scattered in the outer cortex and outer medulla. The data indicate that only a subset of total renal interstitial cells produce EP. During recovery from severe, acute anemia, the numbers of EP- producing cells decreased exponentially as hematocrits rose and correlated with decreases in total renal EP mRNA and serum EP concentrations. These results suggest that following an acute blood loss and during the recovery from a blood loss, the capacity to deliver oxygen, as represented by hematocrit, is the major regulator of EP production.     

Serum immunoreactive erythropoietin in rheumatoid arthritis: impaired response to anemia

Serum immunoreactive erythropoietin (EP) levels were measured in 116 patients with rheumatoid arthritis (RA) and 20 control patients with iron deficiency anemia. Serum EP levels were significantly higher in the 46 anemic RA patients than in the 70 nonanemic RA patients (mean ± 1 SD 31.0 ± 19.8 mU/ml versus 16.8 ± 12.4 mU/ml; P < 0.0001). Furthermore, although a significant inverse correlation between the serum EP level and the hemoglobin value was present in the anemic RA patients (r = −0.57, P < 0.0001), the regression coefficient describing the relationship between serum EP and hemoglobin was significantly lower for the anemic RA patients than for patients with iron deficiency anemia (F = 6.01, P < 0.025).     

Morphological changes in erythroblasts during erythropoietin-induced terminal differentiation in vitro

Immature murine erythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells) differentiate in vitro under the influence of erythropoietin (EP). These cells were used as a model for the examination of morphological changes occurring during terminal erythroid differentiation. FVA cells differentiate more completely in vitro in response to EP than continuous erythroleukemia cell lines do in response to chemical induction. Because they can be isolated in much greater numbers and in much higher purity than bone marrow or spleen cells explanted from anemic mice, FVA cells are an attractive alternative for studies of mammalian terminal erythroid differentiation. FVA cells cultured with EP followed a sequence of differentiation events that included a progressive decrease in cell size, disappearance of nucleoli, condensation of nuclei, and accumulation of hemoglobin. After 45 h of culture most FVA cells enucleated, giving rise to vacuolated reticulocytes and free nuclei that were surrounded by a thin layer of cytoplasm and a plasma membrane. The ratio of nuclear to cytoplasmic volumes increased significantly by 24 h of culture but did not change significantly from 24 through 36 h of culture. Variation in the morphology of enucleating FVA cells indicated that not all cells proceeded through a rigorously defined series of morphological stages prior to enucleation. These results are discussed in terms of previous studies of erythroblast maturation.     

Binding of iodinated erythropoietin to rat bone marrow cells under normal and anemic conditions

Specific binding sites for erythropoietin (Epo) were shown in normal and anemic rat bone marrow cells using [125I]labeled human recombinant Epo. When rats were treated once or several times with phenylhydrazine or malotilate, or by phlebotomy, the serum Epo level determined by RIA began to increase rapidly. Thereafter, both the number of erythroid colony-forming unit (CFU-E)-derived colonies and the Epo binding capacity of bone marrow cells increased almost simultaneously in response to induced anemic states, suggesting that the amount of Epo binding in bone marrow cells may reflect in vivo erythropoiesis. Scatchard analysis of the binding data from normal rats revealed the presence of a single class of binding sites (Kd = 0.18 +/- 0.04 nM, 38 +/- 5 sites/cell). In anemic states, the apparent average receptor number per cell increased (52-62 sites/cell) without changing in binding affinity toward Epo. Furthermore, [125I]Epo was cross-linked to the cell surface molecule of approximately 165 kd in nonreducing conditions and 75 kd in reducing conditions. Autoradiographic analysis indicated that Epo receptors were distributed on immature erythroid cells. Proerythroblasts were the most heavily labeled, whereas orthochromatic erythroblasts and cells of myeloid and lymphoid lineages were not labeled. Calculations based on Scatchard and autoradiographic analysis showed that proerythroblasts have 390 receptor sites per cell, twice as many as basophilic or polychromatophilic erythroblasts have. These results are consistent with the stage-specific action of Epo in physiological differentiation of erythroid cells.     

Receptors for erythropoietin in mouse and human erythroid cells and placenta

High and lower affinity receptors for erythropoietin (EP) were initially identified on a very pure population of EP-responsive erythroblasts obtained from the spleens of mice infected with anemia strain of Friend virus (FVA). The structure of the receptor for EP in these cells was determined to be proteins of 100 and 85 Kd by cross-linking 125I-EP. In this investigation, studies on the receptors for EP were extended to other mouse erythroid cells and human erythroid cells as well as to the placentas of mice and rats. Only lower affinity receptors for EP were detected on erythroblasts purified from the spleens of mice infected with the polycythemia strain of Friend virus and a murine erythroleukemia cell line, both of which are not responsive to EP in culture. Internalization of 125I-EP was observed in both groups of cells. The structure of the receptor determined by cross-linking 125I-EP was two equally labeled proteins of 100 Kd and 85 Kd molecular mass in all these mouse erythroid cells. The structure of the receptor was found to be very similar in human erythroid colony forming cells cultured from normal blood. These cells respond to EP with erythroid maturation and were previously shown to have high and lower affinity receptors. Placentas from mice and rats were found to have only lower affinity receptors for EP, and when placental membranes were cross-linked to 125I-EP, the same 100 Kd and 85 Kd bands were found as seen in mouse and human erythroid cells. The structure of the receptor was similar in cells that have high affinity receptors (FVA-infected and human erythroid colony-forming cells) and nonresponsive erythroid cells and placenta that have lower affinity receptors, but only the cells with the high affinity receptors respond to the addition of EP with erythroid maturation.     

Isolation of erythropoietin-sensitive cells from Friend virus-infected marrow cultures: characteristics of the erythropoietin response

Murine erythroid precursor cells, stimulated to proliferate in vitro in the absence of added erythropoietin (EP) by the anemia strain of Friend virus (FVA), will subsequently respond to EP by complete erythrocyte differentiation. If not exposed to EP, the erythroid cells divide for about 120 hr in culture, and they maintain the potential for full differentiation in response to EP added at any time during the period from 72 to 120 hr. Between 96 and 120 hr of culture without added EP, the EP-sensitive erythroid precursor cells that have formed discrete erythroid bursts can be isolated in relatively large numbers from such cultures by plucking with a Pasteur pipette. The addition of EP initiates the final stages of erythroid differentiation, including heme synthesis in 70%-80% of these isolated cells. With respect to homogeneity of the precursor cells, quantity of EP-responsive cells obtainable, and uniformity of EP responsiveness, this system is uniquely favorable for biochemical studies of the late differentiation effects of EP. The overall changes in gene expression accompanying EP- induced terminal differentiation were examined by two-dimensional gel electrophoresis of proteins labeled for a short time with radioactive amino acids. Several new proteins are synthesized in these erythroid cells during terminal differentiation, but the number is a very small percentage of the total number of proteins being made. Thus, in this system, the effect of EP is to initiate expression of a small group of genes, including those for globins, spectrin, and other proteins involved in the final stages of erythroid differentiation.     

Impaired erythropoietin production in mice treated with cyclosporin A

Because recent data indicate that erythropoietin (Epo) production is defective in allogeneic bone marrow transplant (BMT) patients, we investigated the role of the immunosuppressant, nephrotoxic, agent cyclosporin A (CsA) on renal Epo production using an animal model. Mice were injected with 1.0 to 40.0 mg/kg/d CsA for 15 days. Thereafter, circulating Epo levels were evaluated in both intact animals and in mice made anemic with phenylhydrazine (PHZ). Serum Epo levels measured in CsA-treated animals were then compared with the predicted levels, which had been calculated in a reference population of normal, either intact or anemic, mice. In CsA-treated, intact animals both hematocrit and serum Epo levels were not significantly different from controls. However, serum Epo levels in CsA-treated, anemic mice were significantly lower than those expected in a control population of untreated, anemic mice with similar degrees of anemia. No significant increase in serum creatinine was recorded even at the highest doses of CsA used, nor were we able to document signs of renal toxicity by histologic examination of the kidneys. Therefore, therapeutical doses of CsA appear to affect the production of Epo under conditions in which the demand of the hormone is increased, as in response to anemia. We suggest that a subclinical kidney toxicity produced by CsA might have a role in the pathogenesis of the impaired Epo production observed in BMT patients, and may contribute to a delayed erythroid engraftment in at least some BMT patients.     

Evaluation of stem cell reserve using serial bone marrow transplantation and competitive repopulation in a murine model of chronic hemolytic anemia

Serial transplantation and competitive repopulation were used to evaluate any loss of self-replicative capacity of bone marrow stem cells in a mouse model with increased and persistent hemopoietic demands. Congenic marrows from old control and from young and old mice with hereditary spherocytic anemia (sphha/sphha) were serially transplanted at 35-day intervals into normal irradiated recipients. Old anemic marrow failed or reverted to recipient karyotype at a mean of 3.5 transplants, and young anemic marrow reverted at a mean of 4.0 transplants, whereas controls did so at a mean of 5.0 transplants. In a competitive assay in which a mixture of anemic and control marrow was transplanted, the anemic marrow persisted to 10 months following transplantation; anemic marrow repopulation was greater if anemic marrow sex matched with the host. It is possible that lifelong stress of severe anemia decreases stem cell reserve in the anemic sphha/sphha mouse marrow. However, marginal differences in serial transplantation number and the maintenance of anemic marrow in a competition assay would suggest that marrow stem cells, under prolonged stress, are capable of exhibiting good repopulating and self-replicating abilities.     

Chemotactic action of prostaglandin E2 on mouse mast cells acting via the PGE2 receptor 3

Mast cells are long-lived cells that are principally recognized for their effector function in helminth infections and allergic reactions. These cells are derived from pluripotential hematopoietic stem cells in the bone marrow that give rise to committed mast cell progenitors in the blood and are recruited to tissues, where they mature. Little is known about the chemotactic signals responsible for recruitment of progenitors and localization of mature mast cells. A mouse model was set up to identify possible mast cell progenitor chemoattractants produced during repeated allergen challenge in vivo. After the final challenge, the nasal mucosa was removed to produce conditioned medium, which was tested in chemotaxis assays against 2-wk murine bone marrow-derived c-kit+ mast cells (BMMC). A single peak of chemotactic activity was seen on reverse-phase HPLC with a retention time and electrospray mass spectrum consistent with prostaglandin E2 (PGE2). This lipid was found to be a highly potent chemoattractant for immature (2-wk) and also mature (10-wk) BMMC in vitro. Fluorescently labeled 2-wk c-kit+ BMMC, when injected intravenously, accumulated in response to intradermally injected PGE2. Analysis using TaqMan showed mRNA expression of the PGE2 receptors 3 (EP3) and 4 (EP4) on 2- and 10-wk BMMC. Chemotaxis induced by PGE2 was mimicked by EP3 agonists, blocked by an EP3 receptor antagonist, and partially inhibited by a MAPKK inhibitor. These results show an unexpected function for PGE2 in the chemotaxis of mast cells.     

Normalization of hematocrit in patients with end-stage renal disease on continuous ambulatory peritoneal dialysis: The role of erythropoietin

Observations were made retrospectively and prospectively over one year on all patients on continuous ambulatory peritoneal dialysis (CAPD) to determine the effect of this modality on the hematocrit. Serum erythropoietin and parathyroid hormone levels were measured. Within five months the hematocrit increased 47 to 127 percent up to normal in four of nine patients. Five others remained severely anemic. There was no significant difference in serum creatinine levels among the patients within one month of CAPD. The four patients who responded were anemic while on hemodialysis and other modalities of end-stage renal disease management prior to CAPD. The serum erythropoietin level in the four patients who responded was 9.0 mU/ml or greater with a mean of 28 mU/ml, whereas in those who did not respond it was 5.0 mU/ml or less with a mean of 3 mU/ml. Since uremic toxins in the middle molecule range have been postulated to be responsible for erythropoiesis suppression in end-stage renal disease, and in addition, insufficient erythropoietin production and the clearance of some middle molecular weight substances is six times greater with CAPD than with hemodialysis, it appears that CAPD can normalize the hematocrit in patients with end-stage renal disease who were anemic on other modalities with little or no change in serum creatinine, provided the remnant kidneys are capable of producing sufficient erythropoietin. Parathyroid hormone levels were higher in patients who responded than in patients who did not respond.     

Expression, localization, and signaling of PGE(2) and EP2/EP4 receptors in human nonpregnant endometrium across the menstrual cycle

This study was designed to elucidate the sites of synthesis and action of PGE(2) in the nonpregnant human uterus across the menstrual cycle. The sites of expression of PGE synthase and synthesis of PGE(2) were investigated by immunohistochemistry using full thickness uterine biopsies. Expression of PGE synthase and synthesis of PGE(2) were localized to glandular epithelial and endothelial cells in both basalis and functionalis regions of the human endometrium. By contrast, stromal staining was predominantly localized in the functionalis layer. Some cyclical variation in expression of PGE synthase and PGE(2) synthesis was observed, with reduced expression/synthesis detected in the stromal compartment of the functionalis during the late secretory phase of the menstrual cycle. Subsequently, we assessed the site of action of PGE(2) by investigating the expression of two PGE(2) receptor isoforms, namely EP2 and EP4. Cyclical variation in endometrial EP2 and EP4 receptor mRNA expression was quantified by TaqMan quantitative RT-PCR using RNA isolated from endometrial tissue collected across the menstrual cycle. No differences in EP2 receptor mRNA expression were detected; however, EP4 receptor mRNA expression was significantly higher in late proliferative stage (P < 0.05) than in early, mid, and late secretory stage endometrium. Expression patterns of EP2 and EP4 receptors were localized by nonradioactive in situ hybridization using fluorescein isothiocyanate end- labeled oligonucleotide probes. Expression of both receptors was observed in endometrial glandular epithelial and vascular cells, with no notable spatial or temporal variation. Finally, signaling of EP2/EP4 receptors was assessed by investigating cAMP generation in vitro after stimulation with PGE(2). Endometrial cAMP generation in response to PGE(2) was significantly greater in proliferative tissue compared with early and midsecretory stage tissue (3.77 +/- 0.85 vs. 1.96 +/- 0.28 and 1.38 +/- 0.23, respectively; P < 0.05). In conclusion, this study demonstrates glandular and vascular coexpression of PGE synthase, PGE(2), EP2, and EP4 receptors and suggests an autocrine/paracrine role for PGE(2) in epithelial/endothelial cell function in the human endometrium.     

Impaired fibrin elimination in the kidneys of burned rats

The fibrin elimination rate in the kidneys of burned rats was investigated during the phase of endogenous fibrinolysis inhibition. In rats previously injected with labeled fibrinogen, intravascular fibrin deposition in the kidneys was induced by infusion of thrombin and the fibrin content of the kidneys determined by homogenization and centrifugation of the tissue and radioactivity measurement of the pellet. The kidneys were removed at two different intervals after the thrombin infusion. It was found that the fibrin elimination in the kidneys of burned rats was markedly retarded in comparison to that of control rats.     

Characterization of the neurosecretory activity of hypothalamic beta-endorphin-containing neurons in primary culture

To determine the neurosecretory activity of hypothalamic beta-endorphin (beta EP)-containing neurons, rat fetal hypothalamic cells were mechanically dispersed and maintained in primary cultures for periods up to 24 days; their electrophysiological properties and regulation by depolarization, calcium and sodium channel-active agents were studied. Under culture conditions, the majority of the cells were immunopositive to neurofilament antibody, and a significant number (7-10%) were reactive to beta EP antibody. Cultured cells were often electrically excitable and possessed voltage-activated ionic conductances. In culture, there was a progressive increase in immunoreactive beta EP (IR-beta EP) in both cells and media, reaching maximum values at 12-16 days. The majority of IR-beta EP in both cells and media corresponded to [125I]beta EP on gel chromatography and was similar to the form previously found in the hypothalamus. These findings suggest viability of the beta EP neurons and continuing synthesis of IR-beta EP during the culture period. To evaluate the influence of membrane depolarization on IR-beta EP release, the cells were challenged with 56 mM potassium. This treatment induced a significant increase in medium IR-beta EP. The depolarization-induced IR-beta EP release was dependent upon calcium, since a calcium channel blocker, verapamil (0.1 microM), prevented the release; also a calcium ionophore, A23187 (1 microM), stimulated IR-beta EP release in the cultures. Activation of the sodium channel by veratridine (100 microM) also increased the medium content of IR-beta EP, and this effect was blocked by tetrodotoxin (1 microM). These results suggest that the beta EP neurons in primary culture respond to the well defined physiological challenges and that the culture system can be used in determining the regulation of hypothalamic beta EP activity.     

The effect of 5 alpha-dihydrotestosterone and 5 beta-dihydrotestosterone on erythropoiesis of the newt, Triturus cristatus carnifex (Laur.)

The erythropoietic effects of 5 alpha-dihydrotestosterone (5 alpha-DHT) and 5 beta-dihydrotestosterone (5 beta-DHT) were compared in the newt, Triturus cristatus carnifex (Laur.). In normal animals, 5 beta-DHT was more active than 5 alpha-DHT in increasing the number of circulating erythrocytes. In animals rendered completely anemic, only 5 beta-DHT was effective. The biochemical parameters of delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydratase of new basophilic erythroblasts, and incorporation of labeled glycine into heme and globin of new polychromatic erythroblasts were enhanced by 5 beta-DHT. The results are discussed with reference to a direct erythropoietin-independent action of 5 beta-DHT on erythroid stem cells.     

Contribution of the two Gs-coupled PGE2-receptors EP2-receptor and EP4-receptor to the inhibition by PGE2 of the LPS-induced TNFalpha-formation in Kupffer cells from EP2-or EP4-receptor-deficient mice. Pivotal role for the EP4-receptor in wild type Kupffer cells

BACKGROUND/AIMS: Prostaglandin E2 (PGE2) is known to inhibit the lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFalpha) formation in Kupffer cells via an increase in cAMP. Four receptor-subtypes have been cloned for PGE2 so far. Two of them, the EP2-receptor and the EP4-receptor are linked to stimulatory Gs-proteins and could mediate the inhibition by PGE2 of TNFalpha-formation. METHODS: The significance of both receptors for PGE2-dependent inhibition of LPS-induced TNFalpha-formation was studied using Kupffer cells of mice in which either one of the two receptors had been eliminated by homologous recombination. RESULTS: The mRNAs of both receptors were expressed in wild type mouse Kupffer cells. Exogenous PGE2 inhibited TNFalpha-formation in Kupffer cells lacking either EP2-receptor or EP4-receptor to a similar extent as in control cells, however, 10-fold higher PGE2 concentrations were needed for half maximal inhibition in cells lacking the EP4-receptor than in control or EP2-receptor-deficient cells. The response to endogenous PGE2 was blunted in EP4-receptor-deficient mice only and especially after prolonged incubation. CONCLUSIONS: The data indicate, that PGE2 can inhibit TNFalpha-formation via both the EP2- and the EP4-receptor and that, however, the EP4-receptor appears to be physiologically more relevant in Kupffer cells since it conferred a high affinity response to PGE2.     

Multiple signal transduction pathways through two prostaglandin E receptor EP3 subtype isoforms expressed in human uterus

PGE2 is known to induce uterine contraction by increasing intracellular Ca2+. In the present study, to investigate other functions of PGE2 in human uterus, two EP3 isoforms were isolated by the RT-PCR method using human uterus polyadenylated ribonucleic acid (RNA). These EP3 isoforms, named EP3-V and EP3-VI, are composed of 402 and 393 amino acid residues, respectively, which are unique compared with EP3 isoforms of other species. Their N-terminal 359 amino acid residues are identical to those of previously reported human EP3 isoforms, whereas the two isoforms contained a novel amino acid sequence in their C-terminal tails. The dissociation constant values of EP3-V and EP3-VI for PGE2 were 3.9 and 1.4 nmol/L, respectively, which were consistent with those of previously reported EP3 isoforms. Signaling experiments revealed that M&B28767, an EP3 agonist, not only inhibited forskolin-induced cAMP concentrations, but also activated mitogen-activated protein kinase in Chinese hamster ovary cells stably expressing EP3-V and EP3-VI. These responses were abolished by treatment with pertussis toxin. In addition, M&B28767 increased cAMP concentrations in EP3-VI-expressing cells, whereas it did not in EP3-V-expressing cells. M&B28767 did not stimulate phosphoinositide turnover in EP3-V or EP3-VI-expressing cells. EP3-V and EP3-VI messenger RNAs (mRNAs) were detected abundantly in human uterus, whereas weak, but substantial, bands were detected in the lung and kidney in RT-PCR specific for each mRNA. In situ hybridization revealed EP3-V and EP3-VI mRNAs in the human myometrium, but not in the endometrium. The present study suggests that EP3-V and EP3-VI are possibly involved in the proliferation of cells in human myometrium.     

经血传播病毒在肝脏及肝外组织的感染状况

目的探讨TTV在肝脏及肝外组织中的定位、分布及其意义。方法通过PCR方法扩增TTV基因组ORF2中123bp的DNA片段，构建TTVDNA质粒并克隆，序列分析表明与日本ABO11494序列具有高度同源性。采用地高辛标记TTVDNA探针并与组织进行原位杂交，检测了22例因肝病死亡患者的肝脏、脾脏、肾脏、胃、小肠等组织内TTVDNA感染状况。结果8例肝脏检出TTVDNA，5例肾脏、４例脾脏、２例小肠、２例胃检出TTVDNA。阳性信号在组织中分布呈局灶性或散在胞浆型。肝脏感染度较其它组织高。阳性细胞与组织炎症坏死无固定病理解剖学关系。结论支持TTV有嗜肝性的观点；TTV可以感染肝外多种组织并可能导致TTV持续感染。     

Effect of nephrectomy and cholesterol feeding on the metabolism of intravenously administered DL-mevalonate in the rat

The aim of the investigation was to study the role of the kidney in the metabolism of circulating mevalonate. DL-mevalonate-2-¹⁴C was injected intravenously to sham-operated and nephrectomized rats that were sacrificed after 90 min. The diet given to part of the animals prior to the experiments were supplemented with 5% cholesterol. In intact sham-operated rats given the control diet the labeled non-saponifiable lipids of the kidneys exceeded the amounts of such material recovered in other tissues analyzed. The kidneys also differed from many other organs with regard to the slow rate by which squalene and lanosterol were converted to C27 sterols. Following nephrectomy there was a marked increase of the labeled nonsaponifiable lipids of the liver and blood. As in the intact rats the major part of this label was recovered in the C27 sterol fraction. The radioactivity in the C27 sterol fraction of the liver decreased upon cholesterol feeding. This change was balanced by a reciprocal elevation of labeled squalene and lanosterol whereby the total amounts of the labeled nonsaponifiable lipids was uninfluenced by the supplementation of cholesterol to the diet.