Renal Erythropoietin-producing Cells Research Articles

Cover Image, Volume 237, Number 5, May 2022

Front Cover: The cover image is based on the Research Article Neurogenic and pericytic plasticity of conditionally immortalized cells derived from renal erythropoietin-producing cells by Roland H. Wenger et al., https://doi.org/10.1002/jcp.30677.

Interconnections of fibroblast growth factor 23 and klotho with erythropoietin and hypoxia-inducible factor.

Bone marrow (BM) hematopoiesis is tightly regulated process and bone components such as osteoblasts, extracellular matrix, and minerals influence hematopoiesis via regulation of hematopoietic stem cell function. Erythropoietin (EPO) secreted mostly by renal EPO producing (REP) cells which employ the hypoxia-inducible factor (HIF) pathway. When tissue hypoxia occurs, HIFs bind to hypoxia response element in the EPO promoter and induce EPO production. EPO binds to the EPO receptor on red cell progenitors in the BM and triggers expansion of red cell mass. Fibroblast growth factor-23 (FGF23) which is secreted mostly by osteoblasts and less by BM impacts hematopoiesis by influencing EPO production. Reciprocally, increases of EPO (acute or chronic) influence both FG23 production and cleavage resulting in variation of c fragment FGF23 (cFGF23) and intact FGF23 (iFGF23) ratios. As HIFs stimulate EPO production, they indirectly affect FGF23. Direct stimulation of FGF23 synthesis by binding of HIF on FGF23 promoter is also suggested. FGF23 cleavage by furin is another potential mechanism affecting FGF23 levels. Klotho is present in membrane-bound (transmembrane) and free (circulating) forms. Transmembrane klotho is the co-receptor of FGF23 and forms complexes with FGF23 receptors in the membrane surface and required for FGF23 actions. Recent evidence showed that klotho is also associated with EPO and HIF production suggesting a complex relationship between FGF23, klotho, EPO, and HIF. In this review, we have summarized the connections between FGF23, klotho, HIF, and EPO and their reflections to hematopoiesis.

Neurogenic and pericytic plasticity of conditionally immortalized cells derived from renal erythropoietin-producing cells.

In adult mammals, the kidney is the main source of circulating erythropoietin (Epo), the master regulator of erythropoiesis. In vivo data in mice demonstrated multiple subtypes of interstitial renal Epo‐producing (REP) cells. To analyze the differentiation plasticity of fibroblastoid REP cells, we used a transgenic REP cell reporter mouse model to generate conditionally immortalized REP‐derived (REPD) cell lines. Under nonpermissive conditions, REPD cells ceased from proliferation and acquired a stem cell‐like state, with strongly enhanced hypoxia‐inducible factor 2 (HIF‐2α), stem cell antigen 1 (SCA‐1), and CD133 expression, but also enhanced alpha‐smooth muscle actin (αSMA) expression, indicating myofibroblastic signaling. These cells maintained the “on‐off” nature of Epo expression observed in REP cells in vivo, whereas other HIF target genes showed a more permanent regulation. Like REP cells in vivo, REPD cells cultured in vitro generated long tunneling nanotubes (TNTs) that aligned with endothelial vascular structures, were densely packed with mitochondria and became more numerous under hypoxic conditions. Although inhibition of mitochondrial oxygen consumption blunted HIF signaling, removal of the TNTs did not affect or even enhance the expression of HIF target genes. Apart from pericytes, REPD cells readily differentiated into neuroglia but not adipogenic, chondrogenic, or osteogenic lineages, consistent with a neuronal origin of at least a subpopulation of REP cells. In summary, these results suggest an unprecedented combination of differentiation features of this unique cell type.

Alteration of the DNA Methylation Signature of Renal Erythropoietin-Producing Cells Governs the Sensitivity to Drugs Targeting the Hypoxia-Response Pathway in Kidney Disease Progression.

Chronic kidney disease (CKD) affects more than 10% of the population worldwide and burdens citizens with heavy medical expenses in many countries. Because a vital erythroid growth factor, erythropoietin (EPO), is secreted from renal interstitial fibroblasts [renal EPO-producing (REP) cells], anemia arises as a major complication of CKD. We determined that hypoxia-inducible factor 2α (HIF2α), which is inactivated by HIF-prolyl hydroxylase domain-containing proteins (PHDs) in an oxygen-dependent manner, tightly regulates EPO production in REP cells at the gene transcription level to maintain oxygen homeostasis. HIF2α-mediated disassembly of the nucleosome in the EPO gene is also involved in hypoxia-inducible EPO production. In renal anemia patients, anemic and pathological hypoxia is ineffective toward EPO induction due to the inappropriate over-activation of PHDs in REP cells transformed into myofibroblasts (MF-REP cells) due to kidney damage. Accordingly, PHD inhibitory compounds are being developed for the treatment of renal anemia. However, our studies have demonstrated that the promoter regions of the genes encoding EPO and HIF2α are highly methylated in MF-REP cells, and the expression of these genes is epigenetically silenced with CKD progression. This finding notably indicates that the efficacy of PHD inhibitors depends on the CKD stage of each patient. In addition, a strategy for harvesting renal cells, including REP cells from the urine of patients, is proposed to identify plausible biomarkers for CKD and to develop personalized precision medicine against CKD by a non-invasive strategy.

An immortalized cell line derived from renal erythropoietin-producing (REP) cells demonstrates their potential to transform into myofibroblasts

The erythroid growth factor erythropoietin (Epo) is produced by renal interstitial fibroblasts, called REP (renal Epo-producing) cells, in a hypoxia-inducible manner. In chronic kidney disease (CKD), REP cells lose their Epo-production ability, leading to renal anaemia. Concurrently, REP cells are suggested to be transformed into myofibroblasts, which are the major player of renal fibrosis. Although establishment of cultured cell lines derived from REP cells has been a long-term challenge, we here successfully established a REP-cell-derived immortalized and cultivable cell line (Replic cells) by using a genetically modified mouse line. Replic cells exhibited myofibroblastic phenotypes and lost their Epo-production ability, reflecting the situation in renal fibrosis. Additionally, we found that cell-autonomous TGFβ signalling contributes to maintenance of the myofibroblastic features of Replic cells. Furthermore, the promoters of genes for Epo and HIF2α, a major activator of Epo gene expression, were highly methylated in Replic cells. Thus, these results strongly support our contention that REP cells are the origin of myofibroblasts in fibrotic kidneys and demonstrate that cell-autonomous TGFβ signalling and epigenetic silencing are involved in renal fibrosis and renal anaemia, respectively, in CKD. The Replic cell line is a useful tool to further investigate the molecular mechanisms underlying renal fibrosis.

Pathogenesis and treatment of anemia in chronic kidney disease.

Chronic kidney disease leads to dysfunction of renal erythropoietin-producing cells, resulting in a decrease in erythrocyte production. A decrease in oxygen delivery to vital organs due to anemia results in worse quality of life and is associated with poor prognosis of the patients. Treatment with human recombinant erythropoietin or its improved version of erythropoiesis-stimulating agent (ESA) with a longer half-life is effective. However, some patients show hyporesponsiveness to ESA. Hyporesponsiveness to ESA, which can be induced by factors such as inflammation and uremic toxins, is associated with poor prognosis. Production of erythropoietin in the kidney is regulated by a transcription factor, hypoxia-inducible factor (HIF). PHD regulates HIF activity, and recently developed PHD inhibitors need to be clinically used as drugs to ameliorate anemia in CKD by activating HIF.

DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys.

Renal erythropoietin-producing cells (REPCs) remain in the kidneys of patients with chronic kidney disease, but these cells do not produce sufficient erythropoietin in response to hypoxic stimuli. Treatment with HIF stabilizers rescues erythropoietin production in these cells, but the mechanisms underlying the decreased response of REPCs in fibrotic kidneys to anemic stimulation remain elusive. Here, we show that fibroblast-like FOXD1+ progenitor-derived kidney pericytes, which are characterized by the expression of α1 type I collagen and PDGFRβ, produce erythropoietin through HIF2α regulation but that production is repressed when these cells differentiate into myofibroblasts. DNA methyltransferases and erythropoietin hypermethylation are upregulated in myofibroblasts. Exposure of myofibroblasts to nanomolar concentrations of the demethylating agent 5-azacytidine increased basal expression and hypoxic induction of erythropoietin. Mechanistically, the profibrotic factor TGF-β1 induced hypermethylation and repression of erythropoietin in pericytes; these effects were prevented by 5-azacytidine treatment. These findings shed light on the molecular mechanisms underlying erythropoietin repression in kidney myofibroblasts and demonstrate that clinically relevant, nontoxic doses of 5-azacytidine can restore erythropoietin production and ameliorate anemia in the setting of kidney fibrosis in mice.

Roles of renal erythropoietin-producing (REP) cells in the maintenance of systemic oxygen homeostasis.

Erythropoietic induction is critical for enhancing the efficiency of oxygen delivery during the chronic phase of the systemic hypoxia response. The erythroid growth factor erythropoietin (Epo) triggers the erythropoietic induction through the activation of erythroid genes related to cell survival, differentiation, and iron metabolism. Because Epo is produced in renal Epo-producing (REP) cells in a hypoxia-inducible manner, REP cells serve as a control center for the systemic hypoxia response. In fact, the loss of Epo production in REP cells causes chronic severe anemia in genetically modified mice, and REP cell-specific inactivation of PHD2 (prolyl-hydroxylase domain enzyme 2) results in erythrocytosis via overexpression of the Epo gene due to the constitutive activation of HIF2α (hypoxia-inducible transcription factor 2α). REP cells are located in the interstitial spaces between renal tubules and capillaries, where the oxygen supply is low but oxygen consumption is high, for the highly sensitive detection of decreased oxygen supplies to the body. Under disease conditions, REP cells transform to myofibroblasts and lose their Epo-producing ability. Therefore, elucidation of Epo gene regulation and REP cell features directly contributes to understanding the pathology of chronic kidney disease. To further analyze REP cells, we introduce a newly established mouse line in which REP cells are efficiently labeled with fluorescent protein.

Renal erythropoietin-producing cells in health and disease.

Erythropoietin (Epo) is an indispensable erythropoietic hormone primarily produced from renal Epo-producing cells (REPs). Epo production in REPs is tightly regulated in a hypoxia-inducible manner to maintain tissue oxygen homeostasis. Insufficient Epo production by REPs causes renal anemia and anemia associated with chronic disorders. Recent studies have broadened our understanding of REPs from prototypic hypoxia-responsive cells to dynamic fibrogenic cells. In chronic kidney disease, REPs are the major source of scar-forming myofibroblasts and actively produce fibrogenic molecules, including inflammatory cytokines. Notably, myofibroblast-transformed REPs (MF-REPs) recover their original physiological properties after resolution of the disease insults, suggesting that renal anemia and fibrosis could be reversible to some extent. Therefore, understanding the plasticity of REPs will lead to the development of novel targeted therapeutics for both renal fibrosis and anemia. This review summarizes the regulatory mechanisms how hypoxia-inducible Epo gene expression is attained in health and disease conditions.

Conversion of Renal Erythropoietin-Producing Cells to Myofibroblasts: A Single Mechanism for Both Anemia and Fibrosis in Kidney Disease

Conversion of Renal Erythropoietin-Producing Cells to Myofibroblasts: A Single Mechanism for Both Anemia and Fibrosis in Kidney Disease

Treatment with erythropoiesis-stimulating agents in chronic kidney disease patients with cancer

Treatment of anemia remains an important component in the care of patients with nondialysis chronic kidney disease (CKD) and end-stage renal disease (ESRD). Erythropoietin-stimulating agents (ESAs) remains a key anemia treatment strategy in this patient population. However, anemia management in this group can become more complicated by prior or current history of malignancy. There has been a great deal of work both scientifically and in clinical trials in oncology that have revealed certain concerns and risks of ESA use in patients with cancer. In this review, we will bring together knowledge from nephrology and oncology literature to help nephrologists understand the implications for ESA treatment when CKD/ESRD is complicated by cancer. We also suggest an approach to the management of anemia in this patient group with active or previous malignancy.

Lipopolysaccharide-induced cross-tolerance against renal ischemia–reperfusion injury is mediated by hypoxia-inducible factor-2α-regulated nitric oxide production

Although the protective effect of lipopolysaccharide (LPS) pretreatment on renal ischemia/reperfusion injury is known, a link to hypoxia-inducible factors (HIFs) has not been established. Here we show that LPS treatment led to HIF-2α accumulation in mouse kidneys and endothelial cells, a result of nuclear factor-κB activation. Inactivation of HIF-2α, rather than HIF-1α, completely negated LPS-mediated protection against renal ischemia/reperfusion injury. LPS-stimulated renoprotection was related to inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression, increased production of nitric oxide, and enhanced postischemic microcirculatory recovery. All these effects were lost in HIF-2α knockout mice. Preischemic administration of a nitric oxide donor, rather than erythropoietin, restored the lost preconditioning effect of LPS in HIF-2α knockout mice. In vitro and in vivo studies demonstrated that HIF-2α in endothelial cells, rather than myeloid cells or hepatocytes, was responsible for the LPS-mediated effects. Thus, our results demonstrated that LPS preconditioning protected against renal ischemia/reperfusion injury by HIF-2α activation in endothelial cells that subsequently improved renal microvascular perfusion and reduced ischemic tubular damage.

Fibrosis and anaemia in CKD—two beasts, one ancestor

Renal fibrosis and anaemia are hallmarks of progressive chronic kidney disease (CKD). New evidence demonstrates that these conditions are intimately connected, as injury triggers the phenotypic transition of renal erythropoietin-producing cells into fibrogenic myofibroblasts. Strategies to reverse such a transition may hold promise to alleviate both anaemia and fibrosis in CKD.

A mouse model of adult-onset anaemia due to erythropoietin deficiency

Erythropoietin regulates erythropoiesis in a hypoxia-inducible manner. Here we generate inherited super-anaemic mice (ISAM) as a mouse model of adult-onset anaemia caused by erythropoietin deficiency. ISAM express erythropoietin in the liver but lack erythropoietin production in the kidney. Around weaning age, when the major erythropoietin-producing organ switches from the liver to the kidney, ISAM develop anaemia due to erythropoietin deficiency, which is curable by administration of recombinant erythropoietin. In ISAM severe chronic anaemia enhances transgenic green fluorescent protein and Cre expression driven by the complete erythropoietin-gene regulatory regions, which facilitates efficient labelling of renal erythropoietin-producing cells. We show that the majority of cortical and outer medullary fibroblasts have the innate potential to produce erythropoietin, and also reveal a new set of erythropoietin target genes. ISAM are a useful tool for the evaluation of erythropoiesis-stimulating agents and to trace the dynamics of erythropoietin-producing cells.

Does erythropoietin slow progression of chronic kidney disease?

Implication for health policy/practice/research/medical education:The initiation of erythropoietin therapy for the purpose of renoprotection may need to be sooner than that for erythropoiesis, because erythropoietin may attenuate renal fibrosis through macrophage adjustment and endothelial cell protection by other uncertain mechanisms. Although, agents re-establishing the initial function of renal erythropoietin-producing cells could defer kidney fibrosis, however more studies should be carried out to determine the cellular target of erythropoietin in kidney and developinga new erythropoietin derivative for renal care.

Greened and Gleaned Renal Erythropoietin-Producing Cells Provide Insight into Physiological Control of Erythropoietin Production

Greened and Gleaned Renal Erythropoietin-Producing Cells Provide Insight into Physiological Control of Erythropoietin Production

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 (EpoGFP) with mice bearing an Epo gene allele lacking the 3′ enhancer (EpoΔ3′E). Mice harboring the mutant EpoGFP/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. (282 words)

Isolation and Characterization of Renal Erythropoietin-Producing Cells: Contribution of HIF3α In Vivo.

AbstractAbstract 1541Understanding the nature of renal erythropoietin-producing cells (REPs) remains a central challenge for understanding the mechanisms involved in hypoxia/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 our approach used to isolate and characterize REPs. We bred mice bearing Epo/GFP combined with Epo-3'-enhancer deletion. These mice exhibit perinatal anemia (average Hct = 18% at P4–6). This enabled identification and purification of REPs based on GFP expression in the kidney. Light and confocal microscopy revealed that GFP immunostaining was confined to fibroblasts residing in the peritubular interstitium, which is consistent with our previous observation from the 180-Kb Epo/GFP reporter assay. Flow cytometry revealed that the GFP fraction constituted approximately 0.2% of the whole kidney cells; with most (63%) cells co-expressing CD73, a well-known marker for Epo-expressing cells. qRT-PCR confirmed that Epo expression was increased by approximately 100-fold in the purified REP population compared with that of the unsorted cells or CD73 fraction. Gene expression profiling showed Hif2α and Hif3α enrichment in the purified GFP/REP population. Moreover, in mouse hypoxia experiments, Epo expression was approximately 2.5-fold higher in Hif3α–/– kidney cells than in wild-type cells. The molecular strategy described here provides a means to isolate a pure population of REPs, allowing the analysis of gene expression profile of a defined population of cells essential for Epo production in the kidney. Our findings have led to a novel hypothesis that positive regulation by HIF2α and negative regulation by HIF3α might be necessary for correct renal Epo induction. Disclosures:Yamamoto:Chugai Pharmaceutical, Co., Ltd.: Research Funding.

Erythropoietin production by distal nephron in normal and familial amyloidotic adult human kidneys

the kidney is the major site of erythropoietin production. Many efforts have been made to identify renal erythropoietin-producing cells. Previous studies showed conflicting results, but the predominant localization reported was the peritubular interstitial and tubular epithelial cells. This study was conducted to identify the erythropoietin-producing cells in renal biopsies from 10 cadaveric donors and 45 patients with familial amyloidosis ATTR V30M, thirteen of them with anemia. Familial amyloidosis Type I (FAP-I) is a genetic disorder caused by a transthyretin (TTR) protein variant presenting a single amino acid substitution of methionine for valine at position 30 of the polypeptide chain (TTR V30M). Anemia in FAP-I is associated with inappropriately low serum erythropoietin levels. erythropoietin expression was detected by in situ hybridization (ISH) and confirmed by laser capture microdissection followed by PCR. Renal segments were identified by immunohistochemistry. erythropoietin was mainly expressed by epithelial distal tubular cells and collecting tubules and additionally, in a few biopsies, by glomerular cells. A similar expression pattern was observed in donors and FAP-I patients. No increased mRNA erythropoietin expression was found in anemic patients, all of them presenting only a slight expression in medulla and cortex. these results suggest the distal nephron as the major site of erythropoietin production, and support the notion that an inappropriate erythropoietin production is the cause of anemia in familial amyloidosis ATTR V30M.

Regulated oxygen sensing by protein hydroxylation in renal erythropoietin-producing cells

The kidney is a major site of systemic oxygen sensing, regulating blood erythrocyte and hence oxygen content by hypoxia-inducible erythropoietin (Epo) expression. A constant ratio between blood perfusion and oxygen consumption, a stable corticomedullary oxygen gradient, and a relatively low tissue Po(2) are the prerequisites for the function of renal Epo-producing and oxygen-sensing (REPOS) cells, which are located in the juxtamedullary cortex. In kidney disease, renal oxygen consumption is decreased, leading to an increase in Po(2), dysfunction of REPOS cells, and anemia. The molecular principles of cellular oxygen sensing have been elucidated in the last few years, and genetically altered mouse models as well as hereditary diseases causing erythrocytosis have clarified the oxygen-signaling cascade leading to increased Epo expression in REPOS cells. However, the consequences of a number of recently discovered factors for the regulation of oxygen signaling in REPOS cells are unclear, asking for novel cell culture models which might be hampered by the putative neuron-like nature of this enigmatic cell type.