Probes for INS

PDGFR-β-expressing cells of the kidneys are considered as a relevant site of erythropoietin (EPO) production. The origin of these cells, their contribution to renal EPO production, and if PDGFR-β-positive cells in other organs are also capable to express EPO are less clear. We addressed these questions in mice, in which hypoxia-inducible transcription factors were stabilized in PDGFR-β+ cells by inducible deletion of the von Hippel-Lindau (Vhl) protein. Vhl deletion led to a 600-fold increase of plasma EPO concentration, 170-fold increase of renal EPO messenger RNA (mRNA) levels, and an increase of hematocrit values up to 70 %. Intrarenal localization of EPO-expressing cells coincided with the zonal heterogeneity and distribution of cells expressing PDGFR-β. Amongst a variety of extrarenal organs only adrenal glands showed significant EPO mRNA expression after Vhl deletion in PDGFR-β+ cells. EPO mRNA, plasma EPO, and hematocrit fell to subnormal values if HIF-2α, but not HIF-1α, was deleted either alone or in combination with Vhl in PDGFR-β+ cells. Treatment of mice with a prolyl-hydroxylase inhibitor caused an increase of EPO mRNA abundance and plasma EPO concentrations in wild-type mice and in mice lacking HIF-1α in PDGFR-β+ cells but exerted no effect in mice lacking HIF-2α in PDGFR-β+ cells. These findings suggest that PDGFR-β+ cells are the only relevant site of EPO expression in the kidney and that HIF-2 is the essential transcription factor triggering EPO expression therein. Moreover, our findings suggest that PDGFR-β+cells elaborating EPO might arise from the metanephric mesenchyme, rather than from the neural crest.

Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblastdifferentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease.

Iron is an essential mineral for oxygen delivery and for a variety of enzymatic activities, but excessive iron results in oxidative cytotoxicity. Because iron is primarily used in red blood cells, defective erythropoiesis caused by loss of the erythroid growth factor erythropoietin (Epo) elevates iron storage levels in serum and tissues. Here, we investigated the effects of iron in a mouse model of Epo-deficiency anemia, in which serum iron concentration was significantly elevated. We found that intraperitoneal injection of iron-dextran caused severe iron deposition in renal interstitial fibroblasts, the site of Epo production. Iron overload induced by either intraperitoneal injection or feeding decreased activity of endogenous Epo gene expression by reducing levels of hypoxia-inducible transcription factor 2α (HIF2α), the major transcriptional activator of the Epo gene. Administration of an iron-deficient diet to the anemic mice reduced serum iron to normal concentration and enhanced the ability of renal Epo production. These results demonstrate that iron overload due to Epo deficiency attenuates endogenous Epo gene expression in the kidneys. Thus, iron suppresses Epo production by reducing HIF2α concentration in renal interstitial fibroblasts.

Renal peritubular interstitial fibroblast-like cells are critical for adult erythropoiesis, as they are the main source of erythropoietin (EPO). Hypoxia-inducible factor 2 (HIF-2) controls EPO synthesis in the kidney and liver and is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3, which function as cellular oxygen sensors. Renal interstitial cells with EPO-producing capacity are poorly characterized, and the role of the PHD/HIF-2 axis in renal EPO-producing cell (REPC) plasticity is unclear. Here we targeted the PHD/HIF-2/EPO axis in FOXD1 stroma-derived renal interstitial cells and examined the role of individual PHDs in REPC pool size regulation and renal EPO output. Renal interstitial cells with EPO-producing capacity were entirely derived from FOXD1-expressing stroma, and Phd2 inactivation alone induced renal Epo in a limited number of renal interstitial cells. EPO induction was submaximal, as hypoxia or pharmacologic PHD inhibition further increased the REPC fraction among Phd2-/- renal interstitial cells. Moreover, Phd1 and Phd3 were differentially expressed in renal interstitium, and heterozygous deficiency for Phd1 and Phd3 increased REPC numbers in Phd2-/- mice. We propose that FOXD1 lineage renal interstitial cells consist of distinct subpopulations that differ in their responsiveness to Phd2 inactivation and thus regulation of HIF-2 activity and EPO production under hypoxia or conditions of pharmacologic or genetic PHD inactivation.

In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah −/− mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.

Hair growth disorders often carry a major psychological burden. Therefore, more effective human hair growth-modulatory agents urgently need to be developed. Here, we used the hypertrichosis-inducing immunosuppressant, Cyclosporine A (CsA), as a lead compound to identify new hair growth-promoting molecular targets. Through microarray analysis we identified the Wnt inhibitor, secreted frizzled related protein 1 (SFRP1), as being down-regulated in the dermal papilla (DP) of CsA-treated human scalp hair follicles (HFs) ex vivo. Therefore, we further investigated the function of SFRP1 using a pharmacological approach and found that SFRP1 regulates intrafollicular canonical Wnt/β-catenin activity through inhibition of Wnt ligands in the human hair bulb. Conversely, inhibiting SFRP1 activity through the SFRP1 antagonist, WAY-316606, enhanced hair shaft production, hair shaft keratin expression, and inhibited spontaneous HF regression (catagen) ex vivo. Collectively, these data (a) identify Wnt signalling as a novel, non-immune-inhibitory CsA target; (b) introduce SFRP1 as a physiologically important regulator of canonical β-catenin activity in a human (mini-)organ; and (c) demonstrate WAY-316606 to be a promising new promoter of human hair growth. Since inhibiting SFRP1 only facilitates Wnt signalling through ligands that are already present, this 'ligand-limited' therapeutic strategy for promoting human hair growth may circumvent potential oncological risks associated with chronic Wnt over-activation.