Dynamic Single Cell Transcriptomics Defines Kidney FGF23/KL Bioactivity and Novel Segment-Specific Inflammatory Targets.

FGF23 via its coreceptor αKlotho (KL) provides critical control of phosphate metabolism, which is altered in rare and very common syndromes, however the spatial-temporal mechanisms dictating renal FGF23 functions remain poorly understood. Thus, developing approaches to modify specific FGF23-dictated pathways has proven problematic. Herein, wild type mice were injected with rFGF23 for 1, 4 and 12h and renal FGF23 bioactivity was determined at single cell resolution. Computational analysis identified distinct epithelial, endothelial, stromal, and immune cell clusters, with differential expressional analysis uniquely tracking FGF23 bioactivity at each time point. FGF23 actions were sex independent but critically relied upon constitutive KL expression mapped within proximal tubule (S1-S3) and distal tubule (DCT/CNT) cell sub-populations. Temporal KL-dependent FGF23 responses drove unique and transient cellular identities, including genes in key MAPK- and vitamin D-metabolic pathways via early- (AP-1-related) and late-phase (EIF2 signaling) transcriptional regulons. Combining ATACseq/RNAseq data from a cell line stably expressing KL with the in vivo scRNAseq pinpointed genomic accessibility changes in MAPK-dependent genes, including the identification of FGF23-dependent EGR1 distal enhancers. Finally, we isolated unexpected crosstalk between FGF23-mediated MAPK signaling and pro-inflammatory TNF receptor activation via NF-κB, which blocked FGF23 bioactivity in vitro and in vivo . Collectively, our findings have uncovered novel pathways at the single cell level that likely influence FGF23-dependent disease mechanisms. Translational statement: Inflammation and elevated FGF23 in chronic kidney disease (CKD) are both associated with poor patient outcomes and mortality. However, the links between these manifestations and the effects of inflammation on FGF23-mediated mineral metabolism within specific nephron segments remain unclear. Herein, we isolated an inflammatory pathway driven by TNF/NF-κB associated with regulating FGF23 bioactivity. The findings from this study could be important in designing future therapeutic approaches for chronic mineral diseases, including potential combination therapies or early intervention strategies. We also suggest that further studies could explore these pathways at the single cell level in CKD models, as well as test translation of our findings to interactions of chronic inflammation and elevated FGF23 in human CKD kidney datasets.

Single cell cortical bone transcriptomics define novel osteolineage gene sets altered in chronic kidney disease.

Introduction: Due to a lack of spatial-temporal resolution at the single cell level, the etiologies of the bone dysfunction caused by diseases such as normal aging, osteoporosis, and the metabolic bone disease associated with chronic kidney disease (CKD) remain largely unknown. Methods: To this end, flow cytometry and scRNAseq were performed on long bone cells from Sost-cre/Ai9+ mice, and pure osteolineage transcriptomes were identified, including novel osteocyte-specific gene sets. Results: Clustering analysis isolated osteoblast precursors that expressed Tnc, Mmp13, and Spp1, and a mature osteoblast population defined by Smpd3, Col1a1, and Col11a1. Osteocytes were demarcated by Cd109, Ptprz1, Ramp1, Bambi, Adamts14, Spns2, Bmp2, WasI, and Phex. We validated our in vivo scRNAseq using integrative in vitro promoter occupancy via ATACseq coupled with transcriptomic analyses of a conditional, temporally differentiated MSC cell line. Further, trajectory analyses predicted osteoblast-to-osteocyte transitions via defined pathways associated with a distinct metabolic shift as determined by single-cell flux estimation analysis (scFEA). Using the adenine mouse model of CKD, at a time point prior to major skeletal alterations, we found that gene expression within all stages of the osteolineage was disturbed. Conclusion: In sum, distinct populations of osteoblasts/osteocytes were defined at the single cell level. Using this roadmap of gene assembly, we demonstrated unrealized molecular defects across multiple bone cell populations in a mouse model of CKD, and our collective results suggest a potentially earlier and more broad bone pathology in this disease than previously recognized.

Osteocyte Egln1/Phd2 links oxygen sensing and biomineralization via FGF23.

Osteocytes act within a hypoxic environment to control key steps in bone formation. FGF23, a critical phosphate-regulating hormone, is stimulated by low oxygen/iron in acute and chronic diseases, however the molecular mechanisms directing this process remain unclear. Our goal was to identify the osteocyte factors responsible for FGF23 production driven by changes in oxygen/iron utilization. Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHI) which stabilize HIF transcription factors, increased Fgf23 in normal mice, as well as in osteocyte-like cells; in mice with conditional osteocyte Fgf23 deletion, circulating iFGF23 was suppressed. An inducible MSC cell line ('MPC2') underwent FG-4592 treatment and ATACseq/RNAseq, and demonstrated that differentiated osteocytes significantly increased HIF genomic accessibility versus progenitor cells. Integrative genomics also revealed increased prolyl hydroxylase Egln1 (Phd2) chromatin accessibility and expression, which was positively associated with osteocyte differentiation. In mice with chronic kidney disease (CKD), Phd1-3 enzymes were suppressed, consistent with FGF23 upregulation in this model. Conditional loss of Phd2 from osteocytes in vivo resulted in upregulated Fgf23, in line with our findings that the MPC2 cell line lacking Phd2 (CRISPR Phd2-KO cells) constitutively activated Fgf23 that was abolished by HIF1α blockade. In vitro, Phd2-KO cells lost iron-mediated suppression of Fgf23 and this activity was not compensated for by Phd1 or -3. In sum, osteocytes become adapted to oxygen/iron sensing during differentiation and are directly sensitive to bioavailable iron. Further, Phd2 is a critical mediator of osteocyte FGF23 production, thus our collective studies may provide new therapeutic targets for skeletal diseases involving disturbed oxygen/iron sensing.