Inhibition of RANKL improves the skeletal phenotype of adenine-induced chronic kidney disease in mice

AUTHORS

Corinne E Metzger, Mizuho Kittaka, Alec N LaPlant, Yasuyoshi Ueki, Matthew R Allen

ABSTRACT

Skeletal fragility and high fracture rates are common in CKD. A key component of bone loss in CKD with secondary hyperparathyroidism is high bone turnover and cortical bone deterioration through both cortical porosity and cortical thinning. We hypothesized that RANKL drives high bone resorption within cortical bone leading to the development of cortical porosity in CKD (study 1) and that systemic inhibition of RANKL would mitigate the skeletal phenotype of CKD (study 2). In study 1, we assessed the skeletal properties of male and female Dmp1-cre RANKLfl/fl (cKO) and control genotype (Ranklfl/fl; Con) mice after 10 wk of adenine-induced CKD (AD; 0.2% dietary adenine). All AD mice regardless of sex or genotype had elevated blood urea nitrogen and high PTH. Con AD mice in both sexes had cortical porosity and lower cortical thickness as well as high osteoclast-covered trabecular surfaces and higher bone formation rate. cKO mice had preserved cortical bone microarchitecture despite high circulating PTH as well as no CKD-induced increases in osteoclasts. In study 2, male mice with established AD CKD were either given a single injection of an anti-RANKL antibody (5 mg/kg) 8 wk post-induction of CKD or subjected to 3×/wk dosing with risedronate (1.2 μg/kg) for 4 wk. Anti-RANKL treatment significantly reduced bone formation rate as well as osteoclast surfaces at both trabecular and cortical pore surfaces; risedronate treatment had little effect on these bone parameters. In conclusion, these studies demonstrate that bone-specific RANKL is critical for the development of high bone formation/high osteoclasts and cortical bone loss in CKD with high PTH. Additionally, systemic anti-RANKL ligand therapy in established CKD may help prevent the propagation of cortical bone loss via suppression of bone turnover.

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

AUTHORS

Rafiou Agoro, Intawat Nookaew, Megan L. Noonan, Yamil G. Marambio, Sheng Liu, Wennan Chang1, Hongyu Gao, Lainey M. Hibbard, Corinne E. Metzger, Daniel Horan, William R. Thompson, Xiaoling Xuei, Yunlong Liu, Chi Zhang, Alexander G. Robling, Lynda F. Bonewald, Jun Wan, Kenneth E. White

ABSTRACT

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.

Bone volume, mineral density, and fracture risk after kidney transplantation

AUTHORS

Satu Keronen, Leena Martola, Patrik Finne, Inari S. Burton, Xiaoyu F. Tong, Heikki Kröger, Eero Honkanen

ABSTRACT

Background

Disordered mineral metabolism reverses incompletely after kidney transplantation in numerous patients. Post-transplantation bone disease is a combination of pre-existing chronic kidney disease and mineral disorder and often evolving osteoporosis. These two frequently overlapping conditions increase the risk of post-transplantation fractures.

Material and methods

We studied the prevalence of low bone volume in bone biopsies obtained from kidney transplant recipients who were biopsied primarily due to the clinical suspicion of persistent hyperparathyroidism between 2000 and 2015 at the Hospital District of Helsinki and Uusimaa. Parameters of mineral metabolism, results of dual-energy x-ray absorptiometry scans, and the history of fractures were obtained concurrently.

One hundred nine bone biopsies taken at a median of 31 (interquartile range, IQR, 18–70) months after transplantation were included in statistical analysis. Bone turnover was classified as high in 78 (72%) and normal/low in 31 (28%) patients. The prevalence of low bone volume (n = 47, 43%) was higher among patients with low/normal turnover compared to patients with high turnover [18 (58%) vs. 29 (37%), P = 0.05]. Thirty-seven fragility fractures in 23 (21%) transplant recipients corresponding to fracture incidence 15 per 1000 person-years occurred during a median follow-up 9.1 (IQR, 6.3–12.1) years. Trabecular bone volume did not correlate with incident fractures. Accordingly, low bone mineral density at the lumbar spine correlated with low trabecular bone volume, but not with incident fractures. The cumulative corticosteroid dose was an important determinant of low bone volume, but not of incident fractures.

Conclusions

Despite the high prevalence of trabecular bone loss among kidney transplant recipients, the number of fractures was limited. The lack of association between trabecular bone volume and fractures suggests that the bone cortical compartment and quality are important determinants of bone strength and post-transplantation fracture.

Diosmin, a citrus fruit-derived phlebotonic bioflavonoid protects rats from chronic kidney disease-induced loss of bone mass and strength without deteriorating the renal function

AUTHORS

Shivani Sharma, Konica Porwal, Chirag Kulkarni, Subhashis Pal, Praveer Sihota, Saroj Kumar, Mahesh Chandra Tiwari, Roshan Katekar, Ashish Kumar, Priya Singh, Swati Rajput, Rajdeep Guha, Navin Kumar, Jiaur R. Gayen, and Naibedya Chattopadhyay

ABSTRACT

Kidney Disease Improving Global Outcomes (KDIGO) 2017 Clinical Practice Guideline has recommended treatment decisions for patients with chronic kidney disease (CKD) with osteoporosis and/or high risk of fracture. Bisphosphonates, the first-line anti-osteoporosis drugs have the concern of worsening kidney functions. Moreover, despite impaired bone formation in CKD patients, teriparatide, the formation-stimulating drug is not recommended. Thus, there is an urgent need for safe and effective treatment of osteoporosis in CKD patients. Here, in CKD rats, we tested the osteoprotective effect of diosmin, a citrus-derived bioflavonoid used as a phlebotonic in chronic venous insufficiency and has a renoprotective effect. CKD was developed by 5/6th nephrectomy and diosmin at the human equivalent dose (100 mg kg−1) did not advance renal failure but reduced blood pressure to the level of sham control. Fibroblast growth factor-23 and parathyroid hormone were increased in CKD and diosmin suppressed both. CKD reduced bone mass and deteriorated the microarchitecture of trabecular bones, and diosmin maintained both to control levels. Bone formation and strength were impaired in the CKD and diosmin maintained these levels to control levels. Nanoindentation of bone showed that diosmin significantly increased tissue hardness over the control. Diosmetin, the metabolic surrogate of diosmin had comparable pharmacokinetic profiles between the control and CKD groups. Furthermore, diosmetin (50 mg kg−1) protected against CKD-induced bone loss. These data suggest that diosmin and its metabolic surrogate, diosmetin protect against CKD-induced osteopenia. Since diosmin has no renal adverse effect and protected bone mass and strength in CKD rats, we propose assessing its anti-osteoporosis effect in CKD patients.

Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation

AUTHORS

Kensuke Sasaki, Andrew S. Terker, Yu Pan, Zhilian Li, Shirong Cao, Yinqiu Wang, Aolei Niu, Suwan Wang, Xiaofeng Fan, Ming-Zhi Zhang and Raymond C. Harris

ABSTRACT

Background AKI is characterized by abrupt and reversible kidney dysfunction, and incomplete recovery leads to chronic kidney injury. Previous studies by us and others have indicated that macrophage infiltration and polarization play key roles in recovery from AKI. The role in AKI recovery played by IFN regulatory factor 4 (IRF4), a mediator of polarization of macrophages to the M2 phenotype, is unclear.

Methods We used mice with myeloid or macrophage cell–specific deletion of Irf4 (MΦ Irf4 −/−) to evaluate Irf4’s role in renal macrophage polarization and development of fibrosis after severe AKI.

Results Surprisingly, although macrophage Irf4 deletion had a minimal effect on early renal functional recovery from AKI, it resulted in decreased renal fibrosis 4 weeks after severe AKI, in association with less-activated macrophages. Macrophage Irf4 deletion also protected against renal fibrosis in unilateral ureteral obstruction. Bone marrow–derived monocytes (BMDMs) from MΦ Irf4 −/− mice had diminished chemotactic responses to macrophage chemoattractants, with decreased activation of AKT and PI3 kinase and increased PTEN expression. PI3K and AKT inhibitors markedly decreased chemotaxis in wild-type BMDMs, and in a cultured macrophage cell line. There was significant inhibition of homing of labeled Irf4 −/− BMDMs to postischemic kidneys. Renal macrophage infiltration in response to AKI was markedly decreased in MΦ Irf4 −/− mice or in wild-type mice with inhibition of AKT activity.

Conclusions Deletion of Irf4 from myeloid cells protected against development of tubulointerstitial fibrosis after severe ischemic renal injury in mice, due primarily to inhibition of AKT-mediated monocyte recruitment to the injured kidney and reduced activation and subsequent polarization into a profibrotic M2 phenotype.