Background and Aims The prevention/attenuation of graft ischemic injury is a challenge in kidney transplantation. We developed two rat models to investigate the impact of mesenchymal stromal cells (MSCs) in the ischemic preconditioning of kidneys from Donors after Circulatory Death (DCD) and Donors after Brain Death (DBD). Method Under general anesthesia, rats underwent iv injection of saline (S-groups) or 1.5 106 MSCs (MSC-groups) followed by either DBD (6hr of brain death) or DCD (6hr of anesthesia and 20min warm ischemia) models, resulting in 4 groups (S-DBD, S-DCD, MSC-DBD, MSC-DCD). Kidneys were then procured after IGL1 flush. One kidney was directly fixed and the other one immersed for 14 hours in IGL1 at 4°C. Serum samples were collected before treatment (baseline) and at the time of kidney collection. Urine samples were collected by bladder puncture at the time of kidney collection. Renal function was evaluated. Kidney histology was assessed by PAS staining and KIM1 immunostaining. Total RNA was extracted from S-DCD vs S-DBD kidneys for RNAseq. Results BUN was increased after 6h of anesthesia (DCD) or brain death (DBD) (p<0.01). SCr increased in both S-DBD and MSC-DBD but was lower in MSC-treated rats (MSC-DBD 0.5±0.2mg/dL vs S-DBD 0.7±0.1mg/dL; p = 0.037). Urinary KIM1 was lower in MSC-treated DBD (S-DBD 10.9±4.5 vs MSC-DBD 7.1±1.7; p = 0.03). Acute Tubular Injury (ATI) and KIM1 expression were higher in S-DBD (ATI: S-DBD 65±24% of surface vs S-DCD 39±27% of surface (p = 0.03) and KIM1: S-DBD 0.39±0.24% of surface vs S-DCD 0.10±0.09% of surface (p = 0.0002)). In MSC groups, there was no difference in both ATI extension and KIM1 expression. RNAseq showed that proinflammatory and proapoptotic pathways were upregulated in DBD, whereas transmembrane transport and metabolic pathways were downregulated, compared to DCD. Conclusion The RNA profiles of the kidneys are different upon donor types, which may impact the response to MSC-based ischemic preconditioning.
Abnormal subchondral bone remodeling leading to sclerosis is a main feature of osteoarthritis (OA) and Osteomodulin (OMD), a proteoglycan involved in extracellular matrix mineralization, is associated to the sclerotic phenotype. However, the functions of OMD remain poorly understood, specifically in vivo. We used knock-out and overexpressing male mice for Omd and mutant zebrafish to study its roles in bone and cartilage metabolism and in the development of OA. The expression of Omd is deeply correlated to bone and cartilage microarchitectures affecting the bone volume and the onset of subchondral bone sclerosis and spontaneous cartilage lesions. Mechanistically, OMD binds to RANKL and inhibits osteoclastogenesis; thus controlling the balance of the bone remodeling. In conclusions, OMD is a key factor in subchondral bone sclerosis associated with OA. It participates in bone and cartilage homeostasis acting on the regulation of osteoclastogenesis. Targeting OMD may be a promising new and personalized approach for OA.
BackgroundOsteoarthritis (OA) is associated with metabolic and structural changes in all joint tissues. Subchondral bone sclerosis, cartilage degradation, and synovial inflammation are the main hallmarks of OA [1–3]. OMD is a keratan sulfate proteoglycan, a member of the small leucine-rich proteoglycan (SLRP) family. OMD was first identified in bone where it is involved in the mineralization process [4,5]. Our previous work demonstrated that in the secretome of osteoblasts, OMD was one of the most differentiating factors between osteoblasts originating from the sclerotic and non-sclerotic zone of OA subchondral bone [3]. OMD levels were lower in the supernatant of osteoblasts coming from the sclerotic area.ObjectivesThe present work examined if OMD is involved in bone and cartilage changes occurring during skeletal development and in OA.MethodsWe usedOmdknock-out (KO) or overexpressing male mice and mutant zebrafish to studyin vivothe impact of OMD on skeletal development and aging. We investigated the influence of OMD on the severity of cartilage and bone damage induced by destabilization of the medial meniscus in these mice. We also analyzed the animals’ gait using the CatWalk XT system. The effect of OMD on gene expression by human trabecular osteoblasts in monolayer culture was analyzed by RNA sequencing method. Finally, OMD binding to RANKL was assessed using a solid phase binding assay.ResultsIn wild-type mice, we identified OMD mainly in bone and calcified cartilage. Tibial growth plate significantly decreased in all genotypes with age but to a lesser extent in the KO mice than in other genotypes. In KO mice, the calcified cartilage layer was thinner in the medial tibial plateau and thicker in the tibial lateral plateau than in the wild-type, while total cartilage thickness was not different between genotypes. We also demonstrated thatOmddeficiency led to thicker and less porous bone and subchondral bone sclerosis.Omdknock-out mice spontaneously developed more severe OA cartilage lesions in the medial tibial plateau than the wild-type during aging. In contrast, OMD production did not influence cartilage and bone changes induced by median meniscus destabilization. The gait pattern of mice was abnormal in KO compared to the wild-type genotype with a shorter swing and a smaller paw contact intensity.omd-/-zebrafish developed more severe cartilage lesions than wild-type. In osteoblasts culture, OMD down-regulated some genes involved in the extracellular matrix organization and up-regulated other genes responsible for the collagen network degradation. Finally, OMD bound to RANKL and inhibited osteoclastogenesis.ConclusionAlterations of the OMD expression modify bone and cartilage metabolism and structure. OMD helps to preserve bone and cartilage integrity and a local decrease in its production leads to the development of OA mainly by increasing subchondral bone sclerosis and thinning the calcified cartilage.References[1]Maruotti N, Corrado A, Cantatore FP. Osteoblast role in osteoarthritis pathogenesis. J Cell Physiol. 2017;232. doi:10.1002/jcp.25969[2]Stewart HL, Kawcak CE. The importance of subchondral bone in the pathophysiology of osteoarthritis. Front Vet Sci. 2018;5. doi:10.3389/fvets.2018.00178[3]Sanchez C, Mazzucchelli G, Lambert C,et al.Comparison of secretome from osteoblasts derived from sclerotic versus non-sclerotic subchondral bone in OA: A pilot study. Published Online First: 2018. doi:10.1371/journal.pone.0194591[4]Wendel M, Sommarin Y, Heinegård D. Characterization of osteoadherin - A novel, cell binding keratan sulfate proteoglycan from bone. Acta Orthop. 1995. doi:10.3109/17453679509157655[5]Sommarin Y, Wendel M, Shen Z,et al.Osteoadherin, a cell-binding keratan sulfate proteoglycan in bone, belongs to the family of leucine-rich repeat proteins of the extracellular matrix.Journal of Biological ChemistryPublished Online First: 1998. doi:10.1074/jbc.273.27.16723Acknowledgements:NIL.Disclosure of InterestsNone Declared.
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