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Chronic Kidney Disease (CKD) patients present a micro inflammation state due to failure renal function. The calcitriol has been described as an anti-inflammatory factor that might modulates the inflammatory response in CKD patients. However, these patients have deficiency of Calcitriol due to failure renal function. But, synthesis of this vitamin has been reported in extra renal production, as in monocytes. In this context, it has been reported that the supplementation with 25 vitamin D (calcidiol or inactive form of vitamin D) induces monocytes to downregulate inflammation, due to the intracellular 1α-hidroxilase that converts calcidiol to calcitriol in these cells. Besides some reports used RT-qPCR, Western Blot or immunofluorescence techniques to investigate the expression of inflammatory and vitamin D machinery biomarkers in several disease, in the present study we used flow cytometry technique to evaluate the effect of 25 vitamin D on CD14, Toll-like receptor 4 (TLR4), vitamin D receptor (VDR), 1-α hydroxylase (CYP27), 24 hydroxylase (CYP24) in monocytes lineage (U937). The U937 culture was incubated with healthy or CKD serum and treatment with/without 25-vitamin D (50 ng/ml for 24 h) to evaluate CD14, TRL4, VDR, CYP27 and CYP24 expression. This protocol showed the advantage to investigate the effect of treatment with 25 vitamin D on the intracellular and cell membrane biomarkers expression quickly and simultaneously. In addition, this technique is not laborious, but easy to perform and to interpret compared to RT-qPCR, western blot or immunofluorescence.

ABSTRACT A main obstacle to diagnose and manage renal osteodystrophy (ROD) is the identification of intracortical bone turnover type (low, normal, high). The gold standard, tetracycline‐labeled transiliac crest bone biopsy, is impractical to obtain in most patients. The Kidney Disease Improving Global Outcomes Guidelines recommend PTH and bone‐specific alkaline phosphatase (BSAP) for the diagnosis of turnover type. However, PTH and BSAP have insufficient diagnostic accuracy to differentiate low from non‐low turnover and were validated for trabecular turnover. We hypothesized that four circulating microRNAs (miRNAs) that regulate osteoblast (miRNA‐30b, 30c, 125b) and osteoclast development (miRNA‐155) would provide superior discrimination of low from non‐low turnover than biomarkers in clinical use. In 23 patients with CKD 3‐5D, we obtained tetracycline‐labeled transiliac crest bone biopsy and measured circulating levels of intact PTH, BSAP, and miRNA‐30b, 30c, 125b, and 155. Spearman correlations assessed relationships between miRNAs and histomorphometry and PTH and BSAP. Diagnostic test characteristics for discriminating low from non‐low intracortical turnover were determined by receiver operator curve analysis; areas under the curve (AUC) were compared by χ2 test. In CKD rat models of low and high turnover ROD, we performed histomorphometry and determined the expression of bone tissue miRNAs. Circulating miRNAs moderately correlated with bone formation rate and adjusted apposition rate at the endo‐ and intracortical envelopes (ρ = 0.43 to 0.51; p