Advancing parathyroid anatomy understanding through single-cell RNA sequencing in uremic secondary hyperparathyroidism.

This commentary explores the recent application of single-cell RNA sequencing in the study of uremic secondary hyperparathyroidism, shedding light on the cellular dynamics within parathyroid glands. The use of single-cell RNA sequencing reveals new insights into the differentiation processes of chief and oxyphil cells, challenging traditional views and highlighting the potential of this technology in advancing our understanding of parathyroid anatomy.

Posttranscriptional regulation of the prostaglandin E receptor spliced-isoform EP3-γ and its implication in pancreatic ß-cell failure.

In Type 2 diabetes (T2D), elevated lipid levels have been suggested to contribute to insulin resistance and ß-cell dysfunction. We previously reported that the expression of the PGE2 receptor EP3 is elevated in islets of T2D individuals and is preferentially stimulated by palmitate, leading to ß-cell failure. The mouse EP3 receptor generates three isoforms by alternative splicing which differ in their C-terminal domain and are referred to as mEP3α, mEP3ß, and mEP3γ. We bring evidence that the expression of the mEP3γ isoform is elevated in islets of diabetic db/db mice and is selectively upregulated by palmitate. Specific knockdown of the mEP3γ isoform restores the expression of ß-cell-specific genes and rescues MIN6 cells from palmitate-induced dysfunction and apoptosis. This study indicates that palmitate stimulates the expression of the mEP3γ by a posttranscriptional mechanism, compared to the other spliced isoforms, and that the de novo synthesized ceramide plays an important role in FFA-induced mEP3γ expression in ß-cells. Moreover, induced levels of mEP3γ mRNA by palmitate or ceramide depend on p38 MAPK activation. Our findings suggest that mEP3γ gene expression is regulated at the posttranscriptional level and defines the EP3 signaling axis as an important pathway mediating ß-cell-impaired function and demise.

Customizing EV-CATCHER to Purify Placental Extracellular Vesicles from Maternal Plasma to Detect Placental Pathologies.

Placenta Accreta Spectrum (PAS) is a life-threatening condition in which placental trophoblastic cells abnormally invade the uterus, often up to the uterine serosa and, in extreme cases, tissues beyond the uterine wall. Currently, there is no clinical assay for the non-invasive detection of PAS, and only ultrasound and MRI can be used for its diagnosis. Considering the subjectivity of visual assessment, the detection of PAS necessitates a high degree of expertise and, in some instances, can lead to its misdiagnosis. In clinical practice, up to 50% of pregnancies with PAS remain undiagnosed until delivery, and it is associated with increased risk of morbidity/mortality. Although many studies have evaluated the potential of fetal biomarkers circulating in maternal blood, very few studies have evaluated the potential of circulating placental extracellular vesicles (EVs) and their miRNA contents for molecular detection of PAS. Thus, to purify placental EVs from maternal blood, we customized our robust ultra-sensitive immuno-purification assay, termed EV-CATCHER, with a monoclonal antibody targeting the membrane Placental Alkaline Phosphatase (PLAP) protein, which is unique to the placenta and present on the surface of placental EVs. Then, as a pilot evaluation, we compared the miRNA expression profiles of placental EVs purified from the maternal plasma of women diagnosed with placenta previa (controls, n = 16); placenta lying low in uterus but not invasive) to those of placental EVs purified from the plasma of women with placenta percreta (cases, n = 16), PAS with the highest level of invasiveness. Our analyses reveal that miRNA profiling of PLAP+ EVs purified from maternal plasma identified 40 differentially expressed miRNAs when comparing these two placental pathologies. Preliminary miRNA pathway enrichment and gene ontology analysis of the top 14 upregulated and top nine downregulated miRNAs in PLAP+ EVs, purified from the plasma of women diagnosed with placenta percreta versus those diagnosed with placenta previa, suggests a potential role in control of cellular invasion and motility that will require further investigation.

A universal cell-free DNA approach for response prediction to preoperative chemoradiation in rectal cancer.

The standard treatment approach for stage II/III rectal cancer is neoadjuvant chemoradiation therapy (nCRT) followed by surgery. In recent years, new treatment approaches have led to higher rates of complete tumor eradication combined with organ-preservation strategies. However, better tools are still needed to personalize therapy for the individual patient. In this prospective observational study, we analyzed colon-derived cell-free (cf)DNA (c-cfDNA) using a tissue-specific DNA methylation signature, and its association with therapy outcomes. Analyzing plasma samples (n = 303) collected during nCRT from 37 patients with locally advanced rectal cancer (LARC), we identified colon-specific methylation markers that discriminated healthy individuals from patients with untreated LARC (area under the curve, 0.81; 95% confidence interval, 0.70-0.92; P < .0001). Baseline c-cfDNA predicted tumor response, with increased levels linked to larger residual cancer. c-cfDNA measured after the first week of therapy identified patients with maximal response and complete cancer eradication, who had significantly lower c-cfDNA compared with those who had residual disease (8.6 vs 57.7 average copies/ml, respectively; P = .013). Increased c-cfDNA after 1 week of therapy was also associated with disease recurrence. Methylation-based liquid biopsy can predict nCRT outcomes and facilitate patient selection for escalation and de-escalation strategies.

Kidney Failure Alters Parathyroid Pin1 Phosphorylation and Parathyroid Hormone mRNA-Binding Proteins, Leading to Secondary Hyperparathyroidism.

BACKGROUND: Secondary hyperparathyroidism (SHP) is a common complication of CKD that increases morbidity and mortality. In experimental SHP, increased parathyroid hormone (PTH) expression is due to enhanced PTH mRNA stability, mediated by changes in its interaction with stabilizing AUF1 and destabilizing KSRP. The isomerase Pin1 leads to KSRP dephosphorylation, but in SHP parathyroid Pin1 activity is decreased and hence phosphorylated KSRP fails to bind PTH mRNA, resulting in high PTH mRNA stability and levels. The up- and downstream mechanisms by which CKD stimulates the parathyroid glands remain elusive. METHODS: Adenine-rich high-phosphate diets induced CKD in rats and mice. Parathyroid organ cultures and transfected cells were incubated with Pin1 inhibitors for their effect on PTH expression. Mass spectrometry was performed on both parathyroid and PTH mRNA pulled-down proteins. RESULTS: CKD led to changes in rat parathyroid proteome and phosphoproteome profiles, including KSRP phosphorylation at Pin1 target sites. Furthermore, both acute and chronic kidney failure led to parathyroid-specific Pin1 Ser16 and Ser71 phosphorylation, which disrupts Pin1 activity. Pharmacologic Pin1 inhibition, which mimics the decreased Pin1 activity in SHP, increased PTH expression ex vivo in parathyroid glands in culture and in transfected cells through the PTH mRNA-protein interaction element and KSRP phosphorylation. CONCLUSIONS: Kidney failure leads to loss of parathyroid Pin1 activity by inducing Pin1 phosphorylation. This predisposes parathyroids to increase PTH production through impaired PTH mRNA decay that is dependent on KSRP phosphorylation at Pin1-target motifs. Pin1 and KSRP phosphorylation and the Pin1-KSRP-PTH mRNA axis thus drive SHP.

MiRNA expression deregulation correlates with the Oncotype DX® DCIS score.

BACKGROUND: Current clinical criteria do not discriminate well between women who will or those who will not develop ipsilateral invasive breast cancer (IBC), or a DCIS recurrence after a ductal carcinoma in situ (DCIS) diagnosis. The 12-gene Oncotype DX® DCIS assay (RT qPCR gene-based scoring system) was established and shown to predict the risk of subsequent ipsilateral IBC or DCIS recurrence. Recent studies have shown that microRNA (miRNA) expression deregulation can contribute to the development of IBC, but very few have evaluated miRNA deregulation in DCIS lesions. In this study, we sought to determine whether specific miRNA expression changes may correlate with Oncotype DX® DCIS scores. METHODS: For this study, we used archived formalin-fixed, paraffin-embedded (FFPE) specimens from 41 women diagnosed with DCIS between 2012 and 2018. The DCIS lesions were stratified into low (n = 26), intermediate (n = 10), and high (n = 5) risk score groups using the Oncotype DX® DCIS assay. Total RNA was extracted from DCIS lesions by macro-dissection of unstained FFPE sections, and next-generation small-RNA sequencing was performed. We evaluated the correlation between miRNA expression data and Oncotype score, as well as patient age. RT-qPCR validations were performed to validate the topmost differentially expressed miRNAs identified between the different risk score groups. RESULTS: MiRNA sequencing of 32 FFPE DCIS specimens from the three different risk group scores identified a correlation between expression deregulation of 17 miRNAs and Oncotype scores. Our analyses also revealed a correlation between the expression deregulation of 9 miRNAs and the patient's age. Based on these results, a total of 15 miRNAs were selected for RT-qPCR validation. Of these, miR-190b (p = 0.043), miR-135a (p = 0.05), miR-205 (p = 0.00056), miR-30c (p = 0.011), and miR-744 (p = 0.038) showed a decreased expression in the intermediate/high Oncotype group when compared to the low-risk score group. A composite risk score was established using these 5 miRNAs and indicated a significant association between miRNA expression deregulation and the Oncotype DX® DCIS Score (p < 0.0021), between high/intermediate and low risk groups. CONCLUSIONS: Our analyses identified a subset of 5 miRNAs able to discriminate between Oncotype DX® DCIS score subgroups. Together, our data suggest that miRNA expression analysis may add value to the predictive and prognostic evaluation of DCIS lesions.

Dialysis Therapy for Volume Overload: A Feasible Option to Reduce Heart Failure Hospitalizations in Advanced Heart Failure.

BACKGROUND: Advanced heart failure (HF) carries a high rate of recurrent HF hospitalizations and a very high mortality rate. Mechanical devices and heart transplantation are limited to a select few. Dialysis may be a good alternative for advanced HF patients with volume overload despite maximal pharmacological therapy. OBJECTIVES: To assess the net clinical outcome of peritoneal dialysis or hemodialysis in patients with advanced HF. METHODS: We analyzed all advanced HF patients who were referred for dialysis due to volume overload in our institution. Patients were followed for complications, HF hospitalizations, and survival. RESULTS: We assessed 35 patients; 10 (29%) underwent peritoneal dialysis and 25 (71%) underwent hemodialysis; 71% were male; median (interquartile range) age was 74 (67-78) years. Estimated glomerular filtration rate was 20 (13-32) ml/min per 1.73 m2. New York Heart Association functional capacity was III. Median follow-up time was 719 days (interquartile range 658-780). One-year mortality rate was 8/35 (23%) and overall mortality rate was 16/35 (46%). Three patients (9%) died during the first year due to line or peritoneal dialysis related sepsis, and 6 (17%) died during the entire follow-up. The median number of HF hospitalizations was significantly reduced during the year on dialysis compared to the year prior to dialysis (0.0 [0.0-1.0] vs. 2.0 [0.0-3.0], P < 0.001). CONCLUSIONS: Dialysis is reasonably safe and significantly reduced HF hospitalization in advanced HF patients. Dialysis could be a good alternative for advanced HF patients with intractable volume overload.

MiR-199a-3p Induces Mesenchymal to Epithelial Transition of Keratinocytes by Targeting RAP2B.

Cutaneous squamous cell carcinoma (CSCC) is an epidermal skin cancer that evolves from normal epidermis along several pre-malignant stages. Previously we found specific miRNAs alterations in each step along these stages. miR-199a-3p expression decreases at the transition to later stages. A crucial step for epithelial carcinoma cells to acquire invasive capacity is the disruption of cell-cell contacts and the gain of mesenchymal motile phenotype, a process known as epithelial-to-mesenchymal transition (EMT). This study aims to study the role of decreased expression of miR-199a-3p in keratinocytes' EMT towards carcinogenesis. First, we measured miR-199a-3p in different stages of epidermal carcinogenesis. Then, we applied Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) assay to search for possible biochemical targets of miR-199a-3p and verified that Ras-associated protein B2 (RAP2B) is a bona-fide target of miR-199a-3p. Next, we analyzed RAP2B expression, in CSCC biopsies. Last, we evaluated possible mechanisms leading to decreased miR-199a-3p expression. miR-199a-3p induces a mesenchymal to epithelial transition (MET) in CSSC cells. Many of the under-expressed genes in CSCC overexpressing miR-199a-3p, are possible targets of miR-199a-3p and play roles in EMT. RAP2B is a biochemical target of miR-199a-3p. Overexpression of miR-199a-3p in CSCC results in decreased phosphorylated focal adhesion kinase (FAK). In addition, inhibiting FAK phosphorylation inhibits EMT marker genes' expression. In addition, we proved that DNA methylation is part of the mechanism by which miR-199a-3p expression is inhibited. However, it is not by the methylation of miR-199a putative promoter. These findings suggest that miR-199a-3p inhibits the EMT process by targeting RAP2B. Inhibitors of RAP2B or FAK may be effective therapeutic agents for CSCC.

Plasma microRNA Interindividual Variability in Healthy Individuals, Pregnant Women, and an Individual with a Stably Altered Neuroendocrine Phenotype.

BACKGROUND: Extracellular RNAs (exRNAs) in biofluids are amenable to quantitative analysis and proposed as noninvasive biomarkers for monitoring organ function. Cell-lineage-specific microRNAs (miRNAs) are present in plasma as soluble ribonucleoproteins or enclosed in exRNA carriers and transported through the vasculature. However, more extensive studies of healthy individuals are needed to gain insights into the variability of plasma miRNA abundance and composition. METHODS: The exRNA composition of platelet-depleted plasma collected twice from 236 healthy individuals was characterized by small RNA sequencing. Plasma of pregnant women featuring dramatically increased placental miRNAs and samples from subject P12 with noticeably increased epithelial- and neuroendocrine-origin miRNAs were included for comparison. The miRNA content of 10 000g and 100 000g pellet fractions of plasma generated by ultracentrifugation was also determined. Data analysis methods included Pearson correlation, differential gene expression, and unsupervised clustering. RESULTS: The abundance changes for more variable miRNAs in plasma of normal individuals correlated between coexpressed cell-lineage-specific miRNAs of the liver, neuroendocrine organs, epithelial cells, and muscle. ExRNA of pellet fractions contained <2% of total plasma miRNA with modest enrichment of lineage-specific and variable miRNAs compared to supernatant. The abundance fold changes of miRNAs observed in pregnancy and P12 compared to normal exceeded interquartile variability of healthy individuals. The neuroendocrine miRNA signature of P12 persisted for more than 4 years and was absent in other individuals. CONCLUSIONS: This study defines the framework and effect size for screening of extensive plasma collections for miRNA phenotypes and biomarker discovery.

IL6R is a target of miR-197 in human keratinocytes.

Psoriasis is a chronic inflammatory disorder with cutaneous and systemic manifestations and substantial negative effects on patients' quality of life. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play a role in the pathogenesis of psoriasis. Previously studies, from others and by us, highlighted specific miRNAs that are dysregulated in psoriatic lesions. MicroRNA-197-3p (miR-197) expression is downregulated in psoriatic lesions compared to normal or uninvolved skin in patients with psoriasis. We have previously reported that miR-197 could modulate IL-22 and IL-17 signalling in psoriasis. Herein, we identify additional biochemical targets of miR-197 in psoriasis. We applied a transcriptome-wide biochemical approach, Protein argonaute-2 photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (Ago2 PAR-CLIP), to search for new targets of miR-197 in live keratinocytes, and validated its results using reporter assay and analysing by Western blot protein levels in cells overexpressing miR-197. Ago2 PAR-CLIP identified biochemical targets of miR-197, including the alpha subunit of the IL-6 receptor (IL6R). This work provides evidence that IL6R in bona-fide biochemical target of miR-197. IL6R is known to be up-regulated in psoriasis and even was considered as a possible therapeutic target. From the present data and our previous studies, it appears that miR-197 is a major regulator of the interaction between immune system cells and keratinocytes.

Human plasma and serum extracellular small RNA reference profiles and their clinical utility.

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell-specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell-specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies.

[CATHETER-BASED RENAL DENERVATION FOR THE TREATMENT OF HYPERTENSION].

INTRODUCTION: Hypertension is a significant risk factor for cardiovascular morbidity and mortality. Despite a large variety of pharmacological treatment options, many patients remain uncontrolled. Medical inertia and patients' non-adherence to medications are the main reasons for lack of control. Experimental evidence shows that increased renal sympathetic nerve activity increases blood pressure and surgical denervation lowers blood pressure. In studies published between the years 2010-2013, radiofrequency endovascular ablation of renal sympathetic nerves (RDN) seemed to produce antihypertensive effects. However, a randomized sham-control study failed to prove active treatment-specific benefit. One of the main reasons for failure was the small number of ablations in each procedure. In recent years, technological progress has been made with the possibility to quadruple the number of ablation sites and extend them to the branches of the main renal arteries. Small sham-controlled studies were conducted in patients with grade 1-2 hypertension, either untreated or treated with up to three drugs. At three to six months follow-up, modest yet significant decrease of blood pressure was found, both in office and ambulatory measurements. Data from the Global SYMPLICITY Registry, which collects data from consecutive patients undergoing RDN with Medtronic radiofrequency ablation catheters, most of them with resistant hypertension, also showed significant improvement in blood pressure. However, in the absence of a control group, these real-life results should be interpreted with caution. Selection criteria of patients more likely to benefit from RDN have not been defined and will be discussed in the current review. In summary, RDN is a promising new treatment for hypertension. Randomized sham-controlled studies with a longer follow-up are still needed to confirm long-lasting treatment-specific effects and to show cardiovascular protection and safety.

[RENAL DENERVATION IN REFRACTORY HYPERTENSION].

INTRODUCTION: Resistant hypertension is a commonplace condition among patients referred to specialty hypertension clinics, which is associated with increased morbidity and mortality. Refractory hypertension however is a rare extreme subtype of resistant hypertension in which blood pressure is uncontrolled despite treatment with five antihypertensive drug classes including a diuretic and a mineralocorticoid receptor antagonist, and is associated with even worse prognosis. We herein describe a 40-year-old woman with severe refractory hypertension and target organ damage for who percutaneous renal sympathetic denervation successfully reduced blood pressure to normal levels and alleviated chronic headaches. Renal denervation should be considered in patients with refractory hypertension, especially when sympathetic over-activity is suspected.

Dysregulated miRNA biogenesis downstream of cellular stress and ALS-causing mutations: a new mechanism for ALS.

Interest in RNA dysfunction in amyotrophic lateral sclerosis (ALS) recently aroused upon discovering causative mutations in RNA-binding protein genes. Here, we show that extensive down-regulation of miRNA levels is a common molecular denominator for multiple forms of human ALS. We further demonstrate that pathogenic ALS-causing mutations are sufficient to inhibit miRNA biogenesis at the Dicing step. Abnormalities of the stress response are involved in the pathogenesis of neurodegeneration, including ALS. Accordingly, we describe a novel mechanism for modulating microRNA biogenesis under stress, involving stress granule formation and re-organization of DICER and AGO2 protein interactions with their partners. In line with this observation, enhancing DICER activity by a small molecule, enoxacin, is beneficial for neuromuscular function in two independent ALS mouse models. Characterizing miRNA biogenesis downstream of the stress response ties seemingly disparate pathways in neurodegeneration and further suggests that DICER and miRNAs affect neuronal integrity and are possible therapeutic targets.

Hyperphosphatemia is required for initiation but not propagation of kidney failure-induced calcific aortic valve disease.

High serum levels of phosphate are associated with uremia-induced calcific aortic valve disease (CAVD). However, it is not clear whether hyperphosphatemia is required in all phases of the process. Our aim was to determine the effects of phosphate and phosphate depletion at different phases of valve disease. The experimental design consisted of administering a uremia-inducing diet, with or without phosphate enrichment, to rats for 7 wk. Forty-two rats were fed with a phosphate-enriched uremic regimen that caused renal insufficiency and hyperphosphatemia. Another 42 rats were fed with a phosphate-depleted uremic regimen, which induces similar severity of renal insufficiency, but without its related mineral disorder. Aortic valves were evaluated at several points during the time of diet administration. In the second part, additional 54 rats were fed a phosphate-enriched diet for various time periods and were then switched to a phosphate-depleted diet to complete 7 wk of uremic diet. Osteoblast-like phenotype, inflammation, and eventually valve calcification were observed only in rats that were fed with a phosphate-enriched regimen. Significant valve calcification was observed only in rats that were fed a phosphate-enriched diet for at least 4 wk. Valve calcification was observed only when the switch to a phosphate-depleted regimen occurred after osteoblast markers and activation of Akt and ERK intracellular signaling pathways had already been found in the valve. Phosphate is essential for the initiation of the calcification process. However, when osteoblast markers are already expressed in valve tissue, phosphate depletion will not halt the disease.NEW & NOTEWORTHY High serum levels of phosphate are associated with uremia-induced calcific aortic valve disease. However, it is not clear whether hyperphosphatemia is required in all phases of the process. Our aim was to determine the effects of phosphate and phosphate depletion at different phases of valve disease. Our findings indicated that phosphate is essential for the initiation of the process that includes macrophage accumulation and osteoblast phenotype. Furthermore, hyperphosphatemia is dispensable beyond a certain phase of the process, a point of "no return" after which phosphate depletion does not prevent calcification. This point is relatively early in the course of calcification, when no calcification is apparent, but the inflammation, osteoblast markers, and activation of ERK and Akt pathways have already been identified. Our findings emphasize the complexity of the calcification process and suggest that different mediators might be required during different phases and that the role of phosphate precedes the actual calcification.

Ambulatory monitoring unmasks hypertension among kidney transplant patients: single center experience and review of the literature.

BACKGROUND: Disagreements between clinic and ambulatory blood pressure (BP) measurements are well-described in the general population. Though hypertension is frequent in renal transplant recipients, only a few studies address the clinic-ambulatory discordance in this population. We aimed to describe the difference between clinic and ambulatory BP in kidney transplant patients at our institution. METHODS: We compared the clinic and ambulatory BP of 76 adult recipients of a kidney allograft followed at our transplant center and investigated the difference between these methods, considering confounding by demographic and clinical variables. RESULTS: Clinic systolic BP (SBP) and diastolic BP (DBP) were 128 ± 13/79 ± 9 mmHg. Awake SBP and DBP were 147 ± 18/85 ± 10 mmHg. The clinic-minus-awake SBP and DBP differences were - 18 and - 6 mmHg, respectively. The negative clinic-awake ΔSBP was more pronounced at age > 60 years (p = 0.026) and with tacrolimus use compared to cyclosporine (p = 0.046). Sleep SBP and DBP were 139 ± 21/78 ± 11 mmHg. A non-dipping sleep BP pattern was noted in 73% of patients and was associated with tacrolimus use (p = 0.020). CONCLUSIONS: Our findings suggest pervasive underestimation of BP when measured in the kidney transplant clinic, emphasizes the high frequency of a non-dipping pattern in this population and calls for liberal use of ambulatory BP monitoring to detect and manage hypertension.

Evaluation of methodologies for microRNA biomarker detection by next generation sequencing.

In recent years, microRNAs (miRNAs) in tissues and biofluids have emerged as a new class of promising biomarkers for numerous diseases. Blood-based biomarkers are particularly desirable since serum or plasma is easily accessible and can be sampled repeatedly. To comprehensively explore the biomarker potential of miRNAs, sensitive, accurate and cost-efficient miRNA profiling techniques are required. Next generation sequencing (NGS) is emerging as the preferred method for miRNA profiling; offering high sensitivity, single-nucleotide resolution and the possibility to profile a considerable number of samples in parallel. Despite the excitement about miRNA biomarkers, challenges associated with insufficient characterization of the sequencing library preparation efficacy, precision and method-related quantification bias have not been addressed in detail and are generally underappreciated in the wider research community. Here, we have tested in parallel four commercially available small RNA sequencing kits against a cohort of samples comprised of human plasma, human serum, murine brain tissue and a reference library containing ~ 950 synthetic miRNAs. We discuss the advantages and limits of these methodologies for massive parallel microRNAs profiling. This work can serve as guideline for choosing an adequate library preparation method, based on sensitivity, specificity and accuracy of miRNA quantification, workflow convenience and potential for automation.

Let-7 and MicroRNA-148 Regulate Parathyroid Hormone Levels in Secondary Hyperparathyroidism.

Secondary hyperparathyroidism commonly complicates CKD and associates with morbidity and mortality. We profiled microRNA (miRNA) in parathyroid glands from experimental hyperparathyroidism models and patients receiving dialysis and studied the function of specific miRNAs. miRNA deep-sequencing showed that human and rodent parathyroids share similar profiles. Parathyroids from uremic and normal rats segregated on the basis of their miRNA expression profiles, and a similar finding was observed in humans. We identified parathyroid miRNAs that were dysregulated in experimental hyperparathyroidism, including miR-29, miR-21, miR-148, miR-30, and miR-141 (upregulated); and miR-10, miR-125, and miR-25 (downregulated). Inhibition of the abundant let-7 family increased parathyroid hormone (PTH) secretion in normal and uremic rats, as well as in mouse parathyroid organ cultures. Conversely, inhibition of the upregulated miR-148 family prevented the increase in serum PTH level in uremic rats and decreased levels of secreted PTH in parathyroid cultures. The evolutionary conservation of abundant miRNAs in normal parathyroid glands and the regulation of these miRNAs in secondary hyperparathyroidism indicates their importance for parathyroid function and the development of hyperparathyroidism. Specifically, let-7 and miR-148 antagonism modified PTH secretion in vivo and in vitro, implying roles for these specific miRNAs. These findings may be utilized for therapeutic interventions aimed at altering PTH expression in diseases such as osteoporosis and secondary hyperparathyroidism.