Iddo Z. Ben-Dov

The parathyroid is a target organ for FGF23 in rats

Phosphate homeostasis is maintained by a counterbalance between efflux from the kidney and influx from intestine and bone. FGF23 is a bone-derived phosphaturic hormone that acts on the kidney to increase phosphate excretion and suppress biosynthesis of vitamin D. FGF23 signals with highest efficacy through several FGF receptors (FGFRs) bound by the transmembrane protein Klotho as a coreceptor. Since most tissues express FGFR, expression of Klotho determines FGF23 target organs. Here we identify the parathyroid as a target organ for FGF23 in rats. We show that the parathyroid gland expressed Klotho and 2 FGFRs. The administration of recombinant FGF23 led to an increase in parathyroid Klotho levels. In addition, FGF23 activated the MAPK pathway in the parathyroid through ERK1/2 phosphorylation and increased early growth response 1 mRNA levels. Using both rats and in vitro rat parathyroid cultures, we show that FGF23 suppressed both parathyroid hormone (PTH) secretion and PTH gene expression. The FGF23-induced decrease in PTH secretion was prevented by a MAPK inhibitor. These data indicate that FGF23 acts directly on the parathyroid through the MAPK pathway to decrease serum PTH. This bone-parathyroid endocrine axis adds a new dimension to the understanding of mineral homeostasis.

Predictors of All-Cause Mortality in Clinical Ambulatory Monitoring: Unique Aspects of Blood Pressure During Sleep

The prognostic value of sleep blood pressure reported by recent studies is variable. Our aim was to examine the relationship of sleep blood pressure, measured by 24-hour ambulatory blood pressure monitoring, with all-cause mortality. We studied a cohort of 3957 patients aged 55±16 (58% treated) referred for ambulatory monitoring (1991–2005). Sleep, including daytime sleep, was recorded by diary. Linkage with the national population register identified 303 deaths during 27 750 person-years of follow-up. Hazard ratios (HRs) for mortality in Cox proportional hazards models that included age, sex, hypertension, and diabetes treatment were 1.32 (95% CI: 0.99 to 1.76) for awake hypertension (≥135/85 mm Hg), and 1.67 (95% CI: 1.25 to 2.23) for sleep hypertension (≥120/70 mm Hg). By quintile analysis, the upper fifths of systolic and diastolic dipping during sleep were associated with adjusted HRs of 0.58 (95% CI: 0.41 to 0.82) and 0.68 (95% CI: 0.48 to 0.96), respectively. In a model controlling for awake systolic blood pressure, hazards associated with reduced systolic dipping increased from dippers (>10%; HR: 1.0), through nondippers (0% to 9.9%; HR: 1.30; 95% CI: 1.00 to 1.69) to risers (<0%; HR: 1.96; 95% CI: 1.43 to 2.96). Thus, in practice, ambulatory blood pressure predicts mortality significantly better than clinic blood pressure. The availability of blood pressure measures during sleep and, in particular, the pattern of dipping add clinically predictive information and provide further justification for the use of ambulatory monitoring in patient management.

Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations

We profiled microRNAs (miRNAs) in cell-free serum and plasma samples from human volunteers using deep sequencing of barcoded small RNA cDNA libraries. By introducing calibrator synthetic oligonucleotides during library preparation, we were able to calculate the total as well as specific concentrations of circulating miRNA. Studying trios of samples from newborn babies and their parents we detected placental-specific miRNA in both maternal and newborn circulations and quantitated the relative contribution of placental miRNAs to the circulating pool of miRNAs. Furthermore, sequence variation in the placental miRNA profiles could be traced to the specific placenta of origin. These deep sequencing profiles, which may serve as a model for tumor or disease detection, allow us to define the repertoire of miRNA abundance in the circulation and potential uses as biomarkers.

Parathyroid cell resistance to fibroblast growth factor 23 in secondary hyperparathyroidism of chronic kidney disease

Although fibroblast growth factor 23 (FGF23) acting through its receptor Klotho-FGFR1c decreases parathyroid hormone expression, this hormone is increased in chronic kidney disease despite an elevated serum FGF23. We measured possible factors that might contribute to the resistance of parathyroid glands to FGF23 in rats with the dietary adenine-induced model of chronic kidney disease. Quantitative immunohistochemical and reverse transcription-PCR analysis using laser capture microscopy showed that both Klotho and FGFR1 protein and mRNA levels were decreased in histological sections of the parathyroid glands. Recombinant FGF23 failed to decrease serum parathyroid hormone levels or activate the mitogen-activated protein kinase signaling pathway in the glands of rats with advanced experimental chronic kidney disease. In parathyroid gland organ culture, the addition of FGF23 decreased parathyroid hormone secretion and mRNA levels in control animals or rats with early but not advanced chronic kidney disease. Our results show that because of a downregulation of the Klotho-FGFR1c receptor complex, an increase of circulating FGF23 does not decrease parathyroid hormone levels in established chronic kidney disease. This in vivo resistance is sustained in parathyroid organ culture in vitro.

Increased Parathyroid Hormone Gene Expression in Secondary Hyperparathyroidism of Experimental Uremia Is Reversed by Calcimimetics: Correlation with Posttranslational Modification of the Trans Acting Factor AUF1

Most patients with chronic kidney disease develop secondary hyperparathyroidism with disabling systemic complications. Calcimimetic agents are effective tools in the management of secondary hyperparathyroidism, acting through allosteric modification of the calcium-sensing receptor (CaR) on the parathyroid gland (PT) to decrease parathyroid hormone (PTH) secretion and PT cell proliferation. This study showed that rats that were fed an adenine high-phosphorus diet had increased serum PTH and PTH mRNA levels at 7 and 21 d. For studying the effect of activation of the CaR by the calcimimetics R-568 on PTH gene expression, R-568 was given by gavage to uremic rats for the last 4 d of a 7-d adenine high-phosphorus diet. R-568 decreased both PTH mRNA and serum PTH levels. The effect of the calcimimetic on PTH gene expression was posttranscriptional and correlated with differences in protein-RNA binding and posttranslational modifications of the trans acting factor AUF1 in the PT. The AUF1 modifications as a result of uremia were reversed by treatment with R-568 to those of normal rats. Therefore, uremia and activation of the CaR mediated by calcimimetics modify AUF1 posttranslationally. These modifications in AUF1 correlate with changes in protein-PTH mRNA binding and PTH mRNA levels.

Linear relationship between systolic and diastolic blood pressure monitored over 24 h: assessment and correlates.

Objectives Systolic blood pressure (SBP) and diastolic blood pressure (DBP) frequently display a linear relationship characterized by the systolic-versus-diastolic slope (‘Slope’) or the ‘ambulatory arterial stiffness index’ [AASI = 1 – (diastolic-versus-systolic Slope)] and the correlation coefficient r. We evaluated the effect of using symmetric regression on the AASI and its dependence on clinical characteristics using 24-h ambulatory monitoring. Methods Ambulatory monitoring data of 140 patients (age 56 ± 17 years, 45% men) were retrieved from a service database. Slope and the AASI were evaluated using symmetric regression procedures, and the AASI also by standard regression. Results Correlation between SBP and DBP was r = 0.74 ± 0.14 (r > 0.5 in 95% of patients). Low r-values (when SBP correlates poorly with DBP) were tightly linked with nondipping (P < 0.00001). Use of symmetric rather than standard regression eliminated the bias in slope-related parameters and unmasked their dependence on clinical characteristics. Both symmetric Slope and the AASI were independent of mean arterial pressure and r, increased with pulse pressure (P < 0.01 and P < 0.0001, respectively), with the greater effect of wider pulse pressure in older age (P < 0.005 for both). The symmetric slope was 1.29 ± 0.28, showing bivariate dependence (r = 0.82) on age (exponential, with P < 0.00001) and pulse pressure dipping (P < 0.00001), increased for antihypertensive drug treatment (0.07 ± 0.03, P < 0.05) and diabetes mellitus (0.18 ± 0.06, P < 0.005). Conclusions Application of symmetrical regression provides a more valid estimate of the systolic-on-diastolic slope and the AASI, less influenced by goodness of fit and nocturnal dipping and more sensitive to age and disease states such as hypertension and diabetes, thus providing an improved index of arterial stiffening.

MicroRNA sequence profiles of human kidney allografts with or without tubulointerstitial fibrosis.

Background MicroRNA (miRNA) alterations accompanying interstitial fibrosis and tubular atrophy (IFTA) in kidney allografts may point toward pathologic mechanisms. Small-RNA sequencing provides information on total miRNA abundance and specific miRNA expression and allows analysis of differential expression based on read counts. Methods MiRNA expression profiles of 8 human kidney allograft biopsies (4 IFTA and 4 normal biopsies, discovery set) were characterized using barcoded deep-sequencing of a cDNA library prepared from multiplexed RNA. Statistical analysis of the sequence data guided selection of miRNAs for validation and the levels of selected miRNAs were quantified in 18 biopsies (10 IFTA and 8 normal) using real-time quantitative PCR assays (RT-QPCR). Results Total miRNA content was 50% lower in RNA from IFTA compared with normal biopsies. Global miRNA profiles clustered in partial agreement with diagnosis. Several miRNAs, including miR-21, 142-3p, and 5p and the cluster comprising miR-506 on chromosome X had twofold to sevenfold higher expression in IFTA compared with normal biopsies, whereas miRNA miR-30b and 30c were lower in IFTA biopsies (miR-30a, -30d, -30e, and all respective star sequences showed similar trends). IFTA and normal biopsy distinction was also noted in the pattern of miRNA nucleotide sequence variations. Differentially expressed miRNAs were confirmed on the larger set of allograft biopsies using RT-QPCR, and the levels of miRNAs were found to be associated with allograft function and survival. Conclusion Differentially expressed miRNAs and their predicted targets identified by deep sequencing are candidates for further investigation to decipher the mechanism and management of kidney allograft fibrosis.

Correlation Between Serum Alanine Aminotransferase Activity and Age: An Inverted U Curve Pattern

BACKGROUND:Alanine aminotransferase (ALT) activity is the most widely used laboratory test for the recognition of liver disease. Normality limits for values of serum ALT activity have been questioned lately. One reason for this recent uncertainty may be an unrecognized decline in aminotransferase levels in the aging population.AIMS:Cross-sectional evaluation of the association between age and ALT activity.METHODS:Laboratory data of residents in single home for the aged and of adult subjects in three general practice clinics in Jerusalem, Israel were reviewed, excluding subjects with known liver disease. A single laboratory performed all the tests. We examined the associations of serum aminotransferase levels with age, sex, body-mass-index (BMI), and estimated glomerular filtration rate (GFR). Polynomial regression and analysis of variance (ANOVA) with corrections for multiple comparisons were utilized for the statistical analyses.RESULTS:One hundred and twenty-eight individuals from the home for the aged and 207 individuals from three family practices were included. ALT activity linearly regressed with age (r = 0.22, p < 0.0001). However, polynomial regression revealed a better fit (r = 0.33, p < 0.0001), creating an inverted U curve with a peak at 40–55 yr. According to age groups, serum ALT level was 19 ± 13 U/L in those under 40 yr, 25 ± 19 U/L in 40–55 yr olds, 22 ± 10 U/L in 56–72 yr olds, 17 ± 9 U/L in 73–83 yr olds, and 13 ± 5 U/L in 83–100 yr olds (p < 0.0001). GFR (r = 0.1, p < 0.05) and BMI (r = 0.14, p < 0.01) weakly correlated with ALT. Gender also associated with ALT; 22 ± 15 U/L in men, and 17 ± 11 U/L in women (p < 0.005). Multiple regression analysis including age, gender, GFR, and BMI revealed that age (p = 0.01) and gender (p = 0.04) retained association with ALT activity. No such associations were noted for aspartate aminotransferase (AST) activity.CONCLUSIONS:Our data suggest a significant association between age and serum ALT activity. This association is not a simple linear correlation, but rather an inverted-U-like relation. Thus, when interpreting the laboratory results of a subject suspected of liver disease, age should probably be taken into account. Larger-scale studies are needed to better characterize this issue.

MicroRNA-21 in Glomerular Injury

TGF-β(1) is a pleotropic growth factor that mediates glomerulosclerosis and podocyte apoptosis, hallmarks of glomerular diseases. The expression of microRNA-21 (miR-21) is regulated by TGF-β(1), and miR-21 inhibits apoptosis in cancer cells. TGF-β(1)-transgenic mice exhibit accelerated podocyte loss and glomerulosclerosis. We determined that miR-21 expression increases rapidly in cultured murine podocytes after exposure to TGF-β(1) and is higher in kidneys of TGF-β(1)-transgenic mice than wild-type mice. miR-21-deficient TGF-β(1)-transgenic mice showed increased proteinuria and glomerular extracellular matrix deposition and fewer podocytes per glomerular tuft compared with miR-21 wild-type TGF-β(1)-transgenic littermates. Similarly, miR-21 expression was increased in streptozotocin-induced diabetic mice, and loss of miR-21 in these mice was associated with increased albuminuria, podocyte depletion, and mesangial expansion. In cultured podocytes, inhibition of miR-21 was accompanied by increases in the rate of cell death, TGF-β/Smad3-signaling activity, and expression of known proapoptotic miR-21 target genes p53, Pdcd4, Smad7, Tgfbr2, and Timp3. In American-Indian patients with diabetic nephropathy (n=48), albumin-to-creatinine ratio was positively associated with miR-21 expression in glomerular fractions (r=0.6; P<0.001) but not tubulointerstitial fractions (P=0.80). These findings suggest that miR-21 ameliorates TGF-β(1) and hyperglycemia-induced glomerular injury through repression of proapoptotic signals, thereby inhibiting podocyte loss. This finding is in contrast to observations in murine models of tubulointerstitial kidney injury but consistent with findings in cancer models. The aggravation of glomerular disease in miR-21-deficient mice and the positive association with albumin-to-creatinine ratio in patients with diabetic nephropathy support miR-21 as a feedback inhibitor of TGF-β signaling and functions.

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.