In chronic kidney disease altered cardiac metabolism precedes cardiac hypertrophy.

Conduit arterial disease in chronic kidney disease (CKD) is an important cause of cardiac complications. Cardiac function in CKD has not been studied in the absence of arterial disease. In an Alport syndrome model bred not to have conduit arterial disease, mice at 225 days of life (dol) had CKD equivalent to humans with CKD stage 4-5. Parathyroid hormone (PTH) and FGF23 levels were one log order elevated, circulating sclerostin was elevated, and renal activin A was strongly induced. Aortic Ca levels were not increased, and vascular smooth muscle cell (VSMC) transdifferentiation was absent. The CKD mice were not hypertensive, and cardiac hypertrophy was absent. Freshly excised cardiac tissue respirometry (Oroboros) showed that ADP-stimulated O2 flux was diminished from 52 to 22 pmol/mg (P = 0.022). RNA-Seq of cardiac tissue from CKD mice revealed significantly decreased levels of cardiac mitochondrial oxidative phosphorylation genes. To examine the effect of activin A signaling, some Alport mice were treated with a monoclonal Ab to activin A or an isotype-matched IgG beginning at 75 days of life until euthanasia. Treatment with the activin A antibody (Ab) did not affect cardiac oxidative phosphorylation. However, the activin A antibody was active in the skeleton, disrupting the effect of CKD to stimulate osteoclast number, eroded surfaces, and the stimulation of osteoclast-driven remodeling. The data reported here show that cardiac mitochondrial respiration is impaired in CKD in the absence of conduit arterial disease. This is the first report of the direct effect of CKD on cardiac respiration.NEW & NOTEWORTHY Heart disease is an important morbidity of chronic kidney disease (CKD). Hypertension, vascular stiffness, and vascular calcification all contribute to cardiac pathophysiology. However, cardiac function in CKD devoid of vascular disease has not been studied. Here, in an animal model of human CKD without conduit arterial disease, we analyze cardiac respiration and discover that CKD directly impairs cardiac mitochondrial function by decreasing oxidative phosphorylation. Protection of cardiac oxidative phosphorylation may be a therapeutic target in CKD.

New pathogenic insights inform therapeutic target development for renal osteodystrophy.

In an ancillary analysis of cross-sectional observational studies of bone health in end-stage kidney disease (ESKD), Evenepoel et al. reported that subjects with autosomal-dominant polycystic kidney disease (ADPKD) had a unique phenotype in their renal osteodystrophy. ADPKD caused resistance to parathyroid hormone (PTH) producing lower turnover states and preservation of cortical bone mineral density. PTH resistance was probably produced by increased osteocyte sclerostin levels, which is regulated by mechanical loading sensed through primary cilia sensory function affected by mutation in PKD1 and PKD2.

Androgen exposure potentiates formation of intratubular communities and renal abscesses by Escherichia coli.

Females across their lifespan and certain male populations are susceptible to urinary tract infections (UTI). The influence of female vs. male sex on UTI is incompletely understood, in part because preclinical modeling has been performed almost exclusively in female mice. Here, we employed established and new mouse models of UTI with uropathogenic Escherichia coli (UPEC) to investigate androgen influence on UTI pathogenesis. Susceptibility to UPEC UTI in both male and female hosts was potentiated with 5α-dihydrotestosterone, while males with androgen receptor deficiency and androgenized females treated with the androgen receptor antagonist enzalutamide were protected from severe pyelonephritis. In androgenized females and in males, UPEC formed dense intratubular, biofilm-like communities, some of which were sheltered from infiltrating leukocytes by the tubular epithelium and by peritubular fibrosis. Abscesses were nucleated by small intratubular collections of UPEC first visualized at five days postinfection and briskly expanded over the subsequent 24 hours. Male mice deficient in Toll-like receptor 4, which fail to contain UPEC within abscesses, were susceptible to lethal dissemination. Thus, androgen receptor activation imparts susceptibility to severe upper-tract UTI in both female and male murine hosts. Visualization of intratubular UPEC communities illuminates early renal abscess pathogenesis and the role of abscess formation in preventing dissemination of infection. Additionally, our study suggests that androgen modulation may represent a novel therapeutic route to combat recalcitrant or recurrent UTI in a range of patient populations.

The activin receptor is stimulated in the skeleton, vasculature, heart, and kidney during chronic kidney disease.

We examined activin receptor type IIA (ActRIIA) activation in chronic kidney disease (CKD) by signal analysis and inhibition in mice with Alport syndrome using the ActRIIA ligand trap RAP-011 initiated in 75-day-old Alport mice. At 200 days of age, there was severe CKD and associated Mineral and Bone Disorder (CKD-MBD), consisting of osteodystrophy, vascular calcification, cardiac hypertrophy, hyperphosphatemia, hyperparathyroidism, elevated FGF23, and reduced klotho. The CKD-induced bone resorption and osteoblast dysfunction was reversed, and bone formation was increased by RAP-011. ActRIIA inhibition prevented the formation of calcium apatite deposits in the aortic adventitia and tunica media and significantly decreased the mean aortic calcium concentration from 0.59 in untreated to 0.36 mg/g in treated Alport mice. Aortic ActRIIA stimulation in untreated mice increased p-Smad2 levels and the transcription of sm22α and αSMA. ActRIIA inhibition reversed aortic expression of the osteoblast transition markers Runx2 and osterix. Heart weight was significantly increased by 26% in untreated mice but remained normal during RAP-011 treatment. In 150-day-old mice, GFR was significantly reduced by 55%, but only by 30% in the RAP-011-treated group. In 200-day-old mice, the mean BUN was 100 mg/dl in untreated mice compared to 60 mg/dl in the treated group. In the kidneys of 200-day-old mice, ActRIIA and p-Smad2 were induced and MCP-1, fibronectin, and interstitial fibrosis were stimulated; all were attenuated by RAP-011 treatment. Hence, the activation of ActRIIA signaling during early CKD contributes to the CKD-MBD components of osteodystrophy and cardiovascular disease and to renal fibrosis. Thus, the inhibition of ActRIIA signaling is efficacious in improving and delaying CKD-MBD in this model of Alport syndrome.

Ligand trap of the activin receptor type IIA inhibits osteoclast stimulation of bone remodeling in diabetic mice with chronic kidney disease.

Dysregulation of skeletal remodeling is a component of renal osteodystrophy. Previously, we showed that activin receptor signaling is differentially affected in various tissues in chronic kidney disease (CKD). We tested whether a ligand trap for the activin receptor type 2A (RAP-011) is an effective treatment of the osteodystrophy of the CKD-mineral bone disorder. With a 70% reduction in the glomerular filtration rate, CKD was induced at 14 weeks of age in the ldlr-/- high fat-fed mouse model of atherosclerotic vascular calcification and diabetes. Twenty mice with CKD, hyperphosphatemia, hyperparathyroidism, and elevated activin A were treated with RAP-011, wherease 19 mice were given vehicle twice weekly from week 22 until the mice were killed at 28 weeks of age. The animals were then evaluated by skeletal histomorphometry, micro-computed tomography, mechanical strength testing, and ex vivo bone cell culture. Results in the CKD groups were compared with those of the 16 sham-operated ldlr-/- high fat-fed mice. Sham-operated mice had low-turnover osteodystrophy and skeletal frailty. CKD stimulated bone remodeling with significant increases in osteoclast and osteoblast numbers and bone resorption. Compared with mice with CKD and sham-operated mice, RAP-011 treatment eliminated the CKD-induced increase in these histomorphometric parameters and increased trabecular bone fraction. RAP-011 significantly increased cortical bone area and thickness. Activin A-enhanced osteoclastogenesis was mediated through p-Smad2 association with c-fos and activation of nuclear factor of activated T cells c1 (NFATc1). Thus, an ActRIIA ligand trap reversed CKD-stimulated bone remodeling, likely through inhibition of activin-A induced osteoclastogenesis.

Androgens Enhance Male Urinary Tract Infection Severity in a New Model.

Urinary tract infections (UTIs) occur predominantly in females but also affect substantial male patient populations; indeed, morbidity in complicated UTI is higher in males. Because of technical obstacles, preclinical modeling of UTI in male mice has been limited. We devised a minimally invasive surgical bladder inoculation technique that yields reproducible upper and lower UTI in both male and female mice, enabling studies of sex differences in these infections. Acute uropathogenic Escherichia coli (UPEC) cystitis in C57BL/6 and C3H/HeN males recapitulated the intracellular bacterial community pathway previously shown in females. However, surgically infected females of these strains exhibited more robust bladder cytokine responses and more efficient UPEC control than males. Compared with females, C3H/HeN males displayed a striking predilection for chronic cystitis, manifesting as persistent bacteriuria, high-titer bladder bacterial burdens, and chronic inflammation. Furthermore, males developed more severe pyelonephritis and 100% penetrant renal abscess (a complication that is rare in female mice). These phenotypes were sharply abrogated after castration but restored with exogenous testosterone, suggesting that male susceptibility to UTI is strongly influenced by androgen exposure. These data substantiate the long-standing presumption that anatomic differences in urogenital anatomy confer protection from UTI in males; however, as clinically observed, male sex associated with more severe UTI once these traditional anatomic barriers were bypassed. This study introduces a highly tractable preclinical model for interrogating sex differences in UTI susceptibility and pathogenesis, and illuminates an interplay between host sex and UTI that is more complex than previously appreciated.

Ligand trap for the activin type IIA receptor protects against vascular disease and renal fibrosis in mice with chronic kidney disease.

The causes of cardiovascular mortality associated with chronic kidney disease (CKD) are partly attributed to the CKD-mineral bone disorder (CKD-MBD). The causes of the early CKD-MBD are not well known. Our discovery of Wnt (portmanteau of wingless and int) inhibitors, especially Dickkopf 1, produced during renal repair as participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical. In the search for such factors, we studied the effects of activin receptor type IIA (ActRIIA) signaling by using a ligand trap for the receptor, RAP-011 (a soluble extracellular domain of ActRIIA fused to a murine IgG-Fc fragment). In a mouse model of CKD that stimulated atherosclerotic calcification, RAP-011 significantly increased aortic ActRIIA signaling assessed by the levels of phosphorylated Smad2/3. Furthermore, RAP-011 treatment significantly reversed CKD-induced vascular smooth muscle dedifferentiation as assessed by smooth muscle 22α levels, osteoblastic transition, and neointimal plaque calcification. In the diseased kidneys, RAP-011 significantly stimulated αklotho levels and it inhibited ActRIIA signaling and decreased renal fibrosis and proteinuria. RAP-011 treatment significantly decreased both renal and circulating Dickkopf 1 levels, showing that Wnt activation was downstream of ActRIIA. Thus, ActRIIA signaling in CKD contributes to the CKD-MBD and renal fibrosis. ActRIIA signaling may be a potential therapeutic target in CKD.

Fibroblast growth factor-23 and chronic allograft injury in pediatric renal transplant recipients: a Midwest Pediatric Nephrology Consortium study.

The chronic kidney disease-mineral bone disorder (CKD-MBD) produces fibroblast growth factor-23 (FGF-23) and related circulating pathogenic factors that are strongly associated with vascular injury and declining kidney function in native CKD. Similarly, chronic renal allograft injury (CRAI) is characterized by vascular injury and declining allograft function in transplant CKD. We hypothesized that circulating CKD-MBD factors could serve as non-invasive biomarkers of CRAI. We conducted a cross-sectional, multicenter case-control study. Cases (n = 31) had transplant function >20 mL/min/1.73 m(2) and biopsy-proven CRAI. Controls (n = 31) had transplant function >90 mL/min/1.73 m(2) and/or a biopsy with no detectable abnormality in the previous six months. We measured plasma CKD-MBD factors at a single time point using ELISA. Median (range) FGF23 levels were over twofold higher in CRAI vs. controls [106 (10-475) pg/mL vs. 45 (8-91) pg/mL; p < 0.001]. FGF23 levels were inversely correlated with transplant function (r(2) = -0.617, p < 0.001). Higher FGF23 levels were associated with increased odds of biopsy-proven CRAI after adjusting for transplant function, clinical, and demographic factors [OR (95% CI) 1.43 (1.23, 1.67)]. Relationships between additional CKD-MBD factors and CRAI were attenuated in multivariable models. Higher FGF23 levels were independently associated with biopsy-proven CRAI in children.

Pathophysiology of the chronic kidney disease-mineral bone disorder.

PURPOSE OF REVIEW: The causes of excess cardiovascular mortality associated with chronic kidney disease (CKD) have been attributed in part to the CKD-mineral bone disorder syndrome (CKD-MBD), wherein, novel cardiovascular risk factors have been identified. The causes of the CKD-MBD are not well known and they will be discussed in this review RECENT FINDINGS: The discovery of WNT (portmanteau of wingless and int) inhibitors, especially Dickkopf 1, produced during renal repair and participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical, leading to the finding that activin A is a second renal repair factor circulating in increased levels during CKD. Activin A derives from peritubular myofibroblasts of diseased kidneys, where it stimulates fibrosis, and decreases tubular klotho expression. The type 2 activin A receptor, ActRIIA, is decreased by CKD in atherosclerotic aortas, specifically in vascular smooth muscle cells (VSMC). Inhibition of activin signaling by a ligand trap inhibited CKD induced VSMC dedifferentiation, osteogenic transition and atherosclerotic calcification. Inhibition of activin signaling in the kidney decreased renal fibrosis and proteinuria. SUMMARY: These studies demonstrate that circulating renal repair factors are causal for the CKD-MBD and CKD associated cardiovascular disease, and identify ActRIIA signaling as a therapeutic target in CKD that links progression of renal disease and vascular disease.

Plasma FGF23 and Calcified Atherosclerotic Plaque in African Americans with Type 2 Diabetes Mellitus.

BACKGROUND: Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone implicated in disorders of serum phosphorus concentration and vitamin D. The role of FGF23 in vascular calcification remains controversial. METHODS: Relationships between FGF23 and coronary artery calcified atherosclerotic plaque (CAC), aortoiliac calcified plaque (CP), carotid artery CP, volumetric bone mineral density (vBMD), albuminuria, and estimated glomerular filtration rate (eGFR) were determined in 545 African Americans with type 2 diabetes (T2D) and preserved kidney function in African American-Diabetes Heart Study participants. Generalized linear models were fitted to test associations between FGF23 and cardiovascular, bone, and renal phenotypes, and change in measurements over time, adjusting for age, gender, African ancestry proportion, body mass index, diabetes duration, hemoglobin A1c, blood pressure, renin-angiotensin-system inhibitors, statins, calcium supplements, serum calcium, and serum phosphate. RESULTS: The sample was 56.7% female with a mean (SD) age of 55.6 (9.6) years, diabetes duration of 10.3 (8.2) years, eGFR 90.9 (22.1) ml/min/1.73 m2, urine albumin:creatinine ratio (UACR) 151 (588) (median 13) mg/g, plasma FGF23 161 (157) RU/ml, and CAC 637 (1,179) mg. In fully adjusted models, FGF23 was negatively associated with eGFR (p < 0.0001) and positively associated with UACR (p < 0.0001) and CAC (p = 0.0006), but not with carotid CP or aortic CP. Baseline FGF23 concentration did not associate with changes in vBMD or CAC after a mean of 5.1 years follow-up. CONCLUSIONS: Plasma FGF23 concentrations were independently associated with subclinical coronary artery disease, albuminuria, and kidney function in the understudied African American population with T2D. Findings support relationships between FGF23 and vascular calcification, but not between FGF23 and bone mineral density, in African Americans lacking advanced nephropathy.

Clinical phenotype of APOL1 nephropathy in young relatives of patients with end-stage renal disease.

BACKGROUND: Two coding variants--G1 and G2--in the apolipoprotein L-1 (APOL1) gene are associated with increased incidence of end-stage renal disease (ESRD) in the adult African American population. These variants associate with hypertension-attributed renal disease, focal segmental glomerulosclerosis (FSGS), and HIV-associated nephropathy. We hypothesized that as a genetic disease, APOL1 nephropathy has a pediatric phenotype. METHODS: We investigated the incidence of APOL1 variants in young African Americans with hypertension or FSGS and a family history of ESRD by conducting a case-control study of 93 pediatric and young adult African Americans with hypertension or FSGS to determine the association with APOL1 risk variants, G1, and G2 using custom-made TaqMan-based allelic discrimination assays. RESULTS: Forty of the 61 cases (66 %) with a family history of kidney disease had two APOL1 risk variants, significantly higher than the prevalence in controls and the general African American population (p < 0.001); 24 of 29 patients with hypertension-attributed kidney disease had two APOL1 risk variants, while none of nine hypertensive patients without kidney disease had more than one risk allele. CONCLUSIONS: Although it was a small study cohort, our findings strongly suggest for the first time that two APOL1 risk alleles in young hypertensive African Americans with a family history of ESRD are strongly associated with kidney disease.

CKD-induced wingless/integration1 inhibitors and phosphorus cause the CKD-mineral and bone disorder.

In chronic kidney disease, vascular calcification, renal osteodystrophy, and phosphate contribute substantially to cardiovascular risk and are components of CKD-mineral and bone disorder (CKD-MBD). The cause of this syndrome is unknown. Additionally, no therapy addresses cardiovascular risk in CKD. In its inception, CKD-MBD is characterized by osteodystrophy, vascular calcification, and stimulation of osteocyte secretion. We tested the hypothesis that increased production of circulating factors by diseased kidneys causes the CKD-MBD in diabetic mice subjected to renal injury to induce stage 2 CKD (CKD-2 mice). Compared with non-CKD diabetic controls, CKD-2 mice showed increased renal production of Wnt inhibitor family members and higher levels of circulating Dickkopf-1 (Dkk1), sclerostin, and secreted klotho. Neutralization of Dkk1 in CKD-2 mice by administration of a monoclonal antibody after renal injury stimulated bone formation rates, corrected the osteodystrophy, and prevented CKD-stimulated vascular calcification. Mechanistically, neutralization of Dkk1 suppressed aortic expression of the osteoblastic transcription factor Runx2, increased expression of vascular smooth muscle protein 22-α, and restored aortic expression of klotho. Neutralization of Dkk1 did not affect the elevated plasma levels of osteocytic fibroblast growth factor 23 but decreased the elevated levels of sclerostin. Phosphate binder therapy restored plasma fibroblast growth factor 23 levels but had no effect on vascular calcification or osteodystrophy. The combination of the Dkk1 antibody and phosphate binder therapy completely treated the CKD-MBD. These results show that circulating Wnt inhibitors are involved in the pathogenesis of CKD-MBD and that the combination of Dkk1 neutralization and phosphate binding may have therapeutic potential for this disorder.

Expression of DGCR8-dependent microRNAs is indispensable for osteoclastic development and bone-resorbing activity.

Recently, microRNAs (miRs) have been implicated in bone formation and homeostasis. We previously reported that Dicer generated miRs have pivotal roles in differentiation and activity of osteoclasts. However, recent studies have demonstrated that Dicer is implicated in production of endogenous small interfering RNAs, non-canonical miRs, and other small RNAs in mammals. Hence, a challenging question is the extent to which expression of canonical miRs is obligatory for osteoclastic control of bone metabolism. DiGeorge syndrome critical region gene 8 (DGCR8) is exclusively related to expression of miRs by a canonical processing pathway together with the nuclear RNase III enzyme Drosha. Osteoclast-specific deletion of DGCR8 led to impaired osteoclastic development and bone resorption so that bone development was significantly retarded. In culture, the expression levels of osteoclastic phenotype-related genes and proteins were remarkably inhibited during osteoclastogenesis in DGCR8-deficiency. Thus, we have identified that DGCR8-dependent miRs are indispensable for osteoclastic control of bone metabolism.

Early chronic kidney disease-mineral bone disorder stimulates vascular calcification.

The chronic kidney disease-mineral and bone disorder (CKD-MBD) syndrome is an extremely important complication of kidney diseases. Here we tested whether CKD-MBD causes vascular calcification in early kidney failure by developing a mouse model of early CKD in a background of atherosclerosis-stimulated arterial calcification. CKD equivalent in glomerular filtration reduction to human CKD stage 2 stimulated early vascular calcification and inhibited the tissue expression of α-klotho (klotho) in the aorta. In addition, osteoblast transition in the aorta was stimulated by early CKD as shown by the expression of the critical transcription factor Runx2. The ligand associated with the klotho-fibroblast growth factor receptor complex, FGF23, was found to be expressed in the vascular media of sham-operated mice. Its expression was decreased in early CKD. Increased circulating levels of the osteocyte-secreted proteins, FGF23, and sclerostin may have been related to increased circulating klotho levels. Finally, we observed low-turnover bone disease with a reduction in bone formation rates more than bone resorption. Thus, the CKD-MBD, characterized by cardiovascular risk factors, vascular calcification, increased circulating klotho, FGF23 and sclerostin levels, and low-turnover renal osteodystrophy, was established in early CKD. Early CKD caused a reduction of vascular klotho, stimulated vascular osteoblastic transition, increased osteocytic secreted proteins, and inhibited skeletal modeling producing the CKD-MBD.

HDAC dependent transcriptional repression of Bmp-7 potentiates TGF-ß mediated renal fibrosis in obstructive uropathy.

PURPOSE: Recombinant BMP-7 inhibits the pathogenesis of renal injury in response to various stimuli. However, little is known about the molecular regulation of endogenous BMP-7 and its renal protective functions. We examined transcriptional regulation of Bmp-7 and its role in the pathogenesis of renal injury resulting from urinary tract dysfunction. MATERIALS AND METHODS: Obstruction induced renal injury was modeled in vivo in mice by unilateral ureteral obstruction and in vitro in primary kidney cells by treatment with transforming growth factor-ß, a profibrotic cytokine that is increased in the obstructed kidney. RESULTS: Unilateral ureteral obstruction resulted in the loss of BMP-7 expression in conjunction with histone deacetylation and transcriptional repression of the Bmp-7 promoter. The histone deacetylase inhibitor trichostatin A stimulated Bmp-7 expression in primary kidney cells. Trichostatin A also inhibited the expression of transforming growth factor-ß dependent profibrotic genes in a manner that depended on BMP receptor signaling. These findings extended to the obstructed kidney in vivo, in which trichostatin A treatment restored the expression of Bmp-7 along with BMP-7 mediated suppression of transforming growth factor-ß dependent signaling pathways. Finally, trichostatin A stimulated activation of the BMP-7 pathway the ameliorated obstruction induced renal injury by preventing disruption of the renal architecture and the development of renal fibrosis. CONCLUSIONS: These findings show that histone deacetylase dependent repression of Bmp-7 transcription is a critical event during the pathogenesis of renal injury in obstructive uropathy. Accordingly, treatment with histone deacetylase inhibitors represents a potentially effective strategy to restore BMP-7 expression and its renal protective functions during treatment of obstructive uropathy.

Left ventricular mass progression despite stable blood pressure and kidney function in stage 3 chronic kidney disease.

BACKGROUND/AIMS: Progressive chronic kidney disease (CKD) is associated with worsening cardiovascular (CV) risk not explained by traditional risk factors. Left ventricular (LV) hypertrophy (LVH) is an important CV risk factor, but its progression has not been documented in early CKD. We explored whether progression of LVH in early CKD would occur despite stable kidney function. METHODS: We conducted a post hoc analysis of a 12-month study of lanthanum carbonate in stage 3 CKD, which included longitudinal assessments of CV biomarkers. Primary outcome for the analysis was the change in LV mass (LVM) indexed to height in meters(2.7) (LVM/Ht(2.7)). Secondary outcomes were changes in blood pressure (BP), pulse-wave velocity, LV systolic/diastolic function, fibroblast growth factor 23 (FGF23), klotho, and estimated glomerular filtration rate (eGFR). RESULTS: Thirty-one of 38 original subjects had sufficient data for analysis. LVM/Ht(2.7) increased (47 ± 13 vs. 53 ± 13 g/m(2.7), p = 0.006) over 12 months despite stable BP, stable eGFR and normal LV systolic function. Vascular stiffness and LV diastolic dysfunction persisted throughout the study. Klotho levels decreased (748 ± 289 to 536 ± 410 pg/ml, p = 0.03) but were unrelated to changes in LVM/Ht(2.7). The change in FGF23/klotho ratio was strongly correlated with changes in LVM/Ht(2.7) (r2 = 0.582, p = 0.03). CONCLUSION: Subjects with stage 3 CKD exhibited increasing LVM, persistent LV diastolic dysfunction and vascular stiffness despite stable kidney function, BP and LV systolic function. Abnormal FGF23 signaling due to reduced klotho expression may be associated with increasing LVM.

Down-regulation of miR-21 biogenesis by estrogen action contributes to osteoclastic apoptosis.

Estrogen inhibits osteoclastogenesis and induces osteoclastic apoptosis; however, the molecular mechanisms remain controversial. Recently, a group has demonstrated that osteoclasts are a direct target for estrogen because estrogen stimulates transcription of the Fas Ligand (FasL) gene in osteoclasts, which in turn causes cell death through an autocrine mechanism. In contrast, other groups have shown that the cells are an indirect target for estrogen because estrogen fails to stimulate the transcription of that in osteoclasts. Thus, two quite different molecular mechanisms have been suggested to explain the effects of estrogen in osteoclastic apoptosis. Here we show that the proapoptotic effect of estrogen during osteoclastogenesis is regulated by a posttranscriptional increase in FasL production by down-regulated microRNA-21 (miR-21) biogenesis. Previously, we reported that miR-21 is highly expressed in osteoclastogenesis. We found that estrogen down-regulates miR-21 biogenesis so that FasL, the targets of miR-21, protein levels are posttranscriptionally increased that induce osteoclastic apoptosis. Moreover, the gain-of-function of miR-21 rescued the apoptosis. In addition, we failed to detect estrogen-enhanced FasL levels at mRNA levels. Thus, osteoclastic survival is controlled by autocrine actions of FasL regulated by estrogen and miR-21 plays a central role during estrogen-controlled osteoclastogenesis.

A microRNA expression signature of osteoclastogenesis.

MicroRNAs (miRs) are small noncoding RNAs that principally function in the spatiotemporal regulation of protein translation in animal cells. Although emerging evidence suggests that some miRs play important roles in osteoblastogenesis and skeletal homeostasis, much less is known in osteoclastogenesis. Here, we show that receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis is mediated by miR-21. MiR-21 was identified as an miR expression signature of RANKL-induced osteoclastogenesis that down-regulates programmed cell death 4 (PDCD4) protein levels. Diminished PDCD4 removes a repression from c-Fos, a critical transcription factor for osteoclastogenesis and osteoclast-specific downstream target genes. In addition, RANKL-induced c-Fos up-regulates miR-21 gene expression. Bone marrow-derived monocyte/macrophage precursors deficient of DiGeorge syndrome critical region gene 8, an RNA binding protein associated with miR biogenesis, and Dicer, an endoribonuclease in the RNaseIII family associated with miR biogenesis, possessed significantly decreased miR-21 levels and increased PDCD4 protein levels so that RANKL-induced osteoclastogenesis was impaired in those cells. However, forced expression of miR-21 rescued osteoclast development because of down-regulation of PDCD4 protein expression levels. Thus, our studies provide a new molecular mechanism, including a positive feedback loop of c-Fos/miR-21/PDCD4, regulating osteoclastogenesis.

Effects of phosphate binder therapy on vascular stiffness in early-stage chronic kidney disease.

BACKGROUND/AIMS: Cardiovascular disease (CVD) is increased in chronic kidney disease (CKD), and contributed to by the CKD-mineral bone disorder (CKD-MBD). CKD-MBD begins in early CKD and its vascular manifestations begin with vascular stiffness proceeding to increased carotid artery intima-media thickness (cIMT) and vascular calcification (VC). Phosphorus is associated with this progression and is considered a CVD risk factor in CKD. We hypothesized that modifying phosphorus balance with lanthanum carbonate (LaCO3) in early CKD would not produce hypophosphatemia and may affect vascular manifestations of CKD-MBD. METHODS: We randomized 38 subjects with normophosphatemic stage 3 CKD to a fixed dose of LaCO3 or matching placebo without adjusting dietary phosphorus in a 12-month randomized, double-blind, pilot and feasibility study. The primary outcome was the change in serum phosphorus. Secondary outcomes were changes in measures of phosphate homeostasis and vascular stiffness assessed by carotid-femoral pulse wave velocity (PWV), cIMT and VC over 12 months. RESULTS: There were no statistically significant differences between LaCO3 and placebo with respect to the change in serum phosphorus, urinary phosphorus, tubular reabsorption of phosphorus, PWV, cIMT, or VC. Biomarkers of the early CKD-MBD such as plasma fibroblast growth factor-23, Dickkopf-related protein 1 (DKK1), and sclerostin were increased 2- to 3-fold at baseline, but were not affected by LaCO3. CONCLUSION: Twelve months of LaCO3 had no effect on serum phosphorus and did not alter phosphate homeostasis, PWV, cIMT, VC, or biomarkers of CKD-MBD.