Inhibition of the renal apical sodium dependent bile acid transporter prevents cholemic nephropathy in mice with obstructive cholestasis.

BACKGROUND & AIMS: Cholemic nephropathy (CN) is a severe complication of cholestatic liver diseases for which there is no specific treatment. We revisited its pathophysiology with the aim of identifying novel therapeutic strategies. METHODS: Cholestasis was induced by bile duct ligation (BDL) in mice. Bile flux in kidneys and livers was visualized by intravital imaging, supported by MALDI mass spectrometry imaging and liquid chromatography-tandem mass spectrometry. The effect of AS0369, a systemically bioavailable apical sodium-dependent bile acid transporter (ASBT) inhibitor, was evaluated by intravital imaging, RNA-sequencing, histological, blood, and urine analyses. Translational relevance was assessed in kidney biopsies from patients with CN, mice with a humanized bile acid (BA) spectrum, and via analysis of serum BAs and KIM-1 (kidney injury molecule 1) in patients with liver disease and hyperbilirubinemia. RESULTS: Proximal tubular epithelial cells (TECs) reabsorbed and enriched BAs, leading to oxidative stress and death of proximal TECs, casts in distal tubules and collecting ducts, peritubular capillary leakiness, and glomerular cysts. Renal ASBT inhibition by AS0369 blocked BA uptake into TECs and prevented kidney injury up to 6 weeks after BDL. Similar results were obtained in mice with humanized BA composition. In patients with advanced liver disease, serum BAs were the main determinant of KIM-1 levels. ASBT expression in TECs was preserved in biopsies from patients with CN, further highlighting the translational potential of targeting ASBT to treat CN. CONCLUSIONS: BA enrichment in proximal TECs followed by oxidative stress and cell death is a key early event in CN. Inhibiting renal ASBT and consequently BA enrichment in TECs prevents CN and systemically decreases BA concentrations. IMPACT AND IMPLICATIONS: Cholemic nephropathy (CN) is a severe complication of cholestasis and an unmet clinical need. We demonstrate that CN is triggered by the renal accumulation of bile acids (BAs) that are considerably increased in the systemic blood. Specifically, the proximal tubular epithelial cells of the kidney take up BAs via the apical sodium-dependent bile acid transporter (ASBT). We developed a therapeutic compound that blocks ASBT in the kidneys, prevents BA overload in tubular epithelial cells, and almost completely abolished all disease hallmarks in a CN mouse model. Renal ASBT inhibition represents a potential therapeutic strategy for patients with CN.

High-content phenotypic assay for proliferation of human iPSC-derived cardiomyocytes identifies L-type calcium channels as targets.

Over 5 million people in the United States suffer from heart failure, due to the limited ability to regenerate functional cardiac tissue. One potential therapeutic strategy is to enhance proliferation of resident cardiomyocytes. However, phenotypic screening for therapeutic agents is challenged by the limited ability of conventional markers to discriminate between cardiomyocyte proliferation and endoreplication (e.g. polyploidy and multinucleation). Here, we developed a novel assay that combines automated live-cell microscopy and image processing algorithms to discriminate between proliferation and endoreplication by quantifying changes in the number of nuclei, changes in the number of cells, binucleation, and nuclear DNA content. We applied this assay to further prioritize hits from a primary screen for DNA synthesis, identifying 30 compounds that enhance proliferation of human induced pluripotent stem cell-derived cardiomyocytes. Among the most active compounds from the phenotypic screen are clinically approved L-type calcium channel blockers from multiple chemical classes whose activities were confirmed across different sources of human induced pluripotent stem cell-derived cardiomyocytes. Identification of compounds that stimulate human cardiomyocyte proliferation may provide new therapeutic strategies for heart failure.

A novel approach to measuring macrophage-specific reverse cholesterol transport in vivo in humans.

Reverse cholesterol transport (RCT) is thought to be an atheroprotective function of HDL, and macrophage-specific RCT in mice is inversely associated with atherosclerosis. We developed a novel method using 3H-cholesterol nanoparticles to selectively trace macrophage-specific RCT in vivo in humans. Use of 3H-cholesterol nanoparticles was initially tested in mice to assess the distribution of tracer and response to interventions known to increase RCT. Thirty healthy subjects received 3H-cholesterol nanoparticles intravenously, followed by blood and stool sample collection. Tracer counts were assessed in plasma, nonHDL, HDL, and fecal fractions. Data were analyzed by using multicompartmental modeling. Administration of 3H-cholesterol nanoparticles preferentially labeled macrophages of the reticuloendothelial system in mice, and counts were increased in mice treated with a liver X receptor agonist or reconstituted HDL, as compared with controls. In humans, tracer disappeared from plasma rapidly after injection of nanoparticles, followed by reappearance in HDL and nonHDL fractions. Counts present as free cholesterol increased rapidly and linearly in the first 240 min after nadir; counts in cholesteryl ester increased steadily over time. Estimates of fractional transfer rates of key RCT steps were obtained. These results support the use of 3H-cholesterol nanoparticles as a feasible approach for the measurement of macrophage RCT in vivo in humans.

The Cxcl12, periostin, and Ccl9 genes are direct targets for early B-cell factor in OP-9 stroma cells.

The development of blood cells from hematopoietic stem cells in the bone marrow is dependent on communication with bone marrow stroma cells, making these cells central for the appropriate regulation of hematopoiesis. To identify transcription factors that may play a role in gene regulation in stroma cells, we performed comparative gene expression analysis of fibroblastic NIH3T3 cells, unable to support hematopoiesis in vitro, and OP-9 stroma cells, highly efficient in this regard. These experiments revealed that transcription factors of the early B cell factor (EBF) family were highly expressed in OP-9 cells as compared with the NIH3T3 cells. To identify potential targets genes for EBF proteins in stroma cells, we overexpressed EBF in fibroblasts and analyzed the pattern of induced genes by microarray analysis. This revealed that EBF was able to up-regulate expression of among others the Cxcl12, Ccl9, and Periostin genes. The identification of relevant promoters revealed that they all contained functional EBF binding sites able to interact with EBF in OP-9 cells. Furthermore, ectopic expression of a dominant negative EBF protein or antisense EBF-1 RNA in OP-9 stroma cells resulted in reduced expression of these target genes. These data suggest that EBF proteins might have dual roles in hematopoiesis acting both as intrinsic regulators of B-lymphopoiesis and as regulators of genes in bone marrow stroma cells.

Critical role for Ebf1 and Ebf2 in the adipogenic transcriptional cascade.

The Ebf (O/E) family of helix-loop-helix transcription factors plays a significant role in B lymphocyte and neuronal development. The three primary members of this family, Ebf1, 2, and 3, are all expressed in adipocytes, and Ebf1 promotes adipogenesis when overexpressed in NIH 3T3 fibroblasts. Here we report that these three proteins have adipogenic potential in multiple cellular models and that peroxisome proliferator-activated receptor gamma (PPARgamma) is required for this effect, at least in part due to direct activation of the PPARgamma1 promoter by Ebf1. Ebf1 also directly binds to and activates the C/EBPalpha promoter, which exerts positive feedback on C/EBPdelta expression. Despite this, C/EBPalpha is dispensable for the adipogenic action of Ebf proteins. Ebf1 itself is induced by C/EBPbeta and delta, which bind and activate its promoter. Reduction of Ebf1 and Ebf2 proteins by specific short hairpin RNA blocks differentiation of 3T3-L1 cells, suggesting a critical role for these factors and the absence of functional redundancy between members of this family. Altogether, these data place Ebf1 within the known transcriptional cascade of adipogenesis and suggest critical roles for Ebf1 and Ebf2.

Early B-cell factor (O/E-1) is a promoter of adipogenesis and involved in control of genes important for terminal adipocyte differentiation.

Olf-1/early B-cell factor (O/E-1) is a transcription factor important for B-lymphocyte and neuronal gene regulation. Here we report that all three known O/E genes (O/E-1, -2, and -3) are expressed in mouse adipose tissue and are upregulated during adipocyte differentiation. Forced expression of O/E-1 in either the preadipocyte cell line 3T3-L1 or mouse embryonic fibroblasts augmented adipogenesis, and constitutive expression of O/E-1 in uncommitted NIH 3T3 fibroblasts led to initiation of adipocyte differentiation. Furthermore, a dominant negative form of O/E-1 partially suppressed 3T3-L1 adipogenesis, indicating that expression from endogenous O/E target genes is required for 3T3-L1 terminal differentiation. Thus, our data point to the importance of O/E target genes for adipocyte differentiation and suggest a novel role for O/E-1 as an initiator and stimulator of adipogenesis.