Tomokazu Souma

SLCO4C1 Transporter Eliminates Uremic Toxins and Attenuates Hypertension and Renal Inflammation

Hypertension in patients with chronic kidney disease (CKD) strongly associates with cardiovascular events. Among patients with CKD, reducing the accumulation of uremic toxins may protect against the development of hypertension and progression of renal damage, but there are no established therapies to accomplish this. Here, overexpression of human kidney-specific organic anion transporter SLCO4C1 in rat kidney reduced hypertension, cardiomegaly, and inflammation in the setting of renal failure. In addition, SLCO4C1 overexpression decreased plasma levels of the uremic toxins guanidino succinate, asymmetric dimethylarginine, and the newly identified trans-aconitate. We found that xenobiotic responsive element core motifs regulate SLCO4C1 transcription, and various statins, which act as inducers of nuclear aryl hydrocarbon receptors, upregulate SLCO4C1 transcription. Pravastatin, which is cardioprotective, increased the clearance of asymmetric dimethylarginine and trans-aconitate in renal failure. These data suggest that drugs that upregulate SLCO4C1 may have therapeutic potential for patients with CKD.

Plasticity of Renal Erythropoietin-Producing Cells Governs Fibrosis

CKD progresses with fibrosis and erythropoietin (Epo)-dependent anemia, leading to increased cardiovascular complications, but the mechanisms linking Epo-dependent anemia and fibrosis remain unclear. Here, we show that the cellular phenotype of renal Epo-producing cells (REPs) alternates between a physiologic Epo-producing state and a pathologic fibrogenic state in response to microenvironmental signals. In a novel mouse model, unilateral ureteral obstruction-induced inflammatory milieu activated NFκB and Smad signaling pathways in REPs, rapidly repressed the Epo-producing potential of REPs, and led to myofibroblast transformation of these cells. Moreover, we developed a unique Cre-based cell-fate tracing method that marked current and/or previous Epo-producing cells and revealed that the majority of myofibroblasts are derived from REPs. Genetic induction of NFκB activity selectively in REPs resulted in myofibroblastic transformation, indicating that NFκB signaling elicits a phenotypic switch. Reversing the unilateral ureteral obstruction-induced inflammatory microenvironment restored the Epo-producing potential and the physiologic phenotype of REPs. This phenotypic reversion was accelerated by anti-inflammatory therapy. These findings demonstrate that REPs possess cellular plasticity, and suggest that the phenotypic transition of REPs to myofibroblasts, modulated by inflammatory molecules, underlies the connection between anemia and renal fibrosis in CKD.

Erythropoietin Synthesis in Renal Myofibroblasts Is Restored by Activation of Hypoxia Signaling

Erythropoietin (Epo) is produced by renal Epo-producing cells (REPs) in a hypoxia-inducible manner. The conversion of REPs into myofibroblasts and coincident loss of Epo-producing ability are the major cause of renal fibrosis and anemia. However, the hypoxic response of these transformed myofibroblasts remains unclear. Here, we used complementary in vivo transgenic and live imaging approaches to better understand the importance of hypoxia signaling in Epo production. Live imaging of REPs in transgenic mice expressing green fluorescent protein from a modified Epo-gene locus revealed that healthy REPs tightly associated with endothelium by wrapping processes around capillaries. However, this association was hampered in states of renal injury-induced inflammation previously shown to correlate with the transition to myofibroblast-transformed renal Epo-producing cells (MF-REPs). Furthermore, activation of hypoxia-inducible factors (HIFs) by genetic inactivation of HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) selectively in Epo-producing cells reactivated Epo production in MF-REPs. Loss of PHD2 in REPs restored Epo-gene expression in injured kidneys but caused polycythemia. Notably, combined deletions of PHD1 and PHD3 prevented loss of Epo expression without provoking polycythemia. Mice with PHD-deficient REPs also showed resistance to LPS-induced Epo repression in kidneys, suggesting that augmented HIF signaling counterbalances inflammatory stimuli in regulation of Epo production. Thus, augmentation of HIF signaling may be an attractive therapeutic strategy for treating renal anemia by reactivating Epo synthesis in MF-REPs.

Methylglyoxal augments intracellular oxidative stress in human aortic endothelial cells

Abstract Methylglyoxal (MGO) is a non-enzymatic metabolite in the glycolytic pathway and its concentration in blood and tissues is elevated in diabetes and renal failure. MGO induces tissue injuries via ROS; however, the mechanism remains to be clarified. The present study examined the harmful actions of MGO. Human aortic endothelial cells were assessed under real-time fluorescent microscopy with continuous superfusion. Increases in intracellular ROS were measured with fluorescent indicator, 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate acetyl ester (DCFH-DA). The addition of MGO rapidly increased the ROS in a dose-dependent manner. The increment of DCF was entirely abolished by pre-treatment with superoxide anion scavenger and membrane-permeable catalase, indicating that MGO induces superoxide production. The increment was completely inhibited by 2-thenoyltrifluoroacetone or carbonyl cyanide 3-chlorophenylhydrazone and partially inhibited by N-methyl-L-arginine. These data suggest that MGO stimulates superoxide production from mitochondria and partially stimulates nitric oxide synthase in human endothelial cells.

Angiopoietin receptor TEK mutations underlie primary congenital glaucoma with variable expressivity

Primary congenital glaucoma (PCG) is a devastating eye disease and an important cause of childhood blindness worldwide. In PCG, defects in the anterior chamber aqueous humor outflow structures of the eye result in elevated intraocular pressure (IOP); however, the genes and molecular mechanisms involved in the etiology of these defects have not been fully characterized. Previously, we observed PCG-like phenotypes in transgenic mice that lack functional angiopoietin-TEK signaling. Herein, we identified rare TEK variants in 10 of 189 unrelated PCG families and demonstrated that each mutation results in haploinsufficiency due to protein loss of function. Multiple cellular mechanisms were responsible for the loss of protein function resulting from individual TEK variants, including an absence of normal protein production, protein aggregate formation, enhanced proteasomal degradation, altered subcellular localization, and reduced responsiveness to ligand stimulation. Further, in mice, hemizygosity for Tek led to the formation of severely hypomorphic Schlemms canal and trabecular meshwork, as well as elevated IOP, demonstrating that anterior chamber vascular development is sensitive to Tek gene dosage and the resulting decrease in angiopoietin-TEK signaling. Collectively, these results identify TEK mutations in patients with PCG that likely underlie disease and are transmitted in an autosomal dominant pattern with variable expressivity.

A mouse model of adult-onset anaemia due to erythropoietin deficiency

Erythropoietin regulates erythropoiesis in a hypoxia-inducible manner. Here we generate inherited super-anaemic mice (ISAM) as a mouse model of adult-onset anaemia caused by erythropoietin deficiency. ISAM express erythropoietin in the liver but lack erythropoietin production in the kidney. Around weaning age, when the major erythropoietin-producing organ switches from the liver to the kidney, ISAM develop anaemia due to erythropoietin deficiency, which is curable by administration of recombinant erythropoietin. In ISAM severe chronic anaemia enhances transgenic green fluorescent protein and Cre expression driven by the complete erythropoietin-gene regulatory regions, which facilitates efficient labelling of renal erythropoietin-producing cells. We show that the majority of cortical and outer medullary fibroblasts have the innate potential to produce erythropoietin, and also reveal a new set of erythropoietin target genes. ISAM are a useful tool for the evaluation of erythropoiesis-stimulating agents and to trace the dynamics of erythropoietin-producing cells.

Hypoxia Signaling Cascade for Erythropoietin Production in Hepatocytes

ABSTRACT Erythropoietin (Epo) is produced in the kidney and liver in a hypoxia-inducible manner via the activation of hypoxia-inducible transcription factors (HIFs) to maintain oxygen homeostasis. Accelerating Epo production in hepatocytes is one plausible therapeutic strategy for treating anemia caused by kidney diseases. To elucidate the regulatory mechanisms of hepatic Epo production, we analyzed mouse lines harboring liver-specific deletions of genes encoding HIF-prolyl-hydroxylase isoforms (PHD1, PHD2, and PHD3) that mediate the inactivation of HIF1α and HIF2α under normal oxygen conditions. The loss of all PHD isoforms results in both polycythemia, which is caused by Epo overproduction, and fatty livers. We found that deleting any combination of two PHD isoforms induces polycythemia without steatosis complications, whereas the deletion of a single isoform induces no apparent phenotype. Polycythemia is prevented by the loss of either HIF2α or the hepatocyte-specific Epo gene enhancer (EpoHE). Chromatin analyses show that the histones around EpoHE dissociate from the nucleosome structure after HIF2α activation. HIF2α also induces the expression of HIF3α, which is involved in the attenuation of Epo production. These results demonstrate that the total amount of PHD activity is more important than the specific function of each isoform for hepatic Epo expression regulated by a PHD-HIF2α-EpoHE cascade in vivo.

Renal erythropoietin-producing cells in health and disease.

Erythropoietin (Epo) is an indispensable erythropoietic hormone primarily produced from renal Epo-producing cells (REPs). Epo production in REPs is tightly regulated in a hypoxia-inducible manner to maintain tissue oxygen homeostasis. Insufficient Epo production by REPs causes renal anemia and anemia associated with chronic disorders. Recent studies have broadened our understanding of REPs from prototypic hypoxia-responsive cells to dynamic fibrogenic cells. In chronic kidney disease, REPs are the major source of scar-forming myofibroblasts and actively produce fibrogenic molecules, including inflammatory cytokines. Notably, myofibroblast-transformed REPs (MF-REPs) recover their original physiological properties after resolution of the disease insults, suggesting that renal anemia and fibrosis could be reversible to some extent. Therefore, understanding the plasticity of REPs will lead to the development of novel targeted therapeutics for both renal fibrosis and anemia. This review summarizes the regulatory mechanisms how hypoxia-inducible Epo gene expression is attained in health and disease conditions.

Luminal Alkalinization Attenuates Proteinuria-Induced Oxidative Damage in Proximal Tubular Cells

A highly acidic environment surrounds proximal tubular cells as a result of their reabsorption of HCO(3)(-). It is unclear whether this luminal acidity affects proteinuria-induced progression of tubular cell damage. Here, we investigated the contribution of luminal acidity to superoxide (O(2)(·-)) production induced by oleic acid-bound albumin (OA-Alb) in proximal tubular cells. Acidic media significantly enhanced OA-Alb-induced O(2)(·-) production in the HK-2 proximal tubular cell line. Simultaneous treatment with both OA-Alb and acidic media led to phosphorylation of the intracellular pH sensor Pyk2. Highly phosphorylated Pyk2 associated with activation of Rac1, an essential subcomponent of NAD(P)H oxidase. Furthermore, knockdown of Pyk2 with siRNA attenuated the O(2)(·-) production induced by cotreatment with OA-Alb and acid. To assess whether luminal alkalinization abrogates proteinuria-induced tubular damage, we studied a mouse model of protein-overload nephropathy. NaHCO(3) feeding selectively alkalinized the urine and dramatically attenuated the accumulation of O(2)(·-)-induced DNA damage and proximal tubular injury. Overall, these observations suggest that luminal acidity aggravates proteinuria-induced tubular damage and that modulation of this acidic environment may hold potential as a therapeutic target for proteinuric kidney disease.

GATA2 regulates body water homeostasis through maintaining aquaporin 2 expression in renal collecting ducts.

ABSTRACT The transcription factor GATA2 plays pivotal roles in early renal development, but its distribution and physiological functions in adult kidney are largely unknown. We examined the GATA2 expression pattern in the adult kidney by tracing green fluorescent protein (GFP) fluorescence in Gata2GFP/+ mice that recapitulate endogenous GATA2 expression and found a robust GFP expression specifically in the renal medulla. Upon purification of the GFP-positive cells, we found that collecting duct (CD)-specific markers, including aquaporin 2 (Aqp2), an important channel for water reabsorption from urine, were abundantly expressed. To address the physiological function of GATA2 in the CD cells, we generated renal tubular cell-specific Gata2-deficient mice (Gata2-CKO) by crossing Gata2 floxed mice with inducible Pax8-Cre mice. We found that the Gata2-CKO mice showed a significant decrease in Aqp2 expression. The Gata2-CKO mice exhibited high 24-h urine volume and low urine osmolality, two important signs of diabetes insipidus. We introduced biotin-tagged GATA2 into a mouse CD-derived cell line and conducted chromatin pulldown assays, which revealed direct GATA2 binding to conserved GATA motifs in the Aqp2 promoter region. A luciferase reporter assay using an Aqp2 promoter-reporter showed that GATA2 trans activates Aqp2 through the GATA motifs. These results demonstrate that GATA2 regulates the Aqp2 gene expression in CD cells and contributes to the maintenance of the body water homeostasis.