Yu-Juei Hsu

Generation and analysis of the thiazide-sensitive Na+ -Cl- cotransporter (Ncc/Slc12a3) Ser707X knockin mouse as a model of Gitelman syndrome.

Gitelman syndrome (GS) is characterized by salt‐losing hypotension, hypomagnesemia, hypokalemic metabolic alkalosis, and hypocalciuria. To better model human GS caused by a specific mutation in the thiazide‐sensitive Na+‐Cl− cotransporter (NCC) gene SLC12A3, we generated a nonsense Ncc Ser707X knockin mouse corresponding to human p.Ser710X (c.2135C>A), a recurrent mutation with severe phenotypes in Chinese GS patients. Compared with wild‐type or heterozygous littermates, homozygous (Hom) knockin mice fully recapitulated the phenotype of human GS. The markedly reduced Ncc mRNA and virtually absent Ncc protein expression in kidneys of Hom mice was primarily due to nonsense‐mediated mRNA decay (NMD) surveillance mechanisms. Expression of epithelial Na+ channel (Enac), Ca2+ channels (Trpv5 and Trpv6), and K+ channels (Romk1 and maxi‐K) were significantly increased. Late distal convoluted tubules (DCT) volume was increased and DCT cell ultrastructure appeared intact. High K+ intake could not correct hypokalemia but caused a further increase in maxi‐K but not Romk1 expression. Renal tissue from a patient with GS also showed the enhanced TRPV5 and ROMK1 expression in distal tubules. We suggest that the upregulation of TRPV5/6 and of ROMK1 and Maxi‐K may contribute to hypocalciuria and hypokalemia in Ncc Ser707X knockin mice and human GS, respectively. Hum Mutat 31:1–13, 2010.

Resveratrol increases anti-aging Klotho gene expression via the activating transcription factor 3/c-Jun complex-mediated signaling pathway.

The Klotho gene functions as an aging suppressor gene. Evidence from animal models suggests that induction of Klotho expression may be a potential treatment for age-associated diseases. However, the molecular mechanism involved in regulating renal Klotho gene expression remains unclear. In this study, we determined that resveratrol, a natural polyphenol, induced renal Klotho expression both in vivo and in vitro. In the mouse kidney, resveratrol administration markedly increased both Klotho mRNA and protein expression. In resveratrol-treated NRK-52E cells, increased Klotho expression was accompanied by the upregulation and nuclear translocation of activating transcription factor 3 (ATF3) and c-Jun. ATF3 or c-Jun overexpression enhanced the transcriptional activation of Klotho. Conversely, resveratrol-induced Klotho expression was attenuated in the presence of dominant-negative ATF3 or c-Jun. Coimmunoprecipitation and a chromatin immunoprecipitation assay revealed that ATF3 physically interacted with c-Jun and that the ATF3/c-Jun complex directly bound to the Klotho promoter through ATF3- and AP-1-binding elements. c-Jun cotransfection augmented the effects of ATF3 on Klotho transcription in vitro. Although Sirtuin 1 mRNA expression was induced by resveratrol and involved in regulating Klotho mRNA expression, it was not the primary cause for the aforementioned ATF3/c-Jun pathway. In summary, resveratrol enhances the renal expression of the anti-aging Klotho gene, and the transcriptional factors ATF3 and c-Jun functionally interact and coordinately regulate the resveratrol-mediated transcriptional activation of Klotho.

Genotype, Phenotype, and Follow-Up in Taiwanese Patients with Salt-Losing Tubulopathy Associated with SLC12A3 Mutation

CONTEXT AND OBJECTIVE Genotype, phenotype, and follow-up analysis is rarely performed in a large number of patients with Gitelmans syndrome (GS) caused by mutations in SLC12A3 encoding the thiazide-sensitive NaCl cotransporter. DESIGN, SETTING, AND PATIENTS One hundred seventeen Taiwanese GS patients (70 males and 47 females, age 24 ± 10 yr) with SLC12A3 mutations belonging to 92 unrelated families were investigated. Genomic DNA and/or cDNA from blood leukocytes were analyzed for SLC12A3 mutations and haplotype analysis with intragenic and extragenic markers for recurrent SLC12A3 mutations. Clinical symptoms and biochemical studies at the first presentation as well as follow-up were examined. RESULTS Forty different SLC12A3 mutations were identified. Approximately 12% of patients had triple SLC12A3 mutations. Fourteen mutations were recurrent with only one founder effect (S710X). Typical hypocalciuria and hypomagnesemia were not found in seven and nine patients, respectively. In addition to male patients having an earlier age of onset, more severe hypokalemia, and significantly lower serum aldosterone concentration, patients with homozygous and deep mutations in intron 13 (c.1670-191C → T) had more severe phenotype. Seven and five patients had chronic kidney disease (stage III-IV) and type 2 diabetes at the follow-up, respectively. CONCLUSION Screening of recurrent hot spot SLC12A3 mutations may provide an early diagnosis of GS. Besides gender effect, the nature of homozygous and deep intronic mutations may influence the phenotype. Patients with GS may be at increased risk for the development of chronic kidney disease and type 2 diabetes.

Mechanisms for Hypercalciuria in Pseudohypoaldosteronism Type II-Causing WNK4 Knock-In Mice

The mechanisms underlying hypercalciuria in pseudohypoaldosteronism type II (PHAII) caused by WNK4 mutations remain unclear. In this study, we used Wnk4(D561A/+) knock-in mice as a model of human PHAII for investigating the pathogenesis of hypercalciuria in PHAII. Serum and urine biochemistries were obtained from Wnk4(+/+) and Wnk4(D561A/+) littermates. Expression of the epithelial Ca(2+) channels [transient receptor potential channel vanilloid subtype 5 (TRPV5) and TRPV6] and calbindin-D28k (CBP-D28k) in the distal nephron and two upstream Na(+) transporters, Na(+)/H(+) exchanger 3 and Na(+)-K(+)-2Cl(-) cotransporter 2 involved in paracellular Ca(2+) reabsorption, were examined by real-time PCR, immunofluorescent staining, and immunoblotting. Compared with Wnk4(+/+) littermate controls, Wnk4(D561A/+) mice manifested hypercalciuria despite no significant differences in serum creatinine, ionized Ca(2+), PTH, and 1,25 hydroxylvitamin D(3) levels. There was no significant difference in TRPV5 expression, but a significant increase in TRPV6 and CBP-D28k was observed in Wnk4(D561A/+) mice. Despite no significant change in Na(+)/H(+) exchanger 3 expression, Na(+)-K(+)-2Cl(-) cotransporter 2 expression was significantly attenuated and urine Ca(2+) excretion rate in response to furosemide was blunted in Wnk4(D561A/+) mice. Decreased Ca(2+) reabsorption in the upstream nephron, especially in the thick ascending loops of Henle, with a secondary adaptive increase in TRPV6 and CBP-D28k expression in the distal tubules might be involved in the hypercalciuria of PHAII.

Enhanced expression of glucose transporter-1 in vascular smooth muscle cells via the Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) pathway in experimental renal failure

BACKGROUND Chronic renal failure (CRF) is associated with increased cardiovascular mortality, and medial vascular smooth muscle cell (VSMC) hypertrophy, proliferation, and calcification play a pivotal role in uremic vasculopathy. Glucose transporter-1 (GLUT1) facilitates the transport of glucose into VSMCs, and GLUT1 overexpression associated with high glucose influx leads to a stimulation of VSMC proliferation. However, the role of GLUT1 in uremic vasculopathy remains unclear. This study aimed to identify changes in the expression of GLUT1 in VSMCs in the setting of experimental uremia and investigate whether Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) signaling, which plays a crucial role in VSMC proliferation and glucose metabolism, is involved in the regulation of GLUT1 expression. METHODS In vivo experimental CRF was induced in Wistar rats by 5/6 nephrectomy, and the GLUT1 expression in aortic tissue was determined by the reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemical staining. Indoxyl sulfate (IS) is a uremic retention solute proven with pro-proliferative effect on rat VSMCs, and we further studied the expression of GLUT1 in rat A7r5 rat embryonic aortic cells stimulated by IS in the presence or absence of phloretin, a GLUT1 inhibitor, to explore the pathogenic role of GLUT1 in uremic vasculopathy. The contribution of Akt/TSC2/mTOR/S6K signaling in modifying the GLUT1 expression was also assessed. RESULTS Eight weeks after 5/6 nephrectomy, aortic tissue obtained from CRF rats exhibited increased wall thickness and VSMC hypertrophy, hyperplasia, and degeneration. Compared with the sham-operated control group, the messenger (m)RNA and protein abundance of GLUT1 were both markedly increased in CRF rats. In vitro, IS induced a significant increase in expression of GLUT1 protein as well as pro-proliferative cyclin D1 and p21 mRNA and a modest increase in expression of antiapoptotic p53 mRNA in A7r5 cells, whereas inhibition of GLUT1 mediated glucose influx reduced the pro-proliferative and antiapoptotic effects of IS. In addition to increased GLUT1 expression, IS significantly suppressed Akt and TSC2 phosphorylation after 6-hour and 12-hour treatment, but increased S6K phosphorylation after 3-hour treatment. Inactivation of mTOR downstream signaling by rapamycin treatment inhibited S6K phosphorylation and abolished the stimulatory effect of IS on GLUT1 expression. CONCLUSIONS In vivo and in vitro experimental CRF displayed prominent GLUT1 upregulation in VSMCs. The uremic toxin IS stimulated proliferation of VSMCs possibly through induction of GLUT1 expression. The Akt/TSC/mTOR/S6K signaling pathway may be one of the mechanisms underlying the upregulation of GLUT1 expression in uremic VSMCs.

Inhibition of hypoxia inducible factor-1α attenuates abdominal aortic aneurysm progression through the down-regulation of matrix metalloproteinases.

Hypoxia inducible factor-1α (HIF-1α) pathway is associated with many vascular diseases, including atherosclerosis, arterial aneurysms, pulmonary hypertension and chronic venous diseases. Significant HIF-1α expression could be found at the rupture edge at human abdominal aortic aneurysm (AAA) tissues. While our initial in vitro experiments had shown that deferoxamine (DFO) could attenuate angiotensin II (AngII) induced endothelial activations; we unexpectedly found that DFO augmented the severity of AngII-induced AAA, at least partly through increased accumulation of HIF-1α. The findings promoted us to test whether aneurysmal prone factors could up-regulate the expression of MMP-2 and MMP-9 through aberrantly increased HIF-1α and promote AAA development. AngII induced AAA in hyperlipidemic mice model was used. DFO, as a prolyl hydroxylase inhibitor, stabilized HIF-1α and augmented MMPs activities. Aneurysmal-prone factors induced HIF-1α can cause overexpression of MMP-2 and MMP-9 and promote aneurysmal progression. Pharmacological HIF-1α inhibitors, digoxin and 2-ME could ameliorate AngII induced AAA in vivo. HIF-1α is pivotal for the development of AAA. Our study provides a rationale for using HIF-1α inhibitors as an adjunctive medical therapy in addition to current cardiovascular risk-reducing regimens.

Neurotoxicity associated with standard doses of piperacillin in an elderly patient with renal failure.

Piperacillin–tazobactam, a b-lactam–b-lactamase inhibitor combination, is a penicillin derivative with a broad spectrum of antibacterial activity against most Gram-positive and Gram-negative bacteria. Although tazobactam is non-toxic, piperacillin, like other b-lactam antibiotics, is neurotoxic to some extent if excessively accumulated. Neurotoxicity caused by excessive doses of b-lactam antibiotics is well-known [1], but its association with the renal failure package insert dose of piperacillin (4 g/day) has not been reported to date. Herein, we describe the development of acute unexplained encephalopathy in an 87-year-old man with advanced renal failure who had been treated for 3 days with piperacillin/tazobactam 2.25 g every 12 h. Withdrawal of piperacillin/tazobactam and initiation of high-flux hemodialysis rapidly reversed his encephalopathy. This is the first report of piperacillin-induced neurotoxicity caused by the minimum recommended dose in an elderly man with advanced renal failure.

CB1 cannabinoid receptor antagonist attenuates left ventricular hypertrophy and Akt-mediated cardiac fibrosis in experimental uremia.

Cannabinoid receptor type 1 (CB1R) plays an important role in the development of myocardial hypertrophy and fibrosis-2 pathological features of uremic cardiomyopathy. However, it remains unknown whether CB1R is involved in the pathogenesis of uremic cardiomyopathy. Here, we aimed to elucidate the role of CB1R in the development of uremic cardiomyopathy via modulation of Akt signalling. The heart size and myocardial fibrosis were evaluated by echocardiography and immunohistochemical staining, respectively, in 5/6 nephrectomy chronic kidney disease (CKD) mice treated with a CB1R antagonist. CB1R and fibrosis marker expression levels were determined by immunoblotting in H9c2 cells exposed to the uremic toxin indoxyl sulfate (IS), with an organic anion transporter 1 inhibitor or a CB1R antagonist or agonist. Akt phosphorylation was also assessed to examine the signaling pathways downstream of CB1R activation induced by IS in H9c2 cells. CKD mice exhibited marked left ventricular hypertrophy and myocardial fibrosis, which were reversed by treatment with the CB1R antagonist. CB1R, collagen I, transforming growth factor (TGF)-β, and α-smooth muscle actin (SMA) expression showed time- and dose-dependent upregulation in H9c2 cells treated with IS. The inhibition of CB1R by either CB1R antagonist or small interfering RNA-mediated knockdown attenuated the expression of collagen I, TGF-β, and α-SMA in IS-treated H9c2 cells, while Akt phosphorylation was enhanced by CB1R agonist and abrogated by CB1R antagonist in these cells. In summary, we conclude that CB1R blockade attenuates LVH and Akt-mediated cardiac fibrosis in a CKD mouse model. Uremic toxin IS stimulates the expression of CB1R and fibrotic markers and CB1R inhibition exerts anti-fibrotic effects via modulation of Akt signaling in H9c2 myofibroblasts. Therefore, the development of drugs targeting CB1R may have therapeutic potential in the treatment of uremic cardiomyopathy.

Thiazide-Sensitive Na+-Cl− Cotransporter (NCC) Gene Inactivation Results in Increased Duodenal Ca2+ Absorption, Enhanced Osteoblast Differentiation and Elevated Bone Mineral Density

Inactivation of the thiazide‐sensitive sodium chloride cotransporter (NCC) due to genetic mutations in Gitelmans syndrome (GS) or pharmacological inhibition with thiazide diuretics causes hypocalciuria and increased bone mineral density (BMD) with unclear extrarenal calcium (Ca2+) regulation. We investigated intestinal Ca2+ absorption and bone Ca2+ metabolism in nonsense Ncc Ser707X (S707X) homozygous knockin mice (NccS707X/S707X mice). Compared to wild‐type and heterozygous knockin littermates, NccS707X/S707X mice had increased intestinal absorption of 45Ca2+ and expression of the active Ca2+ transport machinery (transient receptor potential vanilloid 6, calbindin‐D9K, and plasma membrane Ca2+ ATPase isoform 1b). NccS707X/S707X mice had also significantly increased Ca2+ content accompanied by greater mineral apposition rate (MAR) in their femurs and higher trabecular bone volume, cortical bone thickness, and BMD determined by μCT. Their osteoblast differentiation markers, such as bone alkaline phosphatase, procollagen I, osteocalcin, and osterix, were also significantly increased while osteoclast activity was unaffected. Analysis of marrow‐derived bone cells, either treated with thiazide or directly cultured from Ncc S707X knockin mice, showed that the differentiation of osteoblasts was associated with increased phosphorylation of mechanical stress‐induced focal adhesion kinase (FAK) and extracellular signal‐regulated kinase (ERK). In conclusion, NCC inhibition stimulates duodenal Ca2+ absorption as well as osteoblast differentiation and bone Ca2+ storage, possibly through a FAK/ERK dependent mechanism.

Muscle Wasting in Hemodialysis Patients: New Therapeutic Strategies for Resolving an Old Problem

Muscle wasting has long been recognized as a major clinical problem in hemodialysis (HD) patients. In addition to its impact on quality of life, muscle wasting has been proven to be associated with increased mortality rates. Identification of the molecular mechanisms underlying muscle wasting in HD patients provides opportunities to resolve this clinical problem. Several signaling pathways and humeral factors have been reported to be involved in the pathogenic mechanisms of muscle wasting in HD patients, including ubiquitin-proteasome system, caspase-3, insulin/insulin-like growth factor-1 (IGF-1) signaling, endogenous glucocorticoids, metabolic acidosis, inflammation, and sex hormones. Targeting the aforementioned crucial signaling and molecules to suppress protein degradation and augment muscle strength has been extensively investigated in HD patients. In addition to exercise training, administration of megestrol acetate has been proven to be effective in improving anorexia and muscle wasting in HD patients. Correction of metabolic acidosis through sodium bicarbonate supplements can decrease muscle protein degradation and hormone therapy with nandrolone decanoate has been reported to increase muscle mass. Although thiazolidinedione has been shown to improve insulin sensitivity, its role in the treatment of muscle wasting remains unclear. This review paper focuses on the molecular pathways and potential new therapeutic approaches to muscle wasting in HD patients.