Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion.

Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk(-/-) mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1(-/-) mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1(+/+) and Romk1(-/-) mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1(+/+), but such regulation by high K intake was diminished with significant hyperkalemia in Romk1(-/-) mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K(+) secretion in the collecting tubule.

Gender difference in kidney electrolyte transport. I. Role of AT1a receptor in thiazide-sensitive Na+-Cl- cotransporter activity and expression in male and female mice.

We studied gender differences in Na+-Cl- cotransporter (NCC) activity and expression in wild-type (WT) and AT1a receptor knockout (KO) mice. In renal clearance experiments, urine volume (UV), glomerular filtration rate, absolute Na+ (ENa) and K+ (EK), and fractional Na+ (FENa) and K+ excretion were measured and compared at peak changes after bolus intravenous injection of hydrochlorothiazide (HCTZ; 30 mg/kg). In WT, females responded more strongly than males to HCTZ, with larger fractional increases of UV (7.8- vs. 3.4-fold), ENa (11.7- vs. 5.7-fold), FENa (7.9- vs. 4.9-fold), and EK (2.8- vs. 1.4-fold). In contrast, there were no gender differences in the responses to the diuretic in KO mice; HCTZ produced greater effects on male KO than on WT but similar effects on females. In WT, total (tNCC) and phosphorylated (pNCC) NCC protein expressions were 1.8- and 4.6-fold higher in females compared with males (P < 0.05), consistent with the larger response to HCTZ. In KO mice, tNCC and pNCC increased significantly in males to levels not different from those in females. There were no gender differences in the expression of the Na+/H+ exchanger (NHE3) in WT; NHE3 protein decreased to similar extents in male and female KO animals, suggesting AT1a-mediated NHE3 expression in proximal tubules. The resulting increase in delivery of NaCl to the distal nephron may underlie increased NCC expression and activity in mice lacking the AT1a receptor.

Olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation.

Olfactory receptors are G protein-coupled receptors that mediate olfactory chemosensation and serve as chemosensors in other tissues. We find that Olfr78, an olfactory receptor expressed in the kidney, responds to short chain fatty acids (SCFAs). Olfr78 is expressed in the renal juxtaglomerular apparatus, where it mediates renin secretion in response to SCFAs. In addition, both Olfr78 and G protein-coupled receptor 41 (Gpr41), another SCFA receptor, are expressed in smooth muscle cells of small resistance vessels. Propionate, a SCFA shown to induce vasodilation ex vivo, produces an acute hypotensive response in wild-type mice. This effect is differentially modulated by disruption of Olfr78 and Gpr41 expression. SCFAs are end products of fermentation by the gut microbiota and are absorbed into the circulation. Antibiotic treatment reduces the biomass of the gut microbiota and elevates blood pressure in Olfr78 knockout mice. We conclude that SCFAs produced by the gut microbiota modulate blood pressure via Olfr78 and Gpr41.

Regulation of glomerulotubular balance: II: impact of angiotensin II on flow-dependent transport.

Underlying glomerulotubular balance (GTB) is the impact of axial flow to regulate Na(+) and HCO(3)(-) transport by modulating Na(+)-H(+) exchanger 3 (NHE3) and H-ATPase activity. It is not known whether the cascade of events following a change in flow relies on local angiotensin (ANG II) generation or receptor availability. Mouse tubules were microperfused in vitro at flows of 5 and 20 nl/min, and net fluid (J(v)) and HCO(3)(-) (J(HCO3)) absorption and cell height were measured. Na(+) (J(Na)) and Cl(-) (J(Cl)) absorption and changes in microvillous torque were estimated. Raising flow increased Na(+) and HCO(3)(-) reabsorption but did not change either Cl(-) transport or cell volume. Losartan reduced absolute Na(+) and HCO(3)(-) absorption at both low and high flows but did not affect fractional flow-stimulated transport. Compared with controls, in AT(1a) knockout (KO) mouse tubules, 53% of flow-stimulated Na(+) absorption was abolished, but flow-stimulated HCO(3)(-) absorption was retained at similar levels. The remaining flow-stimulated J(HCO3) was eliminated by the H-ATPase inhibitor bafilomycin. Inhibition of the AT(2) receptor by PD123319 increased both J(Na) and J(HCO3) but did not affect flow-mediated fractional changes. NHE3 expression at the protein level was reduced in AT(1a) KO mice kidneys. We conclude that 1) although the AT(1a) receptor is necessary for flow to impact NHE3, the effect on H(+)-ATPase is independent of AT(1a); 2) the small flow-mediated changes in cell volume suggest a coordinate flow effect on both luminal and basolateral transporters; and 3) there is no evidence of flow-dependent Cl(-) transport, and thus no evidence for convective paracellular Cl(-) transport in mouse tubules.

Regulation of glomerulotubular balance. I. Impact of dopamine on flow-dependent transport.

In response to volume expansion, locally generated dopamine decreases proximal tubule reabsorption by reducing both Na/H-exchanger 3 (NHE3) and Na-K-ATPase activity. We have previously demonstrated that mouse proximal tubules in vitro respond to changes in luminal flow with proportional changes in Na(+) and HCO(3)(-) reabsorption and have suggested that this observation underlies glomerulotubular balance. In the present work, we investigate the impact of dopamine on the sensitivity of reabsorptive fluxes to changes in luminal flow. Mouse proximal tubules were microperfused in vitro at low and high flow rates, and volume and HCO(3)(-) reabsorption (J(v) and J(HCO3)) were measured, while Na(+) and Cl(-) reabsorption (J(Na) and J(Cl)) were estimated. Raising luminal flow increased J(v), J(Na), and J(HCO3) but did not change J(Cl). Luminal dopamine did not change J(v), J(Na), and J(HCO3) at low flow rates but completely abolished the increments of Na(+) absorption by flow and partially inhibited the flow-stimulated HCO(3)(-) absorption. The remaining flow-stimulated HCO(3)(-) absorption was completely abolished by bafilomycin. The DA1 receptor blocker SCH23390 and the PKA inhibitor H89 blocked the effect of exogenous dopamine and produced a two to threefold increase in the sensitivity of proximal Na(+) reabsorption to luminal flow rate. Under the variety of perfusion conditions, changes in cell volume were small and did not always parallel changes in Na(+) transport. We conclude that 1) dopamine inhibits flow-stimulated NHE3 activity by activation of the DA1 receptor via a PKA-mediated mechanism; 2) dopamine has no effect on flow-stimulated H-ATPase activity; 3) there is no evidence of flow stimulation of Cl(-) reabsorption; and 4) the impact of dopamine is a coordinated modulation of both luminal and peritubular Na(+) transporters.

Acid regulation of NaDC-1 requires a functional endothelin B receptor.

Metabolically generated acid is the major physiological stimulus for increasing proximal tubule citrate reabsorption, which leads to a decrease in citrate excretion. The activity of the Na-citrate cotransporter, NaDC-1, is increased in vivo by acid ingestion and in vitro by an acidic pH medium. In opossum kidney cells the acid stimulatory effect and the ability of endothelin-1 (ET-1) to stimulate NaDC-1 activity are both blocked by the endothelin B (ET(B)) receptor antagonist, BQ788. Acid feeding had no effect on brush border membrane NaDC-1 activity in mice in which ET(B) receptor expression was knocked out, whereas a stimulatory effect was found in wild-type mice. Using ET(A)/ET(B) chimeric and ET(B) C-terminal tail truncated constructs, ET-1 stimulation of NaDC-1 required a receptor C-terminal tail from either ET(A) or ET(B). The ET-1 effect was greatest when either the ET(B) transmembrane domain and C-terminal tail were present or the ET(B) C-terminal tail was linked to the ET(A) transmembrane domain. This effect was smaller when the ET(B) transmembrane domain was linked to the ET(A) C-terminal tail. Thus, the acid-activated pathway mediating stimulation of NaDC-1 activity requires a functional ET(B) receptor in vivo and in vitro, as does acid stimulation of NHE3 activity. Since increased NaDC-1 and NHE3 activities constitute part of the proximal tubule adaptation to an acid load, these studies indicate that there are similarities in the signaling pathway mediating these responses.

Uridine phosphorylase in breast cancer: a new prognostic factor?

Uridine phosphorylase (UPase) is an enzyme that converts the pyrimidine nucleoside uridine into uracil. Upon availability of ribose-1-phosphate, UPase can also catalyze the formation of nucleosides from uracil as well as from 5-fluorouracil, therefore involved in fluoropyrimidine metabolism. UPase gene expression is strictly controlled at the promoter level by oncogenes, tumor suppressor genes, and cytokines. UPase activity is usually elevated in various tumor tissues, including breast cancer, compared to matched normal tissues and this induction appears to contribute to the therapeutic efficacy of fluoropyrimidines in cancer patients. In this review, we will discuss in detail the role of UPase in the activation of fluoropyrimidines and its effect on the prognosis of breast cancer patients.

Impaired self-renewal and increased colitis and dysplastic lesions in colonic mucosa of AKR1B8-deficient mice.

PURPOSE: Ulcerative colitis and colitis-associated colorectal cancer (CAC) is a serious health issue, but etiopathological factors remain unclear. Aldo-keto reductase 1B10 (AKR1B10) is specifically expressed in the colonic epithelium, but downregulated in colorectal cancer. This study was aimed to investigate the etiopathogenic role of AKR1B10 in ulcerative colitis and CAC. EXPERIMENTAL DESIGN: Ulcerative colitis and CAC biopsies (paraffin-embedded sections) and frozen tissues were collected to examine AKR1B10 expression. Aldo-keto reductase 1B8 (the ortholog of human AKR1B10) knockout (AKR1B8(-/-)) mice were produced to estimate its role in the susceptibility and severity of chronic colitis and associated dysplastic lesions, induced by dextran sulfate sodium (DSS) at a low dose (2%). Genome-wide exome sequencing was used to profile DNA damage in DSS-induced colitis and tumors. RESULTS: AKR1B10 expression was markedly diminished in over 90% of ulcerative colitis and CAC tissues. AKR1B8 deficiency led to reduced lipid synthesis from butyrate and diminished proliferation of colonic epithelial cells. The DSS-treated AKR1B8(-/-) mice demonstrated impaired injury repair of colonic epithelium and more severe bleeding, inflammation, and ulceration. These AKR1B8(-/-) mice had more severe oxidative stress and DNA damage, and dysplasias were more frequent and at a higher grade in the AKR1B8(-/-) mice than in wild-type mice. Palpable masses were seen in the AKR1B8(-/-) mice only, not in wild-type. CONCLUSIONS: AKR1B8 is a critical protein in the proliferation and injury repair of the colonic epithelium and in the pathogenesis of ulcerative colitis and CAC, being a new etiopathogenic factor of these diseases.

[Establishment and identification of the gene cell line transfected aldose reductase like gene and its drug resistance to drugs containing carbonyl group].

OBJECTIVE: To investigate the relationship between aldose reductase like protein (ARL-1) gene overexpressed in HCC cells and drug-resistance of the cell to drugs containing carbonyl group. METHODS: To establish ARL-1 stable expression positive cell line, eukaryotic expression vectors containing ARL-1 gene cDNA were transfected into Hep cell mediated by lipofect AMINE. The positive monoclones were determined by PCR and RT-PCR, respectively. Then MTT assay was used to study the drug resistance ability of the cells to drugs containing carbonyl after incubating three days with those drugs. RESULTS: After ARL-1 gene transfection mediated by lipofect AMINE, one positive monoclonal cell overexpressing ARL-1 gene was selected. Compared with the control cell group, drug resistance ability of the positive cells to ADM and MMC which contain carbonyl group increased 2.3 and 3.17 fold, respectively (t=6.39, P=0.016 in ADM group and t=30.06, P=0.001 in MMC group). In the same time, drug resistance ability to 5-FU which has no carbonyl group had no statistical difference between positive monoclonal cell group and control cell group (t=0.684, P=0.531). CONCLUSIONS: The Hep ARL-1 positive cell line with stable expression of ARL-1 gene has been established successfully and the up-regulation of ARL-1 gene may plays an important role in drug resistance of the cells to anticancer drugs containing carbonyl group.

Differential expression of uridine phosphorylase in tumors contributes to an improved fluoropyrimidine therapeutic activity.

Abrogation of uridine phosphorylase (UPase) leads to abnormalities in pyrimidine metabolism and host protection against 5-fluorouracil (5-FU) toxicity. We elucidated the effects on the metabolism and antitumor efficacy of 5-FU and capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) in our UPase knockout (UPase(-/-)) model. Treatment with 5-FU (85 mg/kg) or capecitabine (1,000 mg/kg) five days a week for four weeks caused severe toxicity and structural damage to the intestines of wild-type (WT) mice, but not in UPase(-/-) animals. Capecitabine treatment resulted in a 70% decrease in blood cell counts of WT animals, with only a marginal effect in UPase(-/-) mice. UPase expressing colon 38 tumors implanted in UPase(-/-) mice revealed an improved therapeutic efficacy when treated with 5-FU and capecitabine because of the higher maximum tolerated dose for fluoropyrimidines achievable in UPase(-/-) mice. (19)F-MRS evaluation of capecitabine metabolism in tumors revealed similar activation of the prodrug in UPase(-/-) mice compared with WT. In WT mice, approximately 60% of capecitabine was transformed over three hours into its active metabolites, whereas 80% was transformed in tumors implanted in UPase(-/-) mice. In UPase(-/-) mice, prolonged retention of 5'dFUR allowed a proportional increase in tumor tissue. The similar presence of fluorinated catabolic species confirms that dihydropyrimidine dehydrogenase activity was not altered in UPase(-/-) mice. Overall, these results indicate the importance of UPase in the activation of fluoropyrimidines, the effect of uridine in protecting normal tissues, and the role for tumor-specific modulation of the phosphorolytic activity in 5-FU or capecitabine-based chemotherapy.

Activation of Stat1, IRF-1, and NF-kappaB is required for the induction of uridine phosphorylase by tumor necrosis factor-alpha and interferon-gamma.

Uridine phosphorylase (UPase) has been shown to be induced in various human and murine tumors and could potentially serve as a specific target for the modulation of tumor-selectivity of fluoropyrimidines. However, the signaling mechanisms underlying the regulation of UPase gene expression have not been determined. In this study, we investigated the effects of IFN-gamma on the regulation of TNF-alpha-induced UPase activity and have uncovered the molecular mechanisms of this potentiation, utilizing murine EMT6 breast cancer cells. Our data has shown that IFN-gamma can significantly increase UPase mRNA expression and the enzymatic activity induced by TNF-alpha in a dose-dependent manner, resulting in an enhanced sensitivity to 5-fluorouracil (5-FU) and 5'-Deoxy-5-fluorouridine (5'DFUR). We have previously shown that TNF-alpha activates NF-kappaB through increased translocation of NF-kappaB p65 from the cytoplasm into the nuclei. Exposure to IFN-gamma mainly affects nuclear IRF-1 and STAT1 in EMT6, but inhibits NF-kappaB p65 activity, indicating that the cooperative stimulation was the result of the independent activation of NF-kappaB, STAT1 and IRF-1 transcriptional factors through binding to their unique sites in the UPase promoter. Notably, the activation of NF-kappaB and STAT1 in human breast tissues is consistent with UPase activity; signifying their role in the up-regulation of the UPase gene expression in human tumors.

Modulation of uridine phosphorylase gene expression by tumor necrosis factor-alpha enhances the antiproliferative activity of the capecitabine intermediate 5'-deoxy-5-fluorouridine in breast cancer cells.

Uridine phosphorylase (UPase) has been shown to play an important role in the antineoplastic activity of 5-fluorouracil (5-FU) and in the anabolism of its oral prodrug, capecitabine, through the conversion of 5'-deoxy-5-fluorouridine (5'-DFUR) into 5-FU. In this study, we investigated the effect of tumor necrosis factor-alpha (TNF-alpha) on UPase gene expression and 5'-DFUR antiproliferative activity and elucidated the involved signal transduction pathway. Our data indicate that TNF-alpha significantly induced UPase mRNA expression and its enzymatic activity in EMT6 murine breast cancer cells, leading to an enhanced cytotoxicity of 5'-DFUR. This is further confirmed by an increased incorporation of 5'-DFUR-originated 5-FU nucleotides into nucleic acids. To clarify the mechanism of TNF-alpha-induced UPase expression, we first observed the effect of TNF-alpha on the UPase promoter activity with a series of 5'-deleted promoter-luciferase constructs. Transient transfection analysis showed that the TNF-alpha-inductive pattern in EMT6 cells was consistent with the presence of a nuclear factor-kappaB (NF-kappaB) binding element (-1332/-1312 bp) in the UPase promoter region. Furthermore, electrophoretic mobility shift assays, supershift, and cotransfection assays revealed that the activation of p65 was responsible for UPase induction by TNF-alpha. Finally, the induction of UPase by TNF-alpha could be suppressed by PS-341, a NF-kappaB inhibitor. In summary, TNF-alpha efficiently induces UPase gene expression through a NF-kappaB subunit p65-dependent pathway enhancing cell sensitivity to 5'-DFUR. The elucidation of this regulation mechanism may aid in the clinical use of 5-FU-based chemotherapy.