Maternal Malnutrition Affects Hepatic Metabolism through Decreased Hepatic Taurine Levels and Changes in HNF4A Methylation.

Fetal programming implies that the maternal diet during pregnancy affects the long-term health of offspring. Although maternal diet influences metabolic disorders and non-alcoholic fatty liver disease in offspring, the hepatic mechanisms related to metabolites are still unknown. Here, we investigated the maternal diet-related alterations in metabolites and the biological pathway in male offspring at three months of age. Pregnant rats were exposed to 50% food restriction during the prenatal period or a 45% high-fat diet during the prenatal and postnatal periods. The male offspring exposed to food restriction and high-fat diets had lower birth weights than controls, but had a catch-up growth spurt at three months of age. Hepatic taurine levels decreased in both groups compared to controls. The decreased hepatic taurine levels in offspring affected excessive lipid accumulation through changes in hepatocyte nuclear factor 4 A methylation. Moreover, the alteration of gluconeogenesis in offspring exposed to food restriction was observed to a similar extent as that of offspring exposed to a high fat diet. These results indicate that maternal diet affects the dysregulation in hepatic metabolism through changes in taurine levels and HNF4A methylation, and predisposes the offspring to Type 2 diabetes and non-alcoholic fatty liver disease in later life.

Descending thin limb of the intermediate loop expresses both aquaporin 1 and urea transporter A2 in the mouse kidney.

A new intermediate type of Henle's loop has been reported that it extends into the inner medulla and turns within the first millimeter beyond the outer medulla. This study aimed to identify the descending thin limb (DTL) of the intermediate loop in the adult C57Bl/6 mouse kidney using aquaporin 1 (AQP1) and urea transporter A2 (UT-A2) antibodies. In the upper part of the inner stripe of the outer medulla (ISOM), AQP1 was expressed strongly in the DTL with type II epithelium of the long loop, but not in type I epithelium of the short loop. The DTL of the intermediate loop exhibited weak AQP1 immunoreactivity. UT-A2 immunoreactivity was not observed in the upper part of any DTL type. AQP1 expression was similar in the upper and middle parts of the ISOM. UT-A2 expression was variable, being expressed strongly in the DTL with type I epithelium of the short loop, but not in type II epithelium of the long loop. In the innermost part of the ISOM, AQP1 was expressed only in type III epithelium of the long loop. UT-A2-positive and UT-A2-negative cells were intermingled in type I epithelium of the intermediate loop, but were not observed in type III epithelium of the long loop. UT-A2-positive DTLs of the intermediate loop extended into the UT-A2/AQP1-negative type I epithelium in the initial part of the inner medulla. These results demonstrate that the DTL of the intermediate loop is composed of type I epithelium and expresses both AQP1 and UT-A2. The functional role of the DTL of the intermediate loop may be distinct from the short or long loops.

Bone marrow-derived cells play a major role in kidney fibrosis via proliferation and differentiation in the infiltrated site.

Increase of interstitial cell population, resulting in the expansion of interstitium, excessive production of extracellular matrix, and reduction of functioning tubules, is critical in fibrotic progression in the kidney of patients suffering from chronic renal diseases. Here, we investigated the contribution of bone marrow-derived cells (BMDC) in kidney fibrosis caused by ureteral obstruction (UO) using eGFP bone marrow-reconstituted chimeric mice. UO caused dramatic increases in the numbers of interstitial cells and expansion of the interstitium. Most kidney interstitial cells expressed GFP. Twenty nine percent of interstitial cells were cells that had proliferated and approximately 89% among them were BMDCs. Proliferation of fibroblasts differentiated from BMDCs significantly occurred in the interstitium of UO-kidney. Removal of BMDCs by whole body irradiation after UO resulted in reduction of kidney fibrosis, while injection of RAW264.7 cells, monocytes/macrophages, into irradiated mice induced a reversal of this reduction. Treatment with apocynin, an inhibitor of NADPH oxidase, reduced infiltration of BMDCs into the UO-kidney, leading to reduction of kidney fibrosis. In addition, only a few slow-cycling cells were observed in the interstitium of normal kidney. Even after UO, no change in the number of those cells was observed. Our findings demonstrate that BMDCs are a major source for interstitial expansion during kidney fibrosis via infiltration into damaged sites, differentiation to fibroblasts, and subsequent proliferation, contributing kidney fibrosis. These data provide a clear therapeutic target for treatment of chronic kidney disease.

Effect of collecting duct-specific deletion of both Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg) on renal response to metabolic acidosis.

The Rhesus (Rh) glycoproteins, Rh B and Rh C Glycoprotein (Rhbg and Rhcg, respectively), are ammonia-specific transporters expressed in renal distal nephron and collecting duct sites that are necessary for normal rates of ammonia excretion. The purpose of the current studies was to determine the effect of their combined deletion from the renal collecting duct (CD-Rhbg/Rhcg-KO) on basal and acidosis-stimulated acid-base homeostasis. Under basal conditions, urine pH and ammonia excretion and serum HCO3(-) were similar in control (C) and CD-Rhbg/Rhcg-KO mice. After acid-loading for 7 days, CD-Rhbg/Rhcg-KO mice developed significantly more severe metabolic acidosis than did C mice. Acid loading increased ammonia excretion, but ammonia excretion increased more slowly in CD-Rhbg/Rhcg-KO and it was significantly less than in C mice on days 1-5. Urine pH was significantly more acidic in CD-Rhbg/Rhcg-KO mice on days 1, 3, and 5 of acid loading. Metabolic acidosis increased phosphenolpyruvate carboxykinase (PEPCK) and Na(+)/H(+) exchanger NHE-3 and decreased glutamine synthetase (GS) expression in both genotypes, and these changes were significantly greater in CD-Rhbg/Rhcg-KO than in C mice. We conclude that 1) Rhbg and Rhcg are critically important in the renal response to metabolic acidosis; 2) the significantly greater changes in PEPCK, NHE-3, and GS expression in acid-loaded CD-Rhbg/Rhcg-KO compared with acid-loaded C mice cause the role of Rhbg and Rhcg to be underestimated quantitatively; and 3) in mice with intact Rhbg and Rhcg expression, metabolic acidosis does not induce maximal changes in PEPCK, NHE-3, and GS expression despite the presence of persistent metabolic acidosis.

Nox4-SH3YL1 complex is involved in diabetic nephropathy.

Nox4-derived H2O2 generation plays an important role in the pathogenesis of chronic kidney diseases (CKDs) such as diabetic nephropathy (DN). Here, we showed that SH3 domain-containing Ysc84-like 1 (SH3YL1), a Nox4 cytosolic activator, regulated DN. Streptozotocin (STZ)-induced type Ⅰ diabetic models in SH3YL1 whole-body knockout (KO) mice and podocyte-specific SH3YL1 conditional KO (Nphs2-Cre/SH3YL1fl/fl) mice were established to investigate the function of SH3YL1 in DN. The expression of fibrosis markers and inflammatory cytokines, the generation of oxidative stress, and the loss of podocytes were suppressed in diabetic SH3YL1 KO and Nphs2-Cre/SH3YL1fl/fl mice, compared to diabetic control mice. To extrapolate the observations derived from diabetic mice to clinical implication, we measured the protein level of SH3YL1 in patients DN. In fact, the SH3YL1 level was increased in patients DN. Overall, the SH3YL1-Nox4 complex was identified to play an important role in renal inflammation and fibrosis, resulting in the development of DN.

The role of 70-kDa heat shock protein in dDAVP-induced AQP2 trafficking in kidney collecting duct cells.

It has been reported that several proteins [heat shock protein 70 (Hsp70 and Hsc70), annexin II, and tropomyosin 5b] interact with the Ser(256) residue on the COOH terminus of aquaporin-2 (AQP2), where vasopressin-induced phosphorylation occurs for mediating AQP2 trafficking. However, it remains unknown whether these proteins, particularly Hsp70, play a role in AQP2 trafficking. Semiquantitative immunoblotting revealed that renal expression of AQP2 and Hsp70 was significantly increased in water-restricted or dDAVP-infused rats. In silico analysis of the 5'-flanking regions of AQP2, Hsp70-1, and Hsp70-2 genes revealed that transcriptional regulator binding elements associated with cAMP response were identified at both the Hsp70-1 and Hsp70-2 promoter regions, in addition to AQP2. Luciferase reporter assay demonstrated the significant increase of luminescence after dDAVP stimulation (10(-8) M, 6 h) in the LLC-PK1 cells transfected with luciferase vector containing 1 kb of the 5'-flanking region of Hsp70-2 gene. Hsp70-2 protein expression was also increased in mpkCCDc14 cells treated by dDAVP in a concentration-dependent manner. Cell surface biotinylation analysis demonstrated that forskolin (10(-5) M, 15 min)-induced AQP2 targeting to the apical plasma membrane was significantly attenuated in the mpkCCDc14 cells with Hsp70-2 knockdown. Moreover, forskolin-induced AQP2 phosphorylation (Ser(256)) was not significantly induced in the mpkCCDc14 cells with Hsp70-2 knockdown. In contrast, Hsp70-2 knockdown did not affect the dDAVP-induced AQP2 abundance. In addition, siRNA-directed knockdown of Hsp70 significantly decreased cell viability. The results suggest that Hsp70 is likely to play a role in AQP2 trafficking to the apical plasma membrane, partly through affecting AQP2 phosphorylation at Ser(256) and cell viability.

Intercalated cell-specific Rh B glycoprotein deletion diminishes renal ammonia excretion response to hypokalemia.

The ammonia transporter family member, Rh B Glycoprotein (Rhbg), is an ammonia-specific transporter heavily expressed in the kidney and is necessary for the normal increase in ammonia excretion in response to metabolic acidosis. Hypokalemia is a common clinical condition in which there is increased renal ammonia excretion despite the absence of metabolic acidosis. The purpose of this study was to examine Rhbg's role in this response through the use of mice with intercalated cell-specific Rhbg deletion (IC-Rhbg-KO). Hypokalemia induced by feeding a K(+)-free diet increased urinary ammonia excretion significantly. In mice with intact Rhbg expression, hypokalemia increased Rhbg protein expression in intercalated cells in the cortical collecting duct (CCD) and in the outer medullary collecting duct (OMCD). Deletion of Rhbg from intercalated cells inhibited hypokalemia-induced changes in urinary total ammonia excretion significantly and completely prevented hypokalemia-induced increases in urinary ammonia concentration, but did not alter urinary pH. We conclude that hypokalemia increases Rhbg expression in intercalated cells in the cortex and outer medulla and that intercalated cell Rhbg expression is necessary for the normal increase in renal ammonia excretion in response to hypokalemia.

Renal ammonia excretion in response to hypokalemia: effect of collecting duct-specific Rh C glycoprotein deletion.

The Rhesus factor protein, Rh C glycoprotein (Rhcg), is an ammonia transporter whose expression in the collecting duct is necessary for normal ammonia excretion both in basal conditions and in response to metabolic acidosis. Hypokalemia is a common clinical condition associated with increased renal ammonia excretion. In contrast to basal conditions and metabolic acidosis, increased ammonia excretion during hypokalemia can lead to an acid-base disorder, metabolic alkalosis, rather than maintenance of acid-base homeostasis. The purpose of the current studies was to determine Rhcg's role in hypokalemia-stimulated renal ammonia excretion through the use of mice with collecting duct-specific Rhcg deletion (CD-Rhcg-KO). In mice with intact Rhcg expression, a K(+)-free diet increased urinary ammonia excretion and urine alkalinization and concurrently increased Rhcg expression in the collecting duct in the outer medulla. Immunohistochemistry and immunogold electron microscopy showed hypokalemia increased both apical and basolateral Rhcg expression. In CD-Rhcg-KO, a K(+)-free diet increased urinary ammonia excretion and caused urine alkalinization, and the magnitude of these changes did not differ from mice with intact Rhcg expression. In mice on a K(+)-free diet, CD-Rhcg-KO increased phosphate-dependent glutaminase (PDG) expression in the outer medulla. We conclude that hypokalemia increases collecting duct Rhcg expression, that this likely contributes to the hypokalemia-stimulated increase in urinary ammonia excretion, and that adaptive increases in PDG expression can compensate for the absence of collecting duct Rhcg.

Expression of the ammonia transporter family member, Rh B Glycoprotein, in the human kidney.

The ammonia transporter family member, Rh B Glycoprotein (RhBG/Rhbg), is essential for ammonia transport by the rodent kidney, but in the human kidney mRNA but not protein expression has been reported. Because ammonia transport is fundamental for acid-base homeostasis, the current study addressed RhBG expression in the human kidney. Two distinct RhBG mRNA sequences have been reported, with different numbers of consecutive cytosines at nt1265 and thus encoding different carboxy-tails. Sequencing the region of difference in both human kidney and liver mRNA showed eight sequential cytosines, not seven as in some reports. Knowing the correct mRNA sequence for RhBG, we then assessed RhBG protein expression using antibodies against the correct amino acid sequence. Immunoblot analysis demonstrated RhBG protein expression in human kidney and immunohistochemistry identified basolateral RhBG in connecting segment (CNT) and the cortical and outer medullary collecting ducts. Colocalization of RhBG with multiple cell-specific markers demonstrated that that CNT cells and collecting duct type A intercalated cells express high levels of RhBG, and type B intercalated cells and principal cells do not express detectable RhBG. Thus, these studies identify the correct mRNA and thus protein sequence for human RhBG and show that the human kidney expresses basolateral RhBG protein in CNT, type A intercalated cells, and non-A, non-B cells. We conclude that RhBG can mediate an important role in human renal ammonia transport.

Expression of the rhesus glycoproteins, ammonia transporter family members, RHCG and RHBG in male reproductive organs.

The rhesus glycoproteins, Rh B glycoprotein (RHBG) and Rh C glycoprotein (RHCG), are recently identified ammonia transporters. Rhcg expression is necessary for normal male fertility, but its specific cellular expression is unknown, and Rhbg has not been reported to be expressed in the male reproductive tract. This study sought to determine the specific cellular expression of Rhcg, to determine whether Rhbg is expressed in the male reproductive tract, and, if so, to determine which cells express Rhbg using real-time RT-PCR, immunoblot analysis, and immunohistochemistry. Both Rhbg and Rhcg were expressed throughout the male reproductive tract. In the testis, high levels of Rhbg were expressed in Leydig cells, and Rhcg was expressed in spermatids during the later stages of their maturation (steps 13-16) in stages I-VIII of the seminiferous epithelium cycle. In the epididymis, basolateral Rhbg was present in narrow cells in the initial segment, in principal cells in the upper corpus, and in clear cells throughout the epididymis. Apical Rhcg immunolabel was present in principal cells in the caput and upper corpus epididymidis and in clear cells in the middle and lower corpus and cauda epididymidis. In the vas deferens, apical Rhcg immunolabel and basolateral Rhbg immunolabel were present in some principal cells and colocalized with H(+)-ATPase immunolabel. We conclude that both Rhbg and Rhcg are highly expressed in specific cells in the male reproductive tract where they can contribute to multiple components of male fertility.

Antineoplastic effect of WIN 55,212-2, a cannabinoid agonist, in a murine xenograft model of gastric cancer.

BACKGROUND: We have previously reported the antineoplastic effects of a cannabinoid agonist in gastric cancer cells. Our aim was to evaluate this in a murine xenograft model. METHODS: Animal models were created after injecting AGS gastric cancer cells subcutaneously into the flank of male BALB/c-nude mice. A cannabinoid agonist, WIN 55,212-2 (7 mg/kg body weight) or vehicle was injected around the tumor subcutaneously every 24 h for 14 days. Tumors were explanted for analysis. RESULTS: Tumor volume decreased by 30% in the WIN 55,212-2-treated group compared to the group treated with vehicle (p < 0.05). Apoptotic cells were found more commonly in the WIN 55,212-2 treatment group than in the control on immunohistochemistry. Compared to the control, WIN 55,212-2 treatment significantly increased caspase-3 cleavage and decreased MMP-2, MMP-7 and MMP-9 protein levels significantly (all p < 0.05). VEGF-A protein level was not different between the 2 groups. CONCLUSION: WIN 55,212-2 has antineoplastic effect on the gastric cancers in in vivo model.

High water intake induces primary cilium elongation in renal tubular cells.

Background: The primary cilium protrudes from the cell surface and functions as a mechanosensor. Recently, we found that water intake restriction shortens the primary cilia of renal tubular cells, and a blockage of the shortening disturbs the ability of the kidneys to concentrate urine. Here, we investigate whether high water intake (HWI) alters primary cilia length, and if so, what is its underlying mechanism and its role on kidney urine production. Methods: Experimental mice were given free access to normal water (normal water intake) or 3% sucrose-containing water for HWI for 2 days. Some mice were administered with U0126 (10 mg/kg body weight), an inhibitor of MEK kinase, from 2 days before HWI, daily. The primary cilium length and urine amount and osmolality were investigated. Results: HWI-induced diluted urine production and primary cilium elongation in renal tubular cells. HWI increased the expression of α-tubulin acetyltransferase 1 (αTAT1), leading to the acetylation of α-tubulins, a core protein of the primary cilia. HWI also increased phosphorylated ERK1/2 (p-ERK1/2) and exocyst complex component 5 (EXOC5) expression in the kidneys. U0126 blocked HWI-induced increases in αTAT1, p-ERK1/2, and EXOC5 expression. U0126 inhibited HWI-induced α-tubulin acetylation, primary cilium elongation, urine amount increase, and urine osmolality decrease. Conclusion: These results show that increased water intake elongates the primary cilia via ERK1/2 activation and that ERK inhibition prevents primary cilium elongation and diluted urine production. These data suggest that the elongation of primary cilium length is associated with the production of diluted urine.

Shortening of primary cilia length is associated with urine concentration in the kidneys.

BACKGROUND: The primary cilium, a microtubule-based cellular organelle present in certain kidney cells, functions as a mechano-sensor to monitor fluid flow in addition to various other biological functions. In kidneys, the primary cilia protrude into the tubular lumen and are directly exposed to pro-urine flow and components. However, their effects on urine concentration remain to be defined. Here, we investigated the association between primary cilia and urine concentration. METHODS: Mice either had free access to water (normal water intake, NWI) or were not allowed access to water (water deprivation, WD). Some mice received tubastatin, an inhibitor of histone deacetylase 6 (HDAC6), which regulates the acetylation of α-tubulin, a core protein of microtubules. RESULTS: WD decreased urine output and increased urine osmolality, concomitant with apical plasma membrane localization of aquaporin 2 (AQP2) in the kidney. After WD, compared with after NWI, the lengths of primary cilia in renal tubular epithelial cells were shortened and HDAC6 activity increased. WD induced deacetylation of α-tubulin without altering α-tubulin levels in the kidney. Tubastatin prevented the shortening of cilia through increasing HDAC6 activity and consequently increasing acetylated α-tubulin expression. Furthermore, tubastatin prevented the WD-induced reduction of urine output, urine osmolality increase, and apical plasma membrane localization of AQP2. CONCLUSIONS: WD shortens primary cilia length through HDAC6 activation and α-tubulin deacetylation, while HDAC6 inhibition blocks the WD-induced changes in cilia length and urine output. This suggests that cilia length alterations are involved, at least in part, in the regulation of body water balance and urine concentration.

Effect of hypokalemia on renal expression of the ammonia transporter family members, Rh B Glycoprotein and Rh C Glycoprotein, in the rat kidney.

Hypokalemia is a common electrolyte disorder that increases renal ammonia metabolism and can cause the development of an acid-base disorder, metabolic alkalosis. The ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), are expressed in the distal nephron and collecting duct and mediate critical roles in acid-base homeostasis by facilitating ammonia secretion. In the current studies, the effect of hypokalemia on renal Rhbg and Rhcg expression was examined. Normal Sprague-Dawley rats received either K(+)-free or control diets for 2 wk. Rats receiving the K(+)-deficient diet developed hypokalemia and metabolic alkalosis associated with significant increases in both urinary ammonia excretion and urine pH. Rhcg expression increased in the outer medullary collecting duct (OMCD). In OMCD intercalated cells, hypokalemia resulted in more discrete apical Rhcg expression and a marked increase in apical plasma membrane immunolabel. In principal cells, in the OMCD, hypokalemia increased both apical and basolateral Rhcg immunolabel intensity. Cortical Rhcg expression was not detectably altered by immunohistochemistry, although there was a slight decrease in total expression by immunoblot analysis. Rhbg protein expression was decreased slightly in the cortex and not detectably altered in the outer medulla. We conclude that in rat OMCD, hypokalemia increases Rhcg expression, causes more polarized apical expression in intercalated cells, and increases both apical and basolateral expression in the principal cell. Increased plasma membrane Rhcg expression in response to hypokalemia in the rat, particularly in the OMCD, likely contributes to the increased ammonia excretion and thereby to the development of metabolic alkalosis.

Renal ischemia-reperfusion injury causes intercalated cell-specific disruption of occludin in the collecting duct.

Renal ischemic events open tight junctions and disrupt epithelial polarity. The purpose of this study was to examine the effects of ischemia-reperfusion (IR) injury on expression and distribution of the tight junction proteins, occludin and ZO-1, in the rat kidney. IR injury was induced by clamping both renal pedicles for 30 min and animals were killed at 6 h after the reperfusion. IR injury decreased blood bicarbonate level, but did not persistently alter pH, Na(+), K(+), or Cl(-). In control kidneys, occludin immunoreactivity was intense in the tight junctions in the thick ascending limb, distal convoluted tubule, and collecting duct, moderate in the thin limbs of the loop of Henle, and was not detected in the proximal tubule, glomerulus, and blood vessels. ZO-1 was expressed in the same sites in which occludin was expressed, and additionally was also expressed in the proximal tubule, glomerulus, and vascular endothelial cells. IR kidneys exhibited damaged renal tubular epithelial cells in both proximal tubule and collecting duct segments in the outer medulla. In the collecting duct, the response of intercalated cells and principal cells differed. Following IR injury, intercalated cells, but not principal cells, lost their normal epithelial polarity and were frequently extruded into the tubule lumen. Occludin, instead of being localized to tight junctions, was localized diffusely in the cytoplasm in intercalated cells of IR kidneys. Principal cells, in contrast, were not detectably affected and neither occludin nor ZO-1 expression were altered in response to IR injury. The normal localization of ZO-1 expression to tight junction sites in both the proximal tubule and collecting duct was altered in response to IR, and, instead, ZO-1 expression was present diffusely in the cytoplasm. IR injury did not alter detectably either occludin or ZO-1 localization to the tight junction of the thick ascending limb cells. The abundance of total occludin protein by immunoblot analysis was not changed with IR injury. These results demonstrate that renal IR injury causes tight junction disruptions in both the proximal tubule and the collecting duct, and that altered distribution of the tight junction protein, occludin, may play a critical role in the collecting duct dysfunction which IR induces.

An anatomical study of the saphenous nerve, artery, and artery perforators within the thigh using cadaveric dissection.

Although the saphenous flap has been used in reconstruction as a free flap, there has not yet been an anatomic study about the perforators of the saphenous artery. The aim of this study is to investigate the anatomy of the saphenous artery and the number and locations of its perforators. We dissected parts of 10 legs from 5 cadavers. Measurements of the positions of the dissected saphenous arteries and their perforators were taken from the medial epicondyle of the femur. We observed the origin, end point, and the diameter of each of the arteries, and we investigated the numbers and locations of both septocutaneous and musculocutaneous perforators. The average length of saphenous artery was 14.8 cm, and it was located 12.0 cm above the medial epicondyle of the femur. The average diameter was 1.63 mm. A median average of 4 perforators branched out from a single saphenous artery. There was a median average of 2 septocutaneous perforators and 2 musculocutaneous perforators from the saphenous artery. The perforators were mainly located at 7 cm proximal to the medial epicondyle of the femur. The saphenous artery has many perforators and is therefore useful as the pedicle of the perforator flap. The saphenous artery perforator flap can be designed within 7 cm proximal to the medial epicondyle of the femur. Our results may be helpful in the applications of the neurocutaneous flap using the saphenous artery or the perforator flap based on septocutaneous perforators.

Effect of a synthetic cannabinoid agonist on the proliferation and invasion of gastric cancer cells.

Although cannabinoids are associated with antineoplastic activity in a number of cancer cell types, the effect in gastric cancer cells has not been clarified. In the present study, we investigated the effects of a cannabinoid agonist on gastric cancer cell proliferation and invasion. The cannabinoid agonist WIN 55,212-2 inhibited the proliferation of human gastric cancer cells in a dose-dependent manner and that this effect was mediated partially by the CB(1) receptor. We also found that WIN 55,212-2 induced apoptosis and down-regulation of the phospho-AKT expression in human gastric cancer cells. Furthermore, WIN 55,212-2 treatment inhibited the invasion of gastric cancer cells, and down-regulated the expression of MMP-2 and VEGF-A through the cannabinoid receptors. Our results open the possibilities in using cannabinoids as a new gastric cancer therapy.

Expression of ammonia transporter family members, Rh B glycoprotein and Rh C glycoprotein, in the developing rat kidney.

Ammonia metabolism is a primary component of acid-base homeostasis but is incompletely developed at time of birth. Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg) are recently recognized ammonia transporter family members expressed in the mammalian kidney. This study's purpose was to establish the expression and localization of Rhbg and Rhcg during kidney development. We examined kidneys from fetal days 16 (E16), 18 (E18), and 20 (E20), and from the first 21 days of postnatal development. Rhbg was expressed initially at E18, with expression only in the connecting tubule (CNT); at E20, Rhbg was expressed in both the CNT and the medullary collecting duct (MCD). In contrast, Rhcg was first expressed at E16 with basal expression in the ureteric bud; at E18, it was expressed in a subset of CNT cells with an apical pattern, followed by apical and basolateral expression in the MCD at E20. In the cortex, Rhbg and Rhcg expression increased in the CNT before expression in the cortical collecting duct during fetal development. In the MCD, both Rhbg and Rhcg expression was initially in cells in the papillary tip, with gradual removal from the tip during the late fetal period and transition during the early neonatal period to an adult pattern with predominant expression in the outer MCD and only rare expression in cells in the initial inner MCD. Double-labeling with intercalated cell-specific markers identified that Rhbg and Rhcg were expressed initially in CNT cells, CNT A-type intercalated cells and non-A, non-B intercalated cells, and in MCD A-type intercalated cells. We conclude that expression of Rhbg and Rhcg parallels intercalated cell development and that immature Rhbg and Rhcg expression at birth contributes to incomplete ammonia excretion capacity.

Expression of the gas-transporting proteins, Rh B glycoprotein and Rh C glycoprotein, in the murine lung.

A family of gas-transporting proteins, the Mep/Amt/Rh glycoprotein family, has been identified recently. These are integral membrane proteins, are widely expressed in sites of gas transport, and are known to transport the gaseous molecule, NH(3), and recent evidence indicates they can transport CO(2). Because the mammalian lung is a critical site for gas transport, the current studies examine the expression of the nonerythroid members of this extended family, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), in the normal mouse lung. Real-time RT-PCR and immunoblot analysis demonstrated both Rhbg and Rhcg mRNA and protein expression, respectively. Immunohistochemistry demonstrated both Rhbg and Rhcg were expressed in bronchial and bronchiolar epithelial cells. Rhbg was expressed by Clara cells, specifically, whereas all bronchial/bronchiolar epithelial cells, with the exception of goblet cells, expressed Rhcg. Rhbg expression was basolateral, whereas Rhcg exhibited apical and intracellular immunolabel, polarized expression similar to that observed in Rhbg- and Rhcg-expressing epithelial cells in other organs. There was no detectable expression of either Rhbg or Rhcg in alveolar endothelial or epithelial cells, in pneumocytes or in vascular tissue. In vitro studies using cultured bronchial epithelial cells confirm Rhbg and Rhcg expression, demonstrate that saturable, not diffusive, transport is the primary mechanism of ammonia/methylammonia transport, and show that the saturable transport mechanism has kinetics similar to those demonstrated previously for Rhbg and Rhcg. These findings suggest Rhbg and Rhcg may contribute to bronchial epithelial cell ammonia metabolism and suggest that they do not contribute to pulmonary CO(2) transport.

Fragmentation of kidney epithelial cell primary cilia occurs by cisplatin and these cilia fragments are excreted into the urine.

The primary cilium, which protrudes from the cell surface, is associated with the pathogenesis of various diseases, including acute kidney injury (AKI). Primary cilium length dynamically changes during the progression of diseases. However, its relevance in disease and the underlying mechanism are largely unknown. In this study, we investigated the role of primary cilia in AKI induced by cisplatin, an effective anticancer drug, and the underlying mechanisms. In addition, we evaluated the usefulness of length alteration and deciliation of primary cilia into the urine for the diagnosis of AKI. Cisplatin induced shortening, elongation, and normalization of the primary cilia in kidney epithelial cells over time. During shortening, primary cilia fragments and ciliary proteins were excreted into the urine. During deciliation, cell proliferation and the expression of cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen were not significantly changed. Shortening and deciliation of primary cilia were observed before significant increases in plasma creatinine and blood urea nitrogen concentration occurred. Pretreatment with Mito-Tempo, a mitochondria-targeted antioxidant, prevented cisplatin-induced primary cilium shortening and inhibited the increases in superoxide formation, lipid peroxidation, blood urea nitrogen, and tissue damage. In contrast, isocitrate dehydrogenase 2 (Idh2) gene deletion, which results in defect of the NADPH-associated mitochondrial antioxidant system, exacerbated cisplatin-induced changes in mice. Taken together, our findings demonstrate that cisplatin induces deciliation into the urine and antioxidant treatment prevents this deciliation, renal dysfunction, and tissue damage after cisplatin injection. These results suggest that cisplatin-induced AKI is associated with primary cilia and urine primary cilia proteins might be a non-invasive biomarker of kidney injury.