Phenotypic characterization of lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice. Pathophysiology Of biliary lipid secretion.

The inbred C57L strain but not the AKR strain of mice carry Lith genes that determine cholesterol gallstone susceptibility. When C57L mice are fed a lithogenic diet containing 15% fat, 1% cholesterol, and 0.5% cholic acid, gallbladder bile displays rapid cholesterol supersaturation, mucin gel accumulation, increases in hydrophobic bile salts, and rapid phase separation of solid and liquid crystals, all of which contribute to the high cholesterol gallstone prevalence rates (D. Q-H. Wang, B. Paigen, and M. C. Carey. J. Lipid Res. 1997. 38: 1395;-1411). We have now determined the hepatic secretion rates of biliary lipids in fasting male and female C57L and AKR mice and the intercross (C57L x AKR)F(1) before and at frequent intervals during feeding the lithogenic diet for 56 days. Bile flow and biliary lipid secretion rates were measured in the first hour of an acute bile fistula and circulating bile salt pool sizes were determined by the "washout" technique after cholecystectomy. Compared with AKR mice, we found that i) C57L and F(1) mice on chow displayed significantly higher secretion rates of all biliary lipids, and larger bile salt pool sizes, as well as higher bile salt-dependent and bile salt-independent flow rates; ii) the lithogenic diet further increased biliary cholesterol and lecithin outputs, but bile salt outputs remained constant. Biliary coupling of cholesterol to lecithin increased approximately 30%, setting the biophysical conditions necessary for cholesterol phase separation in the gallbladder; and iii) no gender differences in lipid secretion rates were noted but male mice exhibited significantly more hydrophobic bile salt pools than females.We conclude that in gallstone-susceptible mice, Lith genes determine increased outputs of all biliary lipids but promote cholesterol hypersecretion disproportionately to lecithin and bile salt outputs thereby inducing lithogenic bile formation.     

Lack of adiponectin promotes formation of cholesterol gallstones in mice

Plasma adiponectin levels are reduced in obese people, and hypoadiponectinemia is recently reported to associate with cholesterol gallstone formation in human. The aim of this study was to examine the role of adiponectin in gallstone formation using adiponectin knockout mice. We analyzed male knockout and C57BL6J mice fed normal or lithogenic diet for 6 weeks. On lithogenic diet, the prevalence rate of gallstone was significantly greater in knockout mice than C57BL6J mice. The molar percentages of β and ω-muricholic acid were significantly higher and hepatic sterol 12α-hydroxylase expression (cyp8b1) was significantly lower in knockout mice than C57BL6J mice fed normal diet. The bile apolipoprotein A-I protein levels were decreased in knockout mice. Histological examination showed gallbladder wall thickening and accumulation of glycoprotein in the gallbladder of knockout mice. Gallbladder phospholipase A2-IVA expression was significantly higher in knockout mice than in C57BL6J mice fed lithogenic diet. Our results indicate that lack of adiponectin promotes gallstone formation in mice.     

Aging per se is an independent risk factor for cholesterol gallstone formation in gallstone susceptible mice

Cholesterol gallstones occur rarely in childhood and adolescence and increase linearly with age in both genders. To explore whether aging per se increases cholesterol saturation of bile and gallstone prevalence, and to investigate age-related changes in hepatic and biliary lipid metabolism, we studied gallstone-susceptible C57L mice and resistant AKR mice of both genders fed 8 weeks with a lithogenic diet containing 1% cholesterol, 0.5% cholic acid, and 15% butter fat starting at (young adult) 8, (older adult) 36, and (aged) 50-weeks-of-age. After the 8-week feeding, gallstone prevalence, gallbladder size, biliary lipid secretion rate, and HMG-CoA reductase activity were significantly greater but cholesterol 7alpha-hydroxylase activity was lower in C57L mice of both genders compared with AKR mice. Increasing age augmented biliary secretion and intestinal absorption of cholesterol, reduced hepatic synthesis and biliary secretion of bile salts, and decreased gallbladder contractility, all of which increased susceptibility to cholesterol cholelithiasis in C57L mice. We conclude that aging per se is an independent risk factor for cholesterol gallstone formation. Because aging increases significantly biliary cholesterol hypersecretion and gallstone prevalence in C57L mice carrying Lith genes, it is highly like that Longevity (aging) genes can enhance lithogenesis of Lith (gallstone) genes.     

Phosphorylation and subcellular localization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger isoform 3 (NHE<Subscript>3</Subscript>) are associated with altered gallbladder absorptive function after formation of cholesterol gallstones

Na⁺/H⁺ exchanger isoform 3 (NHE₃) dysfunction is thought to contribute to the altered gallbladder absorption that occurs in cholesterol gallstone disease, but the mechanism is unknown. The current study was undertaken to examine the expression, phosphorylation, and subcellular localization of NHE₃ in gallbladder epithelium cells (GBECs) of male C57BL/6 mice on a control or lithogenic diet. Thirty-six 8-week-old male C57BL/6 mice were randomly assigned to receive a high cholesterol diet or a regular diet for 8 weeks. Gallstone formation was recorded. Gallbladder bile cholesterol, phospholipid, and total bile acids were examined. RT-PCR was used to measure NHE₃ mRNA expression. NHE₃ protein expression and subcellular localization were examined by Western blotting and immunofluorescence microscopy, respectively. Gallstones were formed in all mice fed the lithogenic diet. Despite higher NHE₃ mRNA expression in gallbladders of the mice on the lithogenic diet than in those on the control diet, there was no significant difference in expression of total NHE₃ protein. However, a higher level of NHE₃ phosphorylated at serine-552 (P-NHE₃) was seen on the lithogenic diet. In immunofluorescence studies, NHE₃ protein was expressed both on the apical membrane and in the cytoplasm of mouse GBEC. This pattern of subcellular distribution of NHE₃ strongly corroborates an exchanger trafficking mechanism in NHE₃ activity regulation in mouse GBEC. We conclude that increased phosphorylation of NHE₃ following gallstone formation leads to turnover of the exchanger, resulting in decreased gallbladder concentrating function.     

Helicobacter pylori and cholesterol gallstone formation in C57L/J mice: a prospective study

Recently, we demonstrated that cholesterol gallstone-prone C57L/J mice rarely develop gallstones unless they are infected with certain cholelithogenic enterohepatic Helicobacter species. Because the common gastric pathogen H. pylori has been identified in the hepatobiliary tree of cholesterol gallstone patients, we wanted to ascertain if H. pylori is cholelithogenic, by prospectively studying C57L infected mice fed a lithogenic diet. Weanling, Helicobacter spp.-free male C57L mice were either infected with H. pylori SS1 or sham dosed. Mice were then fed a lithogenic diet (1.0% cholesterol, 0.5% cholic acid, and 15% dairy triglycerides) for 8 wk. At 16 wk of age, mice were euthanatized, the biliary phenotype was analyzed microscopically, and tissues were analyzed histopathologically. H. pylori infection did not promote cholesterol monohydrate crystal formation (20% vs. 10%), sandy stone formation (0% for both), or true gallstone formation (20%) compared with uninfected mice fed the lithogenic diet (10%). Additionally, H. pylori failed to stimulate mucin gel accumulation in the gallbladder or alter gallbladder size compared with uninfected animals. H. pylori-infected C57L mice developed moderate to severe gastritis by 12 wk, and the lithogenic diet itself produced lesions in the forestomach, which were exacerbated by the infection. We conclude that H. pylori infection does not play any role in murine cholesterol gallstone formation. Nonetheless, the C57L mouse develops severe lesions of both the glandular and nonglandular stomach in response to H. pylori infection and the lithogenic diet, respectively.     

Targeted disruption of the murine mucin gene 1 decreases susceptibility to cholesterol gallstone formation

Gallbladder mucins play a critical role in the pathogenesis of cholesterol gallstones because of their ability to bind biliary lipids and accelerate cholesterol crystallization. Mucin secretion and accumulation in the gallbladder is determined by multiple mucin genes. To study whether mucin gene 1 (Muc1) influences susceptibility to cholesterol cholelithiasis, we investigated male Muc1-deficient (Muc1(-/-)) and wild-type mice fed a lithogenic diet containing 1% cholesterol and 0.5% cholic acid for 56 days. Gene expression of the gallbladder Muc1 and Muc5ac was significantly reduced in Muc1(-/-) mice in response to the lithogenic diet. Muc3 and Muc4 levels were upregulated and were similar between Muc1(-/-) and wild-type mice. Little or no Muc2 and Muc5b mRNAs were detected. Muc1(-/-) mice displayed significant decreases in total mucin secretion and accumulation in the gallbladder as well as retardation of crystallization, growth, and agglomeration of cholesterol monohydrate crystals. At 56 days of feeding, gallstone prevalence was decreased by 40% in Muc1(-/-) mice. However, cholesterol saturation indices of gallbladder bile, hepatic secretion of biliary lipids, and gallbladder size were comparable in Muc1(-/-) and wild-type mice. We conclude that decreased gallstone formation in mice with disrupted Muc1 gene results from reduced mucin secretion and accumulation in the gallbladder.     

Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCK-deficient mice

We investigated the effect of gallbladder hypomotility on cholesterol crystallization and growth during the early stage of gallstone formation in CCK knockout mice. Contrary to wild-type mice, fasting gallbladder volumes were enlarged and the response of gallbladder emptying to a high-fat meal was impaired in knockout mice on chow or the lithogenic diet. In the lithogenic state, large amounts of mucin gel and liquid crystals as well as arc-like and tubular crystals formed first, followed by rapid formation of classic parallelogram-shaped cholesterol monohydrate crystals in knockout mice. Furthermore, three patterns of crystal growth habits were observed: proportional enlargement, spiral dislocation growth, and twin crystal growth, all enlarging solid cholesterol crystals. At day 15 on the lithogenic diet, 75% of knockout mice formed gallstones. However, wild-type mice formed very little mucin gel, liquid, and solid crystals, and gallstones were not observed. We conclude that lack of CCK induces gallbladder hypomotility that prolongs the residence time of excess cholesterol in the gallbladder, leading to rapid crystallization and precipitation of solid cholesterol crystals. Moreover, during the early stage of gallstone formation, there are two pathways of liquid and polymorph anhydrous crystals evolving to monohydrate crystals and three modes for cholesterol crystal growth.     

Defective dietary fat processing in transgenic mice lacking aquaporin-1 water channels

Immunocytochemistry showed expression of aquaporin-1 (AQP1) water channels at sites involved in dietary fat processing, including intrahepatic cholangiocytes, gallbladder, pancreatic microvascular endothelium, and intestinal lacteals. To determine whether AQP1 has a role in dietary fat digestion and/or absorption, mice were placed on a diet that contained 50% fat. Whereas wild-type mice (3-3.5 wk of age, 10-12 g) gained 49 +/- 5% (SE, n = 50) body weight in 8 days, and heterozygous mice gained 46 +/- 4%, AQP1 null mice gained only 4 +/- 3%; weights became similar after return to a 6% fat diet after 6 days. The null mice on a high-fat diet acquired an oily appearance, developed steatorrhea with increased stool triglyceride content, and manifested serum hypotriglyceridemia. Supplementation of the high-fat diet with pancreatic enzymes partially corrected the decreased weight gain in null mice. Absorption of [(14)C]oleic acid from small intestine was not affected by AQP1 deletion, as determined by blood radioactivity after duodenal infusion. Lipase activity in feces and small intestine was remarkably greater in AQP1 null than wild-type mice on low- and high-fat diets. Fluid collections done in older mice (that are less sensitive to a high-fat diet) by ductal cannulation showed threefold increased pancreatic fluid flow in response to secretin/cholecystokinin, but volumes, pH, and amylase activities were affected little by AQP1 deletion, nor were bile flow rates and bile salt concentrations. Together, these results establish a dietary fat misprocessing defect in AQP1 null mice.     

Aquaporin deletion in mice reduces intraocular pressure and aqueous fluid production

Aquaporin (AQP) water channels are expressed in the eye at sites of aqueous fluid production and outflow: AQP1 and AQP4 in nonpigmented ciliary epithelium, and AQP1 in trabecular meshwork endothelium. Novel methods were developed to compare aqueous fluid dynamics in wild-type mice versus mice lacking AQP1 and/or AQP4. Aqueous fluid production was measured by in vivo confocal microscopy after transcorneal iontophoretic introduction of fluorescein. Intraocular pressure (IOP), outflow, and anterior chamber compliance were determined from pressure measurements in response to fluid infusions using micropipettes. Aqueous fluid volume and [Cl(-)] were assayed in samples withdrawn by micropipettes. In wild-type mice (CD1 genetic background, age 4-6 wk), IOP was 16.0 +/- 0.4 mmHg (SE), aqueous fluid volume 7.2 +/- 0.3 microl, fluid production 3.6 +/- 0.2 microl/h, fluid outflow 0.36 +/- 0.06 microl/h/mmHg, and compliance 0.036 +/- 0.006 microl/mmHg. IOP was significantly decreased by up to 1.8 mmHg (P < 0.002) and fluid production by up to 0.9 microl/h in age/litter-matched mice lacking AQP1 and/or AQP4 (outbred CD1 and inbred C57/bl6 genetic backgrounds). However, AQP deletion did not significantly affect outflow, [Cl(-)], volume, or compliance. These results provide evidence for the involvement of AQPs in intraocular pressure regulation by facilitating aqueous fluid secretion across the ciliary epithelium. AQP inhibition may thus provide a novel approach for the treatment of elevated IOP.     

High cholesterol absorption efficiency and rapid biliary secretion of chylomicron remnant cholesterol enhance cholelithogenesis in gallstone-susceptible mice

The study of chylomicron pathway through which it exerts its metabolic effects on biliary cholesterol secretion is crucial for understanding how high dietary cholesterol influences cholelithogenesis. We explored a relationship between cholesterol absorption efficiency and gallstone prevalence in 15 strains of inbred male mice and the metabolic fate of chylomicron and chylomicron remnant cholesterol in gallstone-susceptible C57L and gallstone-resistant AKR mice. Our results show a positive and significant (P<0.0001, r=0.87) correlation between percent cholesterol absorption and gallstone prevalence rates. Compared with AKR mice, C57L mice displayed significantly greater absorption of cholesterol from the small intestine, more rapid plasma clearance of chylomicrons and chylomicron remnants, higher activities of lipoprotein lipase and hepatic lipase, greater hepatic uptake of chylomicron remnants, and faster secretion of chylomicron remnant cholesterol from plasma into bile. All of these increased susceptibility to cholesterol gallstone formation in C57L mice. We conclude that genetic variations in cholesterol absorption efficiency are associated with cholesterol gallstone formation in inbred mice and cholesterol absorbed from the intestine provides an important source for biliary hypersecretion. Differential metabolism of the chylomicron remnant cholesterol between C57L and AKR mice clearly plays a crucial role in the formation of lithogenic bile and gallstones.     

Restoration of gallstone susceptibility by leptin in C57BL/6J ob/ob mice

The absence of leptin due to the ob mutation leads to obesity and confers resistance to diet-induced cholesterol gallstone formation in otherwise susceptible C57BL/6J mice. To investigate contributions of obesity and leptin to gallstone susceptibility, C57BL/6J ob/ob mice were treated daily with i.p. saline or recombinant murine leptin at low (1 microgram/g bw) or high (10 microgram/g bw) doses and were pair-fed a lithogenic diet (15% dairy fat, 1.25% cholesterol, 0.5% cholic acid). Weight loss in ob/ob mice increased in proportion to leptin dose, indicating that the lithogenic diet did not impair leptin sensitivity. In a dose-dependent manner, leptin promoted cholesterol crystallization and gallstone formation, which did not occur in saline-treated mice. Notwithstanding, leptin decreased biliary lipid secretion rates without enriching cholesterol in bile. Leptin did not affect bile salt hydrophobicity, but did increase the biliary content of the most abundant molecular species of phosphatidylcholine, 16:0-18:2. Treatment with leptin down-regulated 3-hydroxy-3-methylglutaryl CoA reductase and prevented cholesterol from accumulating in liver. Consistent with increased hepatic clearance, leptin decreased plasma HDL cholesterol concentrations. This was accommodated in liver without up-regulation of cholesterol 7alpha-hydroxylase or Acat. These data suggest that despite the lithogenic diet, endogenous sources constitute a significant proportion of biliary cholesterol during leptin-induced weight loss. Kinetic factors related to cholesterol nucleation, gallbladder contractility, or mucin secretion may have accounted for leptin-induced gallstone formation.     

Genetic factors at the enterocyte level account for variations in intestinal cholesterol absorption efficiency among inbred strains of mice.

Interindividual and interstrain variations in cholesterol absorption efficiency occur in humans and animals. We investigated physiological biliary and small intestinal factors that might determine variations in cholesterol absorption efficiency among inbred mouse strains. We found that there were significant differences in cholesterol absorption efficiency measured by plasma, fecal, and lymphatic methods: <25% in AKR/J, C3H/J, and A/J strains; 25-30% in SJL/J, DBA/2J, BALB/cJ, SWR/J, and SM/J strains; and 31-40% in C57L/J, C57BL/6J, FVB/J, and 129/SvJ strains. In (AKRxC57L)F1 mice, the cholesterol absorption efficiency (31 +/- 6%) mimicked that of the C57L parent (37 +/- 5%) and was significantly higher than in AKR mice (24 +/- 4%). Although biliary bile salt compositions and small intestinal transit times were similar, C57L mice displayed significantly greater bile salt secretion rates and pool sizes than AKR mice. In examining lymphatic cholesterol transport in the setting of a chronic biliary fistula, C57L mice displayed significantly higher cholesterol absorption rates compared with AKR mice. Because biliary and intestinal transit factors were accounted for, we conclude that genetic variations at the enterocyte level determine differences in murine cholesterol absorption efficiency, with high cholesterol absorption likely to be a dominant trait. This study provides baseline information for identifying candidate genes that regulate intestinal cholesterol absorption at the cellular level.     

Aquaporin-5 dependent fluid secretion in airway submucosal glands

Fluid and macromolecule secretion by submucosal glands in mammalian airways is believed to play an important role in airway defense and surface liquid homeostasis and in the pathogenesis of cystic fibrosis. Immunocytochemistry revealed strong expression of aquaporin water channel AQP5 at the luminal membrane of serous epithelial cells in submucosal glands throughout the mouse nasopharynx and upper airways and AQP4 at the contralateral basolateral membrane in some glands. Novel methods were applied to measure secretion rates and composition of gland fluid in wild type mice and knockout mice lacking AQP4 or AQP5. In mice breathing through a tracheotomy, total gland fluid output was measured from the dilution of a volume marker present in the fluid-filled nasopharynx and upper trachea. Pilocarpine-stimulated fluid secretion was 4.3 +/- 0.4 microl/min in wild type mice, 4.9 +/- 0.9 microl/min in AQP4 null mice, and 1.9 +/- 0.3 microl/min in AQP5 null mice (p < 0.001). Similar results were obtained when secreted fluid was collected in the oil-filled nasopharyngeal cavity. Real-time video imaging of fluid droplets secreted from individual submucosal glands near the larynx in living mice showed a 57 +/- 4% reduced fluid secretion rate in AQP5 null mice. Analysis of secreted fluid showed a 2.3 +/- 0.2-fold increase in total protein in AQP5 null mice and a smaller increase in [Cl(-)], suggesting intact protein and salt secretion across a relatively water impermeable epithelial barrier. Submucosal gland morphology and density did not differ significantly in wild type versus AQP5 null mice. These results indicate that AQP5 facilitates fluid secretion in submucosal glands and that the luminal membrane of gland epithelial cells is the rate-limiting barrier to water movement. Modulation of gland AQP5 expression or function might provide a novel approach to treat hyperviscous gland secretions in cystic fibrosis and excessive fluid secretions in infectious or allergic bronchitis/rhinitis.     

Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: integrated activities of hepatic lipid regulatory enzymes.

There is no consensus whether hepatic lipid regulatory enzymes play primary or secondary roles in cholesterol cholelithiasis. We have used inbred mice with Lith genes that determine cholesterol gallstone susceptibility to evaluate the question. We studied activities of regulatory enzymes in cholesterol biosynthesis (HMG-CoA reductase), cholesterol esterification (acyl-CoA:cholesterol acyltransferase) and the "neutral" (cholesterol 7alpha-hydroxylase) and "acidic" (sterol 27-hydroxylase) pathways of bile salt synthesis in strains C57L/J and SWR/J as well as recombinant inbred (AKXL-29) mice, all of which have susceptible Lith alleles, and compared them to AKR/J mice with resistant Lith alleles. We determined hepatic enzyme activities of male mice before and at frequent intervals during feeding a lithogenic diet (15% dairy fat, 1% cholesterol, 0.5% cholic acid) for 12 weeks. Basal activities on chow show significant genetic variations for HMG-CoA reductase, sterol 27-hydroxylase, and acyl-CoA: cholesterol acyltranferase, but not for cholesterol 7alpha-hydroxylase. In response to the lithogenic diet, activities of the regulatory enzymes in the two bile salt synthetic pathways are coordinately down-regulated and correlate inversely with prevalence rates of cholesterol crystals and gallstones. Compared with gallstone-resistant mice, significantly higher HMG-CoA reductase activities together with lower activities of both bile salt synthetic enzymes are hallmarks of the enzymatic phenotype in mice with susceptible Lith alleles. The most parsimonious explanation for the multiple enzymatic alterations is that the primary Lith phenotype induces secondary events to increase availability of cholesterol to supply the sterol to the hepatocyte canalicular membrane for hypersecretion into bile.     

Disruption of the sterol carrier protein 2 gene in mice impairs biliary lipid and hepatic cholesterol metabolism.

Hepatic up-regulation of sterol carrier protein 2 (Scp2) in mice promotes hypersecretion of cholesterol into bile and gallstone formation in response to a lithogenic diet. We hypothesized that Scp2 deficiency may alter biliary lipid secretion and hepatic cholesterol metabolism. Male gallstone-susceptible C57BL/6 and C57BL/6(Scp2(-/-)) knockout mice were fed a standard chow or lithogenic diet. Hepatic biles were collected to determine biliary lipid secretion rates, bile flow, and bile salt pool size. Plasma lipoprotein distribution was investigated, and gene expression of cytosolic lipid-binding proteins, lipoprotein receptors, hepatic regulatory enzymes, and intestinal cholesterol absorption was measured. Compared with chow-fed wild-type animals, C57BL/6(Scp2(-/-)) mice had higher bile flow and lower bile salt secretion rates, decreased hepatic apolipoprotein expression, increased hepatic cholesterol synthesis, and up-regulation of liver fatty acid-binding protein. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in C57BL/6(Scp2(-/-)) mice. When C57BL/6(Scp2(-/-)) mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress cholesterol synthesis and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Scp2 deficiency did not prevent gallstone formation and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein. These results support a role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution.     

Hepatic deletion of SIRT1 decreases hepatocyte nuclear factor 1α/farnesoid X receptor signaling and induces formation of cholesterol gallstones in mice

SIRT1, a highly conserved NAD(+)-dependent protein deacetylase, is a key metabolic sensor that directly links nutrient signals to animal metabolic homeostasis. Although SIRT1 has been implicated in a number of hepatic metabolic processes, the mechanisms by which hepatic SIRT1 modulates bile acid metabolism are still not well understood. Here we report that deletion of hepatic SIRT1 reduces the expression of farnesoid X receptor (FXR), a nuclear receptor that regulates bile acid homeostasis. We provide evidence that SIRT1 regulates the expression of FXR through hepatocyte nuclear factor 1α (HNF1α). SIRT1 deficiency in hepatocytes leads to decreased binding of HNF1α to the FXR promoter. Furthermore, we show that hepatocyte-specific deletion of SIRT1 leads to derangements in bile acid metabolism, predisposing the mice to development of cholesterol gallstones on a lithogenic diet. Taken together, our findings indicate that SIRT1 plays a vital role in the regulation of hepatic bile acid homeostasis through the HNF1α/FXR signaling pathway.     

Physiological roles of AQP7 in the kidney: Lessons from AQP7 knockout mice

The aquaporin7 (AQP7) water channel is known to be a member of the aquaglyceroporins, which allow the rapid transport of glycerol and water. AQP7 is abundantly present at the apical membrane of the proximal straight tubules in the kidney. In this paper, we review the physiological functions of AQP7 in the kidney. To investigate this, we generated AQP7 knockout mice. The water permeability of the proximal straight tubule brush border membrane measured by the stopped flow method was reduced in AQP7 knockout mice compared to wild-type mice (AQP7, 18.0+/-0.4 x 10(-3 )cm/s vs. wild-type, 20.0+/-0.3 x 10(-3) cm/s). Although AQP7 solo knockout mice did not show a urinary concentrating defect, AQP1/AQP7 double knockout mice showed reduced urinary concentrating ability compared to AQP1 solo knockout mice, indicating that the contribution of AQP7 to water reabsorption in the proximal straight tubules is physiologically substantial. On the other hand, AQP7 knockout mice showed marked glycerol in their urine (AQP7, 1.7+/-0.34 mg/ml vs. wild-type, 0.005+/-0.002 mg/ml). This finding identified a novel pathway of glycerol reabsorption that occurs in the proximal straight tubules. In two mouse models of proximal straight tubule injury, the cisplatin-induced acute renal failure (ARF) model and the ischemic-reperfusion ARF model, an increase of urine glycerol was observed (pre-treatment, 0.007+/-0.005 mg/ml; cisplatin, 0.063+/-0.043 mg/ml; ischemia, 0.076+/-0.02 mg/ml), suggesting that urine glycerol could be used as a new biomarker for detecting proximal straight tubule injury.