Comprehensive analysis of mechanism underlying hypouricemic effect of glucosyl hesperidin

Hyperuricemia is caused by hepatic overproduction of uric acid and/or underexcretion of urate from the kidneys and small intestine. Although increased intake of citrus fruits, a fructose-rich food, is associated with increased risk of gout in humans, hesperidin, a flavonoid naturally present in citrus fruits, reportedly reduces serum uric acid (SUA) levels by inhibiting xanthine oxidase (XOD) activity in rats. However, the effects of hesperidin on renal and intestinal urate excretion were previously unknown. In this study, we used glucosyl hesperidin (GH), which has greater bioavailability than hesperidin, to clarify comprehensive mechanisms underlying the hypouricemic effects of hesperidin in vivo. GH dose-dependently decreased SUA levels in mice with hyperuricemia induced by potassium oxonate and a fructose-rich diet, and inhibited XOD activity in the liver. GH decreased renal urate excretion without changes in kidney URAT1, ABCG2 or GLUT9 expressions, suggesting that reducing uric acid pool size by inhibiting XOD decreased renal urate excretion. We also found that GH had no effect on intestinal urate excretion or protein expression of ABCG2. Therefore, we concluded that GH exhibits a hypouricemic effect by inhibiting XOD activity in the liver without increasing renal or intestinal urate excretion. Of note, this is the first study to elucidate the effect of a flavonoid on intestinal urate excretion using a mice model, whose findings should prove useful in future food science research in the area of urate metabolism. Taking these findings together, GH may be useful for preventing hyperuricemia, especially in people with the overproduction type.     

ABCG2 Dysfunction Increases Serum Uric Acid by Decreased Intestinal Urate Excretion

ATP-binding cassette transporter G2 (ABCG2), also known as breast cancer resistance protein (BCRP), is identified as a high-capacity urate exporter and its dysfunction has an association with serum uric acid (SUA) levels and gout/hyperuricemia risk. However, pathophysiologically important pathway(s) responsible for the ABCG2-mediated urate excretion were unknown. In this study, we investigated how ABCG2 dysfunction affected the urate excretion pathways. First, we revealed that mouse Abcg2 mediates urate transport using the membrane vesicle system. The export process by mouse Abcg2 was ATP-dependent and not saturable under the physiological concentration of urate. Then, we characterized the excretion of urate into urine, bile, and intestinal lumen using in vivo mouse model. SUA of Abcg2-knockout mice was significantly higher than that of control mice. Under this condition, the renal urate excretion was increased in Abcg2-knockout mice, whereas the urate excretion from the intestine was decreased to less than a half. Biliary urate excretion showed no significant difference regardless of Abcg2 genotype. From these results, we estimated the relative contribution of each pathway to total urate excretion; in wild-type mice, the renal excretion pathway contributes approximately two-thirds, the intestinal excretion pathway contributes one-third of the total urate excretion, and the urate excretion into bile is minor. Decreased intestinal excretion could account for the increased SUA of Abcg2-knockout mice. Thus, ABCG2 is suggested to have an important role in extra-renal urate excretion, especially in intestinal excretion. Accordingly, increased SUA in patients with ABCG2 dysfunction could be explained by the decreased excretion of urate from the intestine.     

Regulation of urinary thromboxane B2 in man: Influence of urinary flow rate and tubular transport

Thromboxane B₂ (TxB) is excreted in human urine, but the mechanism of renal excretion and the quantitative relationship of urinary TxB to the active parent compound, thromboxane A₂, of renal or extrarenal origin is not established. To determine the effects of vasoactive hormones, uricosuric agents and urinary flow rate on TxB excretion, urinary TxB was measured by radioimmunoassay and mass spectrometry, and renal metabolism of blood TxB was determined by radiochromatography of urine after i.v. [³H]-TxB infusions. Basal TxB was 6.7 ± 1.1 ng/h during an oral water load, and TxB fell with s.q. antidiuretic hormone (to 3.4 ± 0.4 ng/h, P<0.01) and with fluid restriction (to 2.6 ± 0.5 ng/hr, P=0.001) in parallel with urinary volume. Urinary excretion of unmetabolized [³H]-TxB also fell (by 56%) with fluid restriction, implicating altered metabolism rather than synthesis as the mechanism of the urinary flow effect. Angiotensin II infusions slightly reduced both TxB and urine volume, consistent with a flow effect. In contrast, probenecid did not alter urine volume, but increased urinary uric acid (by 244%), TxB (from 5.6 ± 0.9 to 11.1 ± 2.9 ng/h) and urinary excretion of blood [³H]-TxB (by 243%) by similar amounts (all P<0.05), suggesting that TxB is actively reabsorbed in the proximal tubule, similarly to uric acid. Thus, urinary excretion of TxB of renal and extrarenal origin is regulated by proximal and distal tubule factors.     

Selectively reduced expression of thick ascending limb Tamm-Horsfall protein in hypothyroid kidneys

Hypothyroidism causes major changes in the kidney by affecting its growth, structure, function, and biosynthesis of its specific gene products. As a consequence, the nephron shows a selective hypotrophy in the medullary portion of the thick ascending limb (TAL). Contrastingly, we have shown earlier that the abundance of the major ion transporter of the TAL, the furosemide-sensitive Na-K-2Cl cotransporter (NKCC2) was increased when related to kidney function in the hypothyroid organism. Synthesis of Tamm-Horsfall protein (THP), the most abundant urinary protein produced in the TAL, has been suggested to interfere with TAL function. We therefore studied the localization and synthesis rate of THP in the hypothyroid kidney. We used rats chronically treated with methimazole. Kidneys were processed for western blotting and histochemical evaluation. Morphologically the hypothyroid TAL displayed a selectively reduced epithelial cell height of its medullary portion. This was paralleled by decreased THP immunostaining and mRNA abundance. Western blotting indicated a 40% reduction in renal THP content (P<0.005), and daily urinary THP excretion was 68% lower than in controls (P<0.05). T3 substitution restored these parameters. To further confirm that changes were specific for THP and not merely the consequence of reduced kidney growth, the abundance of barttin, another distal tubular protein related to chloride transport, was tested as well. Barttin was increased by 43% in the hypothyroid rats. Together with our previous results showing increased NKCC2 expression in hypothyroidism, these results demonstrate a selective decrease in medullary THP synthesis. We suggest a potential involvement of THP in the renal functional changes associated with hypothyroidism.     

Metabolism and excretion of exogenous adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate. Studies in the isolated perfused rat kidney and in the intact rat.

Isolated rat kidneys were perfused with a recirculating medium containing exogenous adenosine 3':5'-monophosphate (cyclic AMP) or guanosine 3':5'-monophosphate (cyclic GMP) at an initial concentration of 0.1 mM. Both cyclic nucleotides were rapidly removed from the perfusate. Urinary excretion accounted for about 20% and 40% of the respective cyclic AMP and cyclic GMP lost from the perfusate. The metabolism of the cyclic nucleotides was studied by 14C-labeled cyclic nucleotides in the perfusate. During 60 min, 30% of added cyclic [14C]AMP was metabolized to renal [14C]adenine nucleotides (ATP, ADP, and AMP) and 30% to perfusate [14C]uric acid. Similarly, 20% of cyclic[14C]GMP was metabolized to renal [14C]guanine nucleotides (GTP, GDP, and GMP) and 30% to perfusate [14C]uric acid. Urine contained principally unchanged 14C-labeled cyclic nucleotide. Addition of 0.1 mM cyclic AMP to the perfusate elevated the renal ATP and ADP contents 2-fold. Addition of 0.1 mM of either cyclic AMP or cyclic GMP to the perfusate also elevated the renal production of uric acid 2- to 3-fold. The production and distribution of metabolites of exogenous cyclic nucleotides were also studied in the intact rat. Within 60 min after injection, 3.3 mumol of either 14C-labeled cyclic AMP or cyclic GMP was cleared from the plasma. Kidney cortex and liver were the principal tissues for 14C accumulation. Urinary excretion accounted for about 20 and 45% of the cyclic [14C]AMP and cyclic [14C]GMP lost from the plasma, respectively. The 14C found in the kidney and liver was present almost entirely as the respective purine mono-, di-, and trinucleotides. The other principal metabolite was [14C]allantoin, found in the urine and, to a lesser extent, the liver. The urine contained mostly unchanged 14C-labeled cyclic nucleotide. Unlike the findings with the perfused kidney, [14C]uric acid was not a significant metabolite of the 14C-labeled cyclic nucleotides in these in vivo experiments.     

Idiopathic calcium nephrolithiasis. 1. Differences in urine crystalloids,urine saturation with brushite and urine inhibitors of calcification between persons with and persons without recurrent kidney stone formation.

The propensity of urine to promote calcium stone formation was compared in 64 patients with recurrent idiopathic calcium nephrolithiasis and 30 healthy individuals without such a history. The rates of excretion of urine crystalloids, the urine saturation with brushite (CaHPO4-2H2O), the ability of the urine to calcify collagen in vitro, and the concentration of urine inhibitors of collagen calcification were measured. The patients had a reduced urine citrate excretion rate in addition to an increased urine calcium excretion rate, while the rates for urine magnesium, phosphate, uric acid and oxalate were not significantly different in the two groups of subjects. The urine concentration of magnesium, phosphate and uric acid was decreased in the patients because of the higher urine volume. The urine creatinine excretion rate correlated with the rates of excretion of urine calcium, magnesium, phosphate, uric acid and oxalate in both groups, which suggested that increased lean body mass, possibly associated with greater food intake, may be an important determinant of crystalloid excretion. The urine of the patients was significantly more saturated with brushite than the urine of the control subjects and resulted in greater collagen calcification when incubated in vitro. The urine concentration of inhibitors of collagen calcification, however, was not significantly different in the two groups. Thus, the urine of patients with recurrent idiopathic calcium nephrolithiasis is more highly saturated with brushite, largely as a result of an increased urine calcium excretion rate, and contains a lower concentration of magnesium and citrate, substances that tend to prevent the precipitation and growth of crystals in urine.     

The Treatment of Gout and Disorders of Uric Acid Metabolism with Allopurinol

Allopurinol (4-hydroxypyrazolo (3,4-d)-pyrimidine) is a potent xanthine oxidase inhibitor which inhibits the oxidation of naturally occurring oxypurines, thus decreasing uric acid formation. The clinical and metabolic effects of this agent were studied in 80 subjects with primary and secondary gout and other disorders of uric acid metabolism. Allopurinol has been universally successful in lowering the serum uric acid concentration and uric acid excretion to normal levels, while not significantly affecting the clearance of urate or other aspects of renal function. Oxypurine excretion increased concomitantly with the fall in urine uric acid. The agent is particularly valuable in the management of problems of gout with azotemia, acute uric acid nephropathy and uric acid urolithiasis. The minor side effects, clinical indications and theoretical complications are discussed.     

Tamm-Horsfall protein excretion during chronic alterations in urinary concentration and protein intake in the rat.

Tamm-Horsfall protein (THP), a normal constituent of mammalian urine, has been determined in rat urine under various conditions in an attempt to elucidate the physiological role of this glycoprotein. Experiments were designed to assess whether THP production is related to the process of urine concentration or to the transport activity of the thick ascending limb of the loop of Henle (TAL), the nephron segment where it is produced. For this purpose, THP excretion was measured, by radioimmunoassay, in adult male rats under 4 different conditions induced by the following chronic treatments: (1) furosemide (12 mg/day in osmotic minipumps); (2) increased water intake; (3) antidiuretic hormone (ADH) infusion (50 ng DDAVP/day in osmotic minipumps) in rats of the Brattleboro strain with hereditary hypothalamic diabetes insipidus; (4) high-protein (32% casein) versus low-protein diet (10% casein). Each experiment included 6 experimental and 6 control rats. After treatment for 1-3 weeks, 24-h urines were collected for determination of urine flow rate, osmolality, and creatinine and THP concentrations. No significant changes in THP excretion were observed in experiments (1) and (2) despite 5- to 7-fold-differences in urine flow rate. Antidiuretic hormone treatment in (3) slightly lowered THP excretion (287 +/- 53 vs. 367 +/- 41 micrograms/day per 100 g body weight; p less than 0.005), whereas high-protein diet, in experiment (4), led to a 50% increase in THP excretion (446 +/- 57 vs. 304 +/- 79 micrograms/day per 100 g body weight; p less than 0.001). Expressing THP excretion relative to that of creatine did not change these findings. These results show (1) that chronically established changes in the level of diuresis, chronic furosemide-induced blockade of the Na,K,Cl-cotransporter or the absence of ADH in Brattleboro rats have little or no impact on the level of THP production, and (2) that THP production is independent of the intensity of transport in the TAL, since two conditions which both are known to increase the transport rate of solutes in the TAL (ADH infusion and high-protein diet), resulted in opposite changes in THP excretion. It is concluded that the rate of THP synthesis is neither linked to the process of urine concentration nor to the ion transport activity of the TAL.     

几种天然产物对高尿酸血症大鼠血清尿酸水平的影响初探

目的:探讨几种天然产物对高尿酸血症大鼠血清尿酸水平及尿酸排泄的影响.方法:对wistar大鼠灌胃氧嗪酸钾和酵母膏,制作高尿酸血症大鼠动物模型.灌胃给药褐藻糖胶、柠檬酸钾和东哥阿里提取物,2周后采血并进行代谢实验,检测血清尿酸、尿素氮,24小时尿液体积、pH值、尿酸浓度及总量,分析三种活性物质对机体尿酸水平、尿酸排泄、肾脏功能的影响.结果:三种物质均可显著降低高尿酸血症模型大鼠的血清尿酸水平,其中东哥阿里提取物组的24小时排泄尿酸总量较模型组显著降低,褐藻糖胶对实验大鼠的血清尿素氮水平升高有抑制作用.结论:三种活性物质对高尿酸血症大鼠血清尿酸浓度有降低作用,其中褐藻糖胶对肾脏功能有保护作用,从而保证尿酸的顺利排泄,而东哥阿里在降低血尿酸水平的同时,24小时尿液中排泄的尿酸总量也显著低于模型对照组,其机制可能与抑制尿酸生成有关.     

Conditions for solubilization of Tamm-Horsfall protein/uromodulin in human urine and establishment of a sensitive and accurate enzyme-linked immunosorbent assay (ELISA) method

Tamm-Horsfall protein (THP), also referred to as uromodulin, is a major glycoprotein produced by kidney cells. The level of THP in urine is an indicator of renal function. THP levels are difficult to quantify accurately, because the protein forms aggregates in the pH and ion concentrations found in urine. Here, we describe conditions by which THP can be solubilized and kept soluble during measurement using Triton X-100, EDTA, and alkaline pH. Establishing conditions for precise quantification of THP by enzyme-linked immunosorbent assay (ELISA) will aid in studying the functions of THP.     

Disturbed ratio of renal 20-HETE/EETs is involved in androgen-induced hypertension in cytochrome P450 4F2 transgenic mice

We have previously established a cytochrome P450 4F2 (CYP4F2) transgenic mouse model. The present study elucidated the molecular foundation of hypertension by androgen-induction in this model. The renal expression of CYP4F2 in transgenic mice was highly expressed and strongly induced with 5α-dihydrotestosterone (DHT) treatment determined by Western blot. DHT also increased the renal arachidonic acid ω-hydroxylation and urinary 20-hydroxyeicosatetraenoic acid (20-HETE) excretion (P<0.01), and furthermore elevated the systolic blood pressure by 10 and 22 mm Hg (P<0.05) in female and castrated male transgenic mice, respectively. HET0016 completely eliminated the androgen-induced effects (P<0.01). Endogenous Cyp4a ω-hydroxylases, evaluated by real-time quantitative PCR, were significantly suppressed in transgenic mice (P<0.05). Importantly, transgenic mice with increased 20-HETE showed decreased epoxyeicosatrienoic acids (EETs) and increased dihydroxyeicosatetraenoic acids determined by liquid chromatography-tandem mass spectrometry, contributing to significantly raised ratio of 20-HETE/EETs in the urine and kidney homogenate (P<0.01). These data demonstrate that the androgen aggravated hypertension possibly through an altered ratio of 20-HETE/EETs in CYP4F2 transgenic mice.     

Role of renal prostaglandins in the fall in urine osmolality after papillary micropuncture

The effect of micropuncture of the renal papilla through an intact ureter on urinary concentrating ability of rats was examined. Micropuncture of the renal papilla caused a fall in urine osmolality in the punctured kidney from 1718 +/- 106 to 1035 +/- 79 mosmol/kg X H2O. In order to investigate the role of renal prostaglandins in this process, PGE2 excretion was measured and found to increase from 63.4 +/- 14.0 to 205.5 +/- 57.1 pg/min. Urine osmolality and PGE2 excretion from the contralateral kidney were not significantly altered. In animals given meclofenamate (2 mg/kg X hr), renal PGE2 excretion was reduced to 22.3 +/- 5.1 pg/min prior to micropuncture and it remained low at 8.9 +/- 1.8 pg/min after papillary micropuncture. Meclofenamate also blocked the fall in urine osmolality caused by micropuncture of the renal papilla, with urine osmolality averaging 1940 +/- 122 before and 1782 +/- 96 mosmol/kg X H2O after the micropuncture. These results indicated that papillary micropuncture through an intact ureter increased renal PGE2 excretion and that a rise in renal production of PGE2 or some other prostanoid is associated with a fall in urine concentrating ability.     

Effect of allyl isothiocyanate on plasma and urinary concentrations of some biochemical entities in the rat.

Allyl isothiocyanate (AITC) is a constituent of several plants of the family Cruciferae that are commonly used as food. This study investigated the effect of feeding AITC to male Sprague-Dawley rats on their plasma glucose and uric acid levels as well as on the urinary concentrations of glucose, 17-ketosteroids (17-KS), creatinine, and uric acid. Other test compounds included were thyroxine (T4) and thiouracil (TU). AITC caused a highly significant (P smaller than or equal to 0.01) depression in the plasma glucose and uric acid levels compared with the control. TU caused a significant depression only of the plasma glucose. T4, on the other hand, significantly increased the levels of both glucose and uric acid. The AITC-treated rats voided twice as much urine as the controls or those receiving TU or injected with T4. The 24-h excretion of glucose, uric acid, and creatinine was significantly (P smaller than or equal to 0.01) higher in animals fed AITC than in those consuming the control diet, while the excretion of 17-KS was significantly lower. Results on an equal urine volume basis showed that differences in the excretion of glucose and creatinine were related to differences in the urine volume. TU significantly depressed excretion of all the compounds but glucose. The effect of T4 on the excretion of 17-KS and uric acid resembled that of AITC and TU, thus showing that these compounds depressed the androgenic function of the animal.     

Beneficial role of naringin, a flavanoid on nickel induced nephrotoxicity in rats

This study was conducted to investigate the beneficial role of naringin on nickel induced nephrotoxicity. Nickel (Ni) (20 mg/kg body weight (b.w.) was administered intraperitoneally (i.p.) for 20 days. Naringin was administered orally (20, 40 and 80 mg/kg b.w.) with i.p. administration of Ni. Ni administration increased the levels of serum urea, uric acid and creatinine with a significant decrease in creatinine clearance and decreased levels of urea, uric acid and creatinine in urine. The levels of lipid peroxidation markers and nickel concentration in blood and kidney were also increased. While, the activities of enzymic and non-enzymic antioxidants were decreased. Treatment with naringin attenuated the alterations in the renal and urine markers, decreasing lipid peroxidation markers, increasing the antioxidant cascade and decreasing the nickel concentration in blood and kidney. All these changes were supported by histopathological observations. These findings demonstrate that naringin exerts a protective effect against nickel toxicity.     

Diabetic kidney disease in FVB/NJ Akita mice: temporal pattern of kidney injury and urinary nephrin excretion

Akita mice are a genetic model of type 1 diabetes. In the present studies, we investigated the phenotype of Akita mice on the FVB/NJ background and examined urinary nephrin excretion as a marker of kidney injury. Male Akita mice were compared with non-diabetic controls for functional and structural characteristics of renal and cardiac disease. Podocyte number and apoptosis as well as urinary nephrin excretion were determined in both groups. Male FVB/NJ Akita mice developed sustained hyperglycemia and albuminuria by 4 and 8 weeks of age, respectively. These abnormalities were accompanied by a significant increase in systolic blood pressure in 10-week old Akita mice, which was associated with functional, structural and molecular characteristics of cardiac hypertrophy. By 20 weeks of age, Akita mice developed a 10-fold increase in albuminuria, renal and glomerular hypertrophy and a decrease in the number of podocytes. Mild-to-moderate glomerular mesangial expansion was observed in Akita mice at 30 weeks of age. In 4-week old Akita mice, the onset of hyperglycemia was accompanied by increased podocyte apoptosis and enhanced excretion of nephrin in urine before the development of albuminuria. Urinary nephrin excretion was also significantly increased in albuminuric Akita mice at 16 and 20 weeks of age and correlated with the albumin excretion rate. These data suggest that: 1. FVB/NJ Akita mice have phenotypic characteristics that may be useful for studying the mechanisms of kidney and cardiac injury in diabetes, and 2. Enhanced urinary nephrin excretion is associated with kidney injury in FVB/NJ Akita mice and is detectable early in the disease process.     

Urea-selective concentrating defect in transgenic mice lacking urea transporter UT-B.

Urea transporter UT-B has been proposed to be the major urea transporter in erythrocytes and kidney-descending vasa recta. The mouse UT-B cDNA was isolated and encodes a 384-amino acid urea-transporting glycoprotein expressed in kidney, spleen, brain, ureter, and urinary bladder. The mouse UT-B gene was analyzed, and UT-B knockout mice were generated by targeted gene deletion of exons 3-6. The survival and growth of UT-B knockout mice were not different from wild-type littermates. Urea permeability was 45-fold lower in erythrocytes from knockout mice than from those in wild-type mice. Daily urine output was 1.5-fold greater in UT-B- deficient mice (p < 0.01), and urine osmolality (U(osm)) was lower (1532 +/- 71 versus 2056 +/- 83 mosM/kg H(2)O, mean +/- S.E., p < 0.001). After 24 h of water deprivation, U(osm) (in mosM/kg H(2)O) was 2403 +/- 38 in UT-B null mice and 3438 +/- 98 in wild-type mice (p < 0.001). Plasma urea concentration (P(urea)) was 30% higher, and urine urea concentration (U(urea)) was 35% lower in knockout mice than in wild-type mice, resulting in a much lower U(urea)/P(urea) ratio (61 +/- 5 versus 124 +/- 9, p < 0.001). Thus, the capacity to concentrate urea in the urine is more severely impaired than the capacity to concentrate other solutes. Together with data showing a disproportionate reduction in the concentration of urea compared with salt in homogenized renal inner medullas of UT-B null mice, these data define a novel "urea-selective" urinary concentrating defect in UT-B null mice. The UT-B null mice generated for these studies should also be useful in establishing the role of facilitated urea transport in extrarenal organs expressing UT-B.     

Idiopathic calcium nephrolithiasis. 2. Differences between hypercalciuric and normocalciuric persons with recurrent kidney stone formation and persons without such a history.

Normocalciuric and hypercalciuric patients with idiopathic recurrent calcium nephrolithiasis were compared with healthy individuals without such a history to examine the factors that predispose normocalciuric patients to stone formation. The urine calcium excretion rate was higher in the normocalciuric patients than in the control subjects (227 v. 183 mg/24 h; P less than 0.01), but the urine calcium concentration was not significantly different. The urine magnesium and citrate excretion rates and concentrations were lower in the normocalciuric patients than in the control subjects (P less than 0.001), while the urine uric acid and oxalate excretion rates and concentrations and the urine saturation with brushite (CaHPO4-2H2O) were not significantly different. These results suggest the importance of slight increases in the urine calcium excretion rate together with decreased urine magnesium and citrate excretion rates in normocalciuric persons with recurrent calcium stone formation. The urine of the hypercalciuric patients was more highly saturated with brushite than the urine of the normocalciuric patients and the control subjects, and the excretion rates of uric acid and oxalate were increased in the hypercalciuric patients. The hypercalciuric patients had a higher urine creatinine excretion rate than the normocalciuric patients and a higher daily urine volume than the control subjects, which suggests that differences in lean body mass or fluid and food intake, or both, may be important determinants of these differences in crystalloid excretion. As in the normocalciuric patients, the urine citrate excretion rate and concentration were decreased in the hypercalciuric patients compared with the control subjects.     

Uromodulin (Tamm-Horsfall glycoprotein/uromucoid) is a phosphatidylinositol-linked membrane protein

Uromodulin, originally identified as an immunosuppressive glycoprotein in the urine of pregnant women, has been previously shown to be identical to human Tamm-Horsfall glycoprotein (THP). THP is synthesized by the kidney and localizes to the renal thick ascending limb and early distal tubule. It is released into the urine in large quantities and thus represents a potential candidate for a protein secreted in a polarized fashion from the apical plasma membrane of epithelial cells in vivo. After introduction of the full-length cDNA encoding uromodulin/THP into HeLa, Caco-2, and Madin-Darby canine kidney cells by transfection, however, the expressed glycoprotein was almost exclusively cell-associated, as determined by immunoprecipitation after radioactive labeling of the cells. By immunofluorescence, THP was localized to the plasma membranes of transfected cells. In transfected cell extracts, THP also remained primarily in the detergent phase in a Triton X-114 partitioning assay, indicating that it has a hydrophobic character, in contrast to its behavior after isolation from human urine. Triton X-114 detergent-associated THP was redistributed to the aqueous phase after treatment of cell extracts with phosphatidylinositol-specific phospholipase C. Treatment of intact transfected HeLa cells with phosphatidylinositol-specific phospholipase C also resulted in the release of THP into the medium, suggesting that it is a glycosylphosphatidylinositol (GPI)-linked membrane protein. Similar to other known GPI-linked proteins, uromodulin/THP contains a stretch of 16 hydrophobic amino acids at its extreme carboxyl terminus which could function as a GPI addition signal and was shown to label with [3H]ethanolamine. The results indicate that THP is a member of this class of lipid-linked membrane proteins and is released into the urine after the loss of its hydrophobic anchor, probably by the action of a phospholipase or protease.     

Reinvestigation of denervation diuresis and natriuresis in conscious dogs.

The function of innervated and denervated kidney was compared in clearance studies with conscious dogs. The animals were prepared for experiments by unilateral renal denervation and surgical division of the bladder to form two hemibladders enabling separate urine collection from two kidneys. The mean urine flow was 6% higher for the denervated kidney (not significant) while mean differences for osmolar clearance (+ 13%), sodium excretion (+21%) and GFT (+5%) were all significant (P less than 0.05). When corrected to 100 ml GFR, sodium excretion was not significantly higher for the denervated kidney. In most experiments higher sodium excretion on the denefvated side was associated with higher GFR. Thus, contrary to some earlier views, a slight increase in the excretory function which follows denervation of the kidney is demonstrable also in conscious undisturbed animals. The data suggest that increased haemodynamics of the denervated kidney are responsible for higher excretion, but do not exclude a contribution of inhibited tubular reabsorption.