UT-A urea transporter protein expressed in liver: upregulation by uremia.

In perfused rat liver, there is phloretin-inhibitable urea efflux, but whether it is mediated by the kidney UT-A urea transporter family is unknown. To determine whether cultured HepG2 cells transport urea, thiourea influx was measured. HepG2 cells had a thiourea influx rate of 1739 +/- 156 nmol/g protein per min; influx was inhibited 46% by phloretin and 32% by thionicotinamide. Western analysis of HepG2 cell lysate using an antibody to UT-A1, UT-A2, and UT-A4 revealed two protein bands: 49 and 36 kD. The same bands were detected in cultured rat hepatocytes, freshly isolated rat hepatocytes, and in liver from rat, mouse, and chimpanzee. Both bands were present when analyzed by native gel electrophoresis, and deglycosylation of rat liver lysate had no effect on either band. Differential centrifugation of rat liver lysate showed that the 49-kD protein is in the membrane fraction and the 36-kD protein is in the cytoplasm. To determine whether the abundance of these UT-A proteins varies in vivo, rats were made uremic by 5/6 nephrectomy. The 49-kD protein was significantly increased 5.5-fold in livers from uremic rats compared to pair-fed control rats. It is concluded that phloretin-inhibitable urea flux in liver may occur via a 49-kD protein that is specifically detected by a UT-A antibody. Uremia increases the abundance of this 49-kD UT-A protein in rat liver in vivo.     

Acidosis, not azotemia, stimulates branched-chain, amino acid catabolism in uremic rats

To investigate branched-chain, amino acid metabolism (BCAA) in muscle in chronic renal failure (CRF), we studied rats with moderately severe uremia (PUN 110 approximately mg/dl) and spontaneous metabolic acidosis (bicarbonate, 19 +/- 1 mEq/liter). Plasma BCAA levels in CRF compared to pair-fed control rats were approximately 15% lower and muscle valine was 93 microM lower (P less than 0.05). BCAA metabolism was measured in incubated epitrochlearis muscles using L-[1-14C]valine or L-[1-14C]leucine in the presence and absence of insulin. BCAA decarboxylation was increased (P less than 0.05) and insulin-stimulated BCAA incorporation into protein was blunted (P less than 0.05) by CRF. Since we have found that metabolic acidosis, by itself, stimulates muscle branched-chain, ketoacid dehydrogenase activity, another group of CRF and control rats was given NaHCO3 which corrected the acidosis, but not the azotemia. BCAA decarboxylation in muscle was reduced in CRF rats given NaHCO3, and this was reflected in increased plasma and muscle BCAA concentrations. We conclude that in CRF, chronic metabolic acidosis stimulates BCAA decarboxylation in skeletal muscle and this could contribute to the reduced intra- and extracellular concentrations of BCAA. Correction of acidosis should be a goal of therapy in CRF, especially when dietary regimens restrict intake of BCAA.     

Effects of aldosterone on the protein expressions and the urea transport activity of UT-A1 and UT-A3

Objective To investigate the effects of protein expressions and the urea transport activity of aldosterone on urea transporter A1 (UT-A1) and urea transporter A3 (UT-A3) in HEK293 cells and Xenopus laevis oocytes. Methods (1) Western Blot was used to investigate the protein expressions of UT-A1 and UT-A3. (2) Cell surface biotinylation was used to investigate the protein expressions of UT-A1 and UT-A3 on the cell surface of Xenopus laevis oocytes. (3) 14C-urea transport experiment was conducted to investigate the transport activity of UT-A1 and UT-A3 in Xenopus laevis oocytes. Results (1) Compared with UT-A1 or UT-A3 high expression groups, the total protein levels of UT-A1 and UT-A3 were all significantly reduced in aldosterone treatment groups (all P＜0.01). (2) Compared with UT-A1 or UT-A3 high expression groups, the levels of protein expression on cell surface were all significantly reduced in aldosterone groups (all P＜0.01). (3) Compared with UT-A1 or UT-A3 high expression groups, 14C-urea transport experiment results showed that aldosterone treatment groups had significantly reduced the urea transporter activity of UT-A1 (1 min: 94.32±9.044 vs 40.68±4.274, P＜0.01, n=6; 3 min: 165.0±4.7 vs 80.3±0.6, P＜0.01, n=6), and UT-A3 (1 min: 204.6±3.1 vs 176.7±9.1, P＜0.05, n=6; 3 min: 371.4±14.9 vs 318.8±12.0, P＜0.05, n=6). Conclusion Aldosterone can directly down-regulate the protein expressions of UT-A1 and UT-A3 in both total protein and cell surface level, which reduces their urea transport activity.     

Utilization of alpha-ketoisocaproate for protein synthesis in uremic rats

We have recently shown that the nutritional efficiency, R, of alpha-ketoisocaproate (KIC) as a substitute for leucine, defined as the ratio of the dose of leucine to the dose of KIC (on a leucine-free diet) for equal growth, can be evaluated isotopically: 14C-KIC and 3H-leucine are administered p.o.; six hours later, 14C/3H in the leucine of whole body protein, divided by 14C/3H in the injectate, gives a value distinguishable from R assessed in the same animals by growth experiments. To see how chronic uremia affects R, 11/12 nephrectomized rats and sham-operated controls were fed a regular diet for 15 days and then given these isotopes p.o. Six hours later, R, measured in whole body protein, and in the protein of brain, heart, muscle, salivary gland, liver, and the kidney remnant was significantly greater than in sham-operated controls. The greatest difference (39%) was seen in liver protein and the smallest difference (19%) in muscle. Thus chronic uremia increases the efficiency, relative to leucine, with which KIC is utilized for protein synthesis in all of these organs and in the body as a whole. Possible explanations are discussed.     

Lipogenesis from ketone bodies in perfused livers from streptozocin-induced diabetic rats

Production of ketone bodies and their contribution to lipogenesis were measured in isolated livers from normal and streptozocin-induced diabetic (STZ-D) rats perfused with tracer amounts of 3H2O and (R)-3-hydroxy[3-14C]butyrate. Diabetes decreased by 80-95% the total rates of fatty acid and 3-beta-hydroxysterol synthesis in perfused livers and livers of live rats. The activity of cytosolic acetoacetyl-CoA synthetase was slightly (17%) decreased in livers from STZ-D rats. The incorporation of ketone bodies into fatty acids and sterols was markedly inhibited in perfused livers from STZ-D rats despite the stimulation of ketogenesis by diabetes and the presence of oleate. Treatment of the rats with insulin before liver perfusion led to a normalization of the rates of ketogenesis and fatty acid synthesis. The rates of sterol synthesis were only partially normalized by insulin treatment. We conclude that in STZ-D, ketosis does not stimulate hepatic lipogenesis via cytosolic activation of acetoacetate.     

Insulin release from column-perifused isolated islets of uremic rats

In order to examine the insulin secretion in chronic renal failure, isolated pancreatic islets either from uremic rats or from control rats were mixed into a short column of Bio-Gel P-2 polyacrylamide beads and perifused. Uremic rats had higher concentrations of blood urea nitrogen, serum creatinine, and immunoreactive insulin and lower concentration of plasma 1,25-(OH)2D3 than control rats. Although the basal insulin release in the presence of 5.0 mM glucose showed no difference between uremic and control rats, the initial insulin release in the presence of 16.2 mM glucose was significantly lower (p less than 0.05) in uremic than in controls rats. The insulin content in islets was not different between both groups. These findings suggest that there might be impairment of the initial insulin secretion without changes of insulin content in pancreatic islets in uremia.     

Reduction of urea generation and muscle protein degradation by adrenalectomy in acutely uremic rats

The effect of adrenalectomy on the enhanced protein degradation in acute uremia was investigated. Therefore, serum urea nitrogen, urea N appearance and Nt-methylhistidine were followed in bilaterally nephrectomized rats. At 48 h after induction of uremia the animals displayed serum urea nitrogen levels of 223 +/- 9.5 mg/dl as compared to 26.0 +/- 1.0 mg/dl in sham-treated rats. This increment was significantly attenuated in acutely uremic, adrenalectomized animals (176 +/- 6.0 mg/dl). When these rats were substituted with corticosterone (5 mg/kg body weight), serum urea nitrogen readily increased to levels of acutely uremic animals with intact adrenal glands (225 +/- 6.0 mg/dl). The net generation of urea, as determined by the urea N appearance, was significantly increased during acute uremia (370 +/- 26 mg/48 h) as compared to SHAM animals (220 +/- 15 mg/48 h). This increment of urea formation could almost be completely reversed by simultaneous adrenalectomy (238 +/- 20 mg/48 h). When these rats were substituted with corticosterone, the urea N appearance rebounded to values quite comparable to acutely uremic rats with intact adrenal glands (363 +/- 30 mg/48 h). To determine whether skeletal muscle proteins might serve as a source for the enhanced protein degradation in acute uremia, plasma levels of Nt-methylhistidine were measured. Bilaterally nephrectomized rats had Nt-methylhistidine values of 9.6 +/- 1.0 micrograms/ml. In acutely uremic rats without adrenal glands, Nt-methylhistidine levels were found to be significantly decreased (6.0 +/- 0.4 micrograms/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

The possible role of c-fos protein in hypothalamus in sleep disturbance in chronic uremic rats.

Sleep disturbance is very common in patients with chronic renal failure, but its mechanism is not clear. The activity of c-fos protein (FOS) in ventrolateral preoptic neurons (VLPO) is associated with the sleep pattern. The purpose of this study was to evaluate the relationship between sleep disturbance and the expression of FOS in VLPO of chronic uremic rats. Chronic uremia was induced by the 5/6 nephrectomized model. The movements of the rats were measured with infrared monitoring during the daytime (8.00-20.00) and nighttime (20.00-8.00). Rats were killed at 10.00 or 16.00 h for the daytime (uremic rats 7, control 8) and at 22.00 h for the nighttime (uremic rats 7, control 9). The expression of FOS in VLPO was examined with the immunohistochemical method. The number of recorded daytime movements in uremic rats was significantly higher than in control rats (458 +/- 185 vs. 222 +/- 41, p < 0.001), but the number of recorded nighttime movements in uremic rats was lower than in control rats (949 +/- 430 vs. 1,618 +/- 261, p < 0.001). In the daytime, the number of FOS immunoreactive cells in uremic rats was lower than in control rats (18.4 +/- 5.3 vs. 42.8 +/- 6.3, p < 0. 001), but there was no difference between two groups in the nighttime (10.8 +/- 8.4 vs. 12.5 +/- 5.1, p = 0.62). There was a strong negative correlation between the number of recorded movements and the number of FOS immunoreactive cells in VLPO (r = -0.700, p < 0.001). This finding suggests that sleep disturbances in chronic uremic rats might be related to the decreased expression of FOS in VLPO.     

可溶性Tie2融合蛋白对尿毒症腹膜透析大鼠血管新生的影响

目的 观察可溶性Tie2融合蛋白(sTie2/Fc)对尿毒症腹膜透析大鼠腹膜血管新生的影响.方法 48只雄性SD大鼠,按随机数字表法分为以下6组:正常对照组、假手术组、尿毒症非腹透组、4.25％腹透组、sTie2/Fc 2.5 μg/kg干预组、sTie2/Fc 5.0 μg/kg干预组.腹透组按照4.25％腹透液30...     

Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4

Urea transport in the kidney is important for the production of concentrated urine and is mediated by a family of transporter proteins, identified from erythropoietic tissue (UT-B) and from kidney (UT-A). Two isoforms of the renal urea transporter (UT-A) have been cloned so far: UT-A1 and UT-A2. We used rapid amplification of cDNA ends to clone two new isoforms of the rat UT-A transporter: UT-A3 and UT-A4. UT-A3 and UT-A4 are 87% homologous. The UT-A3 cDNA encodes a peptide of 460 amino acids, which corresponds to the amino-terminal half of the UT-A1 peptide and is 62% identical to UT-A2. The UT-A4 cDNA encodes a peptide of 466 amino acids, which is 84% identical to UT-A2. Transient transfection of HEK-293 cells with the UT-A3 or UT-A4 cDNA results in phloretin-inhibitable urea uptake, which is increased by forskolin. Thus, both new isoforms encode functional urea transporters that may be vasopressin-regulated. UT-A3 and UT-A4 mRNA are expressed in the renal outer and inner medulla but not in the cortex; unidentified UT-A isoforms similar to UT-A3 may also be expressed in the testis. It is concluded that there are at least four different rat UT-A urea transporters.     

Nicotinamide metabolites accumulate in the tissues of uremic rats.

OBJECTIVE: N-methyl-2-pyridone-5-carboxamide (Met2PY) and N-methyl-4-pyridone-3-carboxamide (Met4PY) are nicotinamide (NA) metabolites described by our group and considered to be uremic toxins. Plasma concentration of Met2PY and Met4PY in end-stage renal disease (ESRD) is several-fold higher in comparison with healthy controls. However, there are no data about the concentrations of these compounds in different tissues. The aim of this study was to establish whether the observed high concentrations of these compounds were also present in different tissues of uremic animals. METHODS: This study was conducted in 10 5/6 nephrectomized male Wistar rats and 10 sham-operated animals. Concentrations of nicotinamide and its end products were measured using high-performance liquid chromatography and mass spectrometry. RESULTS: We observed significant elevations of Met2PY and Met4PY in tissues of uremic rats. The greatest differences between healthy and uremic rats were observed in the liver, lungs, and skeletal muscles. Accumulation in the kidney and heart was not so pronounced, but in the case of Met4PY was statistically significant. CONCLUSIONS: We found that Met2PY and Met4PY accumulate not only in the blood but also in tissues of uremic rats.     

Enhanced gastrointestinal absorption of aluminum in uremic rats

To investigate the possibility of enhanced gastrointestinal absorption of aluminum in uremia, we measured the urinary aluminum excretion of rats following an oral load of 11 mg aluminum. Rats, in which uremia had been established by the remnant kidney model, excreted 1.5 to 2.2-fold higher amounts of aluminum in their urine over a collection period of five days compared with their controls. Within this period of time up to 0.17 +/- 0.08% of the oral dose of aluminum was recovered in the urine of the uremic animals. Serum concentrations of aluminum were significantly elevated five hours after ingestion of aluminum, but this increase was similar in rats with normal or reduced renal function. Uremic rats excreted significantly less aluminum during the first 24 hours after i.v. administration of 15 micrograms aluminum if the data were corrected for the higher baseline excretion rates. The excretion rate showed a negative correlation with the serum creatinine. Selective parathyroidectomy had no effect on the pattern or amount of urinary aluminum excretion after an oral load in either uremic rats or in rats with normal renal function. We conclude that the gastrointestinal absorption of aluminum is increased in uremic rats, and that parathyroid hormone has no detectable effect on the magnitude of aluminum absorption, regardless of the renal function in this model.     

Catabolic effects of ethanol in chronically uremic rats

Both ethanol consumption and uremia are considered to be associated with wasting, malnutrition and debilitation. The present study was designed to investigate as to whether ethanol exerts a stimulatory effect on the catabolic state of renal failure. Rats underwent 5/6-nephrectomy and were fed either with or without ethanol. The degree of uremia was comparable in both groups. Ethanol-fed uremic rats, however, displayed higher serum levels of urea (+ 103%) and glucose (+29%), as compared to uremic animals without alcohol. Subsequently, the urea N appearance was enhanced (+60%) in uremic rats with alcohol as compared to uremic animals without alcohol. In sham rats urea N appearance was also increased (+39%) following ethanol administration in comparison to sham-operated rats without alcohol, albeit to a lesser degree. Urinary Nt-methylhistidine excretion, an indicator of myofibrillar protein breakdown, was enhanced throughout the experiment in uremic rats receiving ethanol. Finally, ethanol caused higher urinary excretion rates of corticosterone in uremic animals as compared to uremic rats without ethanol. There was a significant correlation between urinary corticosterone excretion and both urea N appearance and urinary Nt-methylhistidine excretion. We conclude that ethanol consumption further aggravates the catabolic state of uremia and that this is mediated by an increment in glucocorticoid production.     

Downregulation of UT-A1/UT-A3 is associated with urinary concentrating defect in glucocorticoid-excess state

Excessive glucocorticoid hormone, as occurs with Cushing syndrome, is known to be associated with altered body water homeostasis, but the molecular mechanisms are unknown. In this study, rats treated with daily dexamethasone (Dex) for 14 d provided a model of Cushing syndrome. Compared with control rats, Dex-treated rats demonstrated increased mean arterial pressure, urine flow rate, and urinary excretion of both sodium and urea. Dex-treated rats had increased abundance of aquaporin 1 (AQP1), AQP3, and Na-K-2Cl co-transporter proteins and a marked reduction of the urea transporters UT-A1 and UT-A3. In response to an acute water load, Dex-treated rats increased water excretion more than control rats, but both groups exhibited similar AQP2 expression. In response to fluid deprivation, Dex-treated rats demonstrated an impaired urinary concentrating capacity: Urine flow rate was higher and urine osmolality was lower than control rats despite an increase in AQP1, AQP3, and Na-K-2Cl co-transporter expression. AQP2 expression was similar between the two groups, but UT-A1 and UT-A3 were decreased and urinary urea excretion was increased in Dex-treated rats. Because Dex-treated rats ingested less food and water compared with controls, paired food and water studies were performed; these substantiated the previous results. In summary, the alterations in body water observed with glucocorticoid excess may be a result, in part, of impaired urinary concentrating capacity; downregulation of UT-A1 and UT-A3 and increased urea excretion may contribute to this impairment.     

Sodium thiosulfate prevents vascular calcifications in uremic rats

Accelerated vascular calcification is a severe complication of chronic kidney disease contributing to high morbidity and mortality in patients undergoing renal replacement therapy. Sodium thiosulfate is increasingly used for the treatment of soft tissue calcifications in calciphylaxis. Therefore, we determined whether it also prevents development of vascular calcifications in chronic kidney disease. We found that uremic rats treated by thiosulfate had no histological evidence of calcification in the aortic wall whereas almost three-fourths of untreated uremic rats showed aortic calcification. Urinary calcium excretion was elevated and the calcium content of aortic, heart, and renal tissue was significantly reduced in the thiosulfate-treated compared to non-treated animals. Sodium thiosulfate treatment transiently lowered plasma ionized calcium and induced metabolic acidosis. It also lowered bone strength in the treated animals compared to their normal controls. Hence, sodium thiosulfate prevented vascular calcifications in uremic rats, likely by enhancing acid- and/or chelation-induced urinary calcium loss. The negative impact on rat bone integrity necessitates a careful risk-benefit analysis before sodium thiosulfate can be used in individual human patients.     

Cinacalcet suppresses calcification of the aorta and heart in uremic rats

High serum parathyroid hormone levels are associated with vascular calcification. Cinacalcet is a calcimimetic agent that inhibits parathyroid hormone secretion and is used to treat patients with secondary hyperparathyroidism. Here we measured the effects of oral cinacalcet on calcification of the aorta and heart in rats with a remnant kidney (5/6 nephrectomy) model of uremia that were fed a high-phosphate diet containing lactose to accelerate the process of aortic calcification. Alizarin red staining showed that the smooth muscle in the aortic arch of rats with a remnant kidney was calcified. The tissue levels of calcium and phosphorus in the aorta and hearts of these rats were significantly increased compared to sham-operated rats. Expression of the osteoblastic lineage genes osteocalcin, osteopontin and runt-related gene 2 were also increased in the aorta of these rats. Cinacalcet suppressed these calcification-related changes by reducing serum parathyroid hormone, calcium, phosphorus, and the calcium-phosphorus product. Parathyroidectomy also suppressed calcification in this model. We suggest that cinacalcet inhibits calcification of the aorta and heart in uremic patients with secondary hyperparathyroidism by decreasing serum parathyroid hormone levels.