Urea Reabsorption Research Articles

Low-protein diet promotes nitrogen retention efficiency via enhanced renal urea reabsorption and microbial hydrogen incorporation in the rumen of goats

Feeding a low-protein diet can enhance urea reabsorption and transfer to the rumen, which might alter microbial protein (MCP) synthesis and redirect molecular hydrogen (H2) flow away from methanogenesis. The aim of this study was to determine the effects of feeding goats a low-protein diet on urea nitrogen (N) salvage and metabolism, and consequence on H2 metabolism for MCP synthesis and methanogenesis. Goats (Xiangdong Black goats, a local breed in southern China,) with body weight of 20.4 ± 2.15 kg (mean ± SD) were fed a control diet (consisted of 70% rice straw and 30% concentrates, with crude protein [CP] content by 108 g kg−1) or a low-protein diet (55.2 g CP/kg). 15NH4Cl and 2H2 were employed to further confirm the incorporation of N and hydrogen for MCP synthesis through in vitro culture. Feeding a low-protein diet resulted in greater proportion of retained N (+156%, P < 0.05), plasma urea N (+81.9%, P < 0.05) or rumen ammonia-N (+125%, P < 0.001) relative to digested N and greater renal urea N reabsorption (+11.2%, P < 0.05), together with upregulated relative protein expression of urea transporter A (UTA) in the kidneys (P < 0.05) and mRNA expression of UTA in rumen epithelium (P < 0.05). Feeding low-protein diet resulted in greater concentration of rumen dissolved hydrogen (+55.6%, P < 0.05), ratio of MCP to ammonia-N (+17.6%, P < 0.05), and lower enteric methane emissions (g/kg digested DM, −18.5%, P < 0.05). Further, in vitro experiments verified that the addition of NH4Cl decreased methane and hydrogen production (P < 0.001), although only a minor quantity of headspace 2H2 gas was incorporated for MCP synthesis. In conclusions, feeding a low-protein diet to goats enhances renal urea reabsorption capacity and facilitates the rumen microbial hydrogen incorporation for MCP synthesis, leading to enhanced N retention efficiency and reduced methane emissions. However, the effect of such an approach on livestock performance should be tested in the future studies.

Segment-Dependent Gene Expression Profiling of the Cartilaginous Fish Nephron Using Laser Microdissection for Functional Characterization of Nephron at Segment Levels.

For adaptation to a high salinity marine environment, cartilaginous fishes have evolved a ureosmotic strategy. They have a highly elaborate "four-loop nephron" in the kidney, which is considered to be important for reabsorption of urea from the glomerular filtrate to maintain a high concentration of urea in the body. However, the function and regulation, generally, of the "four-loop nephron" are still largely unknown due to the complicated configuration of the nephron and its many subdivided segments. Laser microdissection (LMD) followed by RNA-sequencing (RNA-seq) analysis is a powerful technique to obtain segment-dependent gene expression profiles. In the present study, using the kidney of cloudy catshark, Scyliorhinus torazame, we tested several formaldehyde-free and formaldehyde-based fixatives to optimize the fixation methods. Fixation by 1% neutral buffered formalin for 15 min resulted in sufficient RNA and structural integrities, which allowed LMD clipping of specific nephron segments and subsequent RNA-seq analysis. RNA-seq from the LMD samples of the second-loop, the fourth-loop, and the five tubular segments in the bundle zone revealed a number of specific membrane transporter genes that can characterize each segment. Among them, we examined expressions of the Na + -coupled cotransporters abundantly expressed in the second loop samples. Although the proximal II segment of the second loop is known for the elimination of excess solutes, the present results imply that the PII segment is also crucial for reabsorption of valuable solutes. Looking ahead to future studies, the segment-dependent gene expression profiling will be a powerful technique for unraveling the renal mechanisms and regulation in euryhaline elasmobranchs.

Molecular and morphological investigations on the renal mechanisms enabling euryhalinity of red stingray Hemitrygon akajei.

Most cartilaginous fishes live in seawater (SW), but a few exceptional elasmobranchs (sharks and rays) are euryhaline and can acclimate to freshwater (FW) environments. The plasma of elasmobranchs is high in NaCl and urea concentrations, which constrains osmotic water loss. However, these euryhaline elasmobranchs maintain high levels of plasma NaCl and urea even when acclimating to low salinity, resulting in a strong osmotic gradient from external environment to body fluid. The kidney consequently produces a large volume of dilute urine to cope with the water influx. In the present study, we investigated the molecular mechanisms of dilute urine production in the kidney of Japanese red stingray, Hemitrygon akajei, transferred from SW to low-salinity environments. We showed that red stingray maintained high plasma NaCl and urea levels by reabsorbing more osmolytes in the kidney when transferred to low salinity. RNA-seq and qPCR analyses were conducted to identify genes involved in NaCl and urea reabsorption under the low-salinity conditions, and the upregulated gene expressions of Na+-K+-Cl- cotransporter 2 (nkcc2) and Na+/K+-ATPase (nka) were found in the FW-acclimated individuals. These upregulations occurred in the early distal tubule (EDT) in the bundle zone of the kidney, which coils around the proximal and collecting tubules to form the highly convoluted structure of batoid nephron. Considering the previously proposed model for urea reabsorption, the upregulation of nkcc2 and nka not only causes the reabsorption of NaCl in the EDT, but potentially also supports enhanced urea reabsorption and eventually the production of dilute urine in FW-acclimated individuals. We propose advantageous characteristics of the batoid-type nephron that facilitate acclimation to a wide range of salinities, which might have allowed the batoids to expand their habitats.

Kidney functions adaptations of professional soccer players in response to an entire game season.

This study investigated the renal function of soccer players after an entire game-season. Thirty-five athletes recruited to play for the Macae Futebol Clube were invited for this study, of which 18 athletes completed the entire game season. Blood and 24-hour urine were collected at the beginning (Pre-Season) and the end of the game season (Post-Season). Kidney functions were assessed by calculating the urinary excretion, clearance, and fractional excretion of the selected solutes. Plasma creatinine, sodium, total protein, and osmolality were lower in the Post-Season . In contrast, plasma urea was higher in the Post-Season period. Urinary excretion of urea was reduced while albumin excretion was higher in comparison to Pre-Season. The clearances of creatinine, total proteins, and albumin were higher in the Post-Season period. In accordance, the fractional excretion of albumin increased. On the other hand, the clearance and fractional excretion of urea was lower in the Post-Season period. These results show that soccer-associated exercise throughout the entire game-season induces kidney functions adaptations that may prevent dehydration in these athletes through increased urea reabsorption to conserve water. In addition, this data corroborates to increased glomerular permeability to plasma proteins, such as albumin, that soccer players may experience.

Rapid effects of aldosterone and phosphatase inhibition on water and urea permeabilities

Aldosterone promotes renal sodium uptake, thereby increasing water reabsorption. But our previous data showed that aldosterone decreases UT‐A1 urea transporter expression, suggesting aldosterone could inhibit water reabsorption. In this study, we investigated the rapid effect of aldosterone on vasopressin‐stimulated osmotic water permeability in rat inner medullary collecting ducts (IMCDs). In IMCDs from male Sprague Dawley rats weighing 50–75g, we saw that aldosterone significantly decreased osmotic water permeability from 152 ± 21 to 122 ± 17 μm/s (45 min, n=4, p&lt;0.01). Given the rapid nature of the response, we further investigated whether the effect of aldosterone might be through phosphatase‐mediated modulation of transporter phosphorylation. Inhibition of PP1/PP2a phosphatases by calyculin increased basal osmotic water permeability from 58 ± 8 to 92 ± 14 μm/s (n=4, p&lt;0.01). In the presence of vasopressin, stimulation by calyculin remained significant in both male and female rats 77 ± 10 to 119 ± 15 μm/s (30 min, n=6, p&lt;0.01). Addition of aldosterone to the bath in the presence of calyculin reversed the increase in osmotic water permeability from 119 ± 15 to 89 ± 10 μm/s (45 min, p&lt;0.01). Inhibition of pp2B with tacrolimus also stimulated water permeability (88 ± 13 to 108 ± 19 μm/s), which was reversed by addition of aldosterone (81 ± 10 μm/s). Urea permeability was also increased by both calyculin (25 ± 7 × 10−5 to 32 ± 7 × 10−5 cm/s; n=3, p&lt;0.05) and tacrolimus (15 ± 1 × 10−5 to 18 ± 1 × 10− 5 cm/s; n=4, p&lt;0.05). Aldosterone decreased pser256‐ and increased pser261‐AQP2. Inhibition of PP2B by tacrolimus increased pser256‐ and decreased pser261‐AQP2. Calyculin increases pser256‐AQP2 and this was reversed by aldosterone. Treatment of inner medullary tissue with 50 nM calyculin or 62 nM tacrolimus resulted in significantly increased UT‐A1 phosphorylation. We conclude that aldosterone is able to rapidly inhibit water and urea reabsorption, possibly by altering phosphatase activity and changing the phosphorylation states of AQP2 and UT‐A1.Support or Funding InformationAHA 18CDA34060053NIH/NIDDKRO1‐DK41707

UT-A1/A3 knockout mice show reduced fibrosis following unilateral ureteral obstruction.

Renal fibrosis is a major contributor to the development and progression of chronic kidney disease. A low-protein diet can reduce the progression of chronic kidney disease and reduce the development of renal fibrosis, although the mechanism is not well understood. Urea reabsorption into the inner medulla is regulated by inner medullary urea transporter (UT)-A1 and UT-A3. Inhibition or knockout of UT-A1/A3 will reduce interstitial urea accumulation, which may be beneficial in reducing renal fibrosis. To test this hypothesis, the effect of unilateral ureteral obstruction (UUO) was compared in wild-type (WT) and UT-A1/A3 knockout mice. UUO causes increased extracellular matrix associated with increases in transforming growth factor-β, vimentin, and α-smooth muscle actin (α-SMA). In WT mice, UUO increased the abundance of three markers of fibrosis: transforming growth factor-β, vimentin, and α-SMA. In contrast, in UT-A1/A3 knockout mice, the increase following UUO was significantly reduced. Consistent with the Western blot results, immunohistochemical staining showed that the levels of vimentin and α-SMA were increased in WT mice with UUO and that the increase was reduced in UT-A1/A3 knockout mice with UUO. Masson's trichrome staining showed increased collagen in WT mice with UUO, which was reduced in UT-A1/A3 knockout mice with UUO. We conclude that reduced UT activity reduces the severity of renal fibrosis following UUO.

Regenerating re-absorption function of proximal convoluted tubule using microfluidics for kidney-on-chip applications

Human kidney is a sophisticated organ with 1 Million nephrons arranged in subtle form. Kidney has the most failure cases in the world compared to the rest of the body organs. Kidney failure is a severe problem, where cardiac blood output is not filtered. Dialysis is one available substitute for kidney failure, which seems to help the patient incompletely. There is a great necessity for a device (artificial kidney) that can be implanted into the body to resume the kidney function. In replicating kidney function there are many potential challenges, which must be addressed for faithful regeneration. This paper primarily focuses on regenerating the proximal convoluted tubule (PCT) size dependent re-absorption, mimicking this function using microfluidics is not reported earlier. Different structural changes in the design have been adopted and the accomplishments are discussed. It is observed that the total flow (the total of flow through all 1000 channels) in straight is 0.4 × 10−16 m3/s, in the diagonal channel is 0.4 × 10−16 m3/s, in step is 0.32 × 10−16 m3/s and in serpentine is 0.38 × 10−16 m3/s. The size-dependent re-absorption of solutes, proteins, and urea with the help of array of channels has been achieved. The dimensions of the main tubule and channel are selected to replicate cell–cell interactions. The re-absorption rate obtained is around 48%, which is closely reaching the PCT re-absorption rate. The increase in the number of channels shows increase in re-absorption rate. The novelty of reported work lies in regenerating the human kidney proximal tubule cell function of size and shape dependent re-absorption using microfluidics technology. The proposed device performance proves its prevalence in kidney-on-chip applications.

Inner Medullary Urea Transporters Contribute to Development of Renal Fibrosis in Mice With Unilateral Ureteral Obstruction

Fourteen percent of the population of the United States is affected by chronic kidney disease (CKD). Although the mechanisms that are responsible for CKD remain unclear, there are many clinical and animal studies showing that low protein diets reduce the long term effects of CKD. One possible explanation for these benefits is the restricted urea load of the low protein diet suggesting a role for the urea transporters in this process. Unilateral ureteral obstruction (UUO) is a well known model to produce fibrogenic responses such as increased capillary macrophages, pro‐fibrogenic proteins, interstitial matrix, myofibroblasts, etc. in CKD rodents. We hypothesized that the urea transporters contribute to the development of renal fibrosis. We used UUO mice receiving a maximal urea load (high protein diet) to investigate contributions of urea transporters to renal fibrosis. C57Bl6 mice ± genetic ablation of UT‐A1 and UT‐A3 urea transporters (UT‐A1/A3 KO) underwent UUO surgery (ligation of the left proximal ureter). Four days after surgery mice were given a high (40%) protein diet for 10 further days. Mice were killed and obstructed kidneys collected for protein and histochemical analysis. All of the obstructed kidneys showed evidence of hydronephrosis after 14 days. BUN was unchanged by UUO: WT control: 44.2 mg/dl, WT UUO: 46.8 mg/dl, KO control: 43.4, KO UUO: 47.1. TGF‐b was increased in both WT and UT‐A1/A3 KO mice with UUO (302% and 389% respectively). WT with UUO showed a 320% increase in α‐SMA vs mice without UUO. UT‐A1/A3 KO mice with UUO showed a blunted 271% increase in α‐SMA vs unobstructed UT‐A1/A3 KO mice. Vimentin, another marker of fibrosis, was significantly increased 555% by UUO (vs no UUO). UUO in UT‐A1/A3 KO mice did not increase vimentin. H&amp;E staining revealed similar infiltration of inflammatory cells in both UUO WT and UUO UT‐A1/A3 KO mice. Trichrome staining showed higher collagen levels in UUO WT vs UUO UT‐A1/A3 KO mice. Immunohistochemical staining for αSMA and vimentin revealed higher levels in UUO WT vs UUO UT‐A1/A3 KO mice. These data suggest that urea transporters do not prevent inflammation caused by UUO. However, the overall development of fibrosis in UUO mice fed a high protein diet is attenuated by the absence of urea transporters suggesting that urea reabsorption by urea transporters contributes to renal fibrosis. If urea transport inhibitors replicate the effects of genetic knock out, they may represent a promising future therapy.Support or Funding InformationRelypsa Nephrology and Cardiology Fellowship Grant;NIH RO1 DK41707This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tibetan sheep require less energy intake than small-tailed Han sheep for N balance when offered a low protein diet

Tibetan sheep are indigenous to the Qinghai-Tibetan Plateau and graze natural pasture all year round; small-tailed Han sheep were introduced to the plateau, are raised mainly in feedlots and graze natural pasture only in summer. A low energy intake could have a negative effect on N utilization and N balance in ruminants. We hypothesized that Tibetan sheep require less energy for N balance and utilize N more efficiently than Han sheep when offered a low protein and low energy diet. To test this hypothesis, we examined the effect of dietary energy on whole-body N retention and urea kinetics in Tibetan (47.7 ± 2.46 kg BW) and Han (46.2 ± 3.42 kg BW) sheep. Diets were formulated to be low in crude protein (˜7%) with different metabolizable energy levels (6.73, 7.65, 8.57 and 9.49 MJ/kg). Urea kinetics were determined using continuous intrajugular infusion of 15N15N-urea for 72 h. N retention was greater (P < 0.01) in Tibetan than in Han sheep and increased linearly with increasing energy intake (P < 0.001). Tibetan sheep maintained a positive N balance on all dietary energy levels, whereas Han sheep were in a negative N balance at the lowest energy intake. To achieve a N balance of zero, Tibetan sheep required 0.28 MJ/kg0.75, whereas Han sheep required 0.33 MJ/kg0.75 per day. Fecal N excretion was less (P < 0.05) for Tibetan than for Han sheep. Plasma urea concentrations tended to be greater in Tibetan than in Han sheep (P < 0.10), but urinary urea excretion was lower in Tibetan sheep at the lowest energy diet (linear dietary energy × breed; P < 0.05). As energy intake increased, urinary N decreased linearly (P < 0.001) and microbial N production increased linearly (P < 0.001). Urea entry rate and gastrointestinal tract entry rate were greater in Tibetan than in Han sheep (P < 0.01). Urinary purine derivatives excretion, microbial N production, and the portion of microbial N that originated from recycled urea were greater in Tibetan than in Han sheep (P < 0.01). Renal urea reabsorption rate was greater in Tibetan than in Han sheep (P < 0.05) and increased linearly with dietary energy level (P < 0.05). We concluded that: 1) Tibetan sheep were better able to cope with low N diets when energy intake was low; and 2) when N intakes are low, N utilization is improved by supplemental energy.

Резекция печени изменяет механизмы выведения аммиака почками крыс с хроническим тетрахлорметановым гепатитом

Цель исследования - изучение влияния резекции печени (РП) на аммиакэкскретирующую функцию почек при хроническом тетрахлорметановом гепатите. Методика. Опыты выполнены на 265 беспородных белых крысах (самках) массой 180-220 г. Хронический гепатит воспроизводили подкожным введением 50% раствора тетрахлорметана (CCl) на оливковом масле (0,1 мл/100 г массы тела, через сутки, c двумя двухнедельными перерывами между 6, 7 и 13-14 инъекциями). На 65-е сут. (последние) введения тетрахлорметана, удаляли часть левой доли печени (15-20% массы органа). На 3-и, 7-е и 14-е сут. после РП или лапаротомии («ложнооперированные» животные) в почках, артериальной и венозной крови, моче исследовали содержание аммиака, глутамина и мочевины. Результаты. Прогрессирование эндогенной аммиачной интоксикации после РП на фоне тетрахлорметанового гепатита сопровождается повышенной экскрецией аммиака почками. Однако это не устраняет артериальную гипераммониемию и не предотвращает накопление почками аммиака. Инкреция глютамина из почек в кровоток прекращается. К 14-м сут. послеоперационного периода возрастает потребление глютамина из артериальной крови, что приводит к его накоплению в почках. Стимулируя выведение мочевины из организма с мочой, РП одновременно активирует её образование в почках, с дальнейшим поступлением как в кровоток, так и в мочу. В зависимости от сроков послеоперационного периода это сопровождается изменением скорости реабсорбции мочевины в почках. Заключение. Полученные результаты свидетельствуют, что при РП на фоне тетрахлорметанового гепатита почки не предотвращают прогрессирование эндогенной аммиачной интоксикации, патологическое накопление аммиака и глутамина её клетками, но сохраняют способность принимать участие в регуляции повышенного содержания мочевины в артериальной крови.Mechanical (resection) or toxic (hepatitis) liver damage alone has an ambiguous effect on renal ammonia excretion during development of endogenous ammonia intoxication. The aim. The study investigated the effect of liver resection (LR) on renal ammonia excretion in chronic tetrachlorocarbon (CCl)-induced hepatitis. Methods. Experiments were conducted on 240 mongrel white rats (females) weighing 180-220 g. Chronic hepatitis was induced by subcutaneous injection of 50% solution of carbon tetrachloride (CCl) in olive oil (0.1 ml/100g body weight per day with two two-week breaks between injections 6-7 and 13-14). LR with removal of a part of the left lobe (15-20% of body weight) was performed on the 65th (last) day of CCl injections. The animals were examined on the 3rd, 7th and 14th day after LR or laparotomy (sham operation). Contents of ammonia (AM), glutamine (GN), and urea were measured in the kidney, arterial (AB) and venous ( v.renlis ) blood, and urine. Results. Progression of endogenous ammonia intoxication after LR associated with CCl-induced hepatitis and increased renal excretion of Am involves three mechanisms: 1) excretion of Am that is delivered to kidneys in the free form with AB; 2) stimulation of renal tubule secretion of Am that had formed in kidneys by deamidation of «arterial» Gn; and 3) contrary to rules, partial reabsorption of Am from collecting tubules into the blood. However, this does not eliminate arterial hyperammonemia or prevent accumulation of Am in kidneys. The stimulatory effect of LR in CCl-induced hepatitis on Gn incretion from kidneys to the circulation stops by the 14 day after surgery, and the accompanying increased consumption of Gn from AВ results in Gn accumulation in kidneys. LR stimulates urea excretion with urine and simultaneously activates kidney formation of urea, which further enters the bloodstream and urine. Depending on the postoperative period this is associated with changes in the rate of urea reabsorption in kidneys. Conclusions. In RP associated with CCl-induced hepatitis, kidneys cannot prevent progression of endogenous ammonia intoxication and pathological accumulation of ammonia and glutamine in kidney cells but retain the ability to participate in the regulation of the increased urea level in AB.

The Effect of Hyperbaric Oxygenation on the Circulation of Urea in Rats Following Experimental Partial Hepatectomy

Material and methods. The studies were performed on 75 white mature rats (females) weighing 180—220g. Partial hepatectomy was performed by resection of a part of the left lobe of the liver (15—20% of the organ weight). Hyperbaric oxygenation (HBO) was performed three times (3 ata, 50 min). The first, second and third sessions were performed 4—8 hours, 24 hours and 48 hours after the surgery, respectively. The urea level was determined in the tissues of visceral organs, as well as in arterial blood (aorta), portal blood, blood from renal and hepatic veins, bile from common bile duct, and in urine on the 1st, 4th and 11th days of posthyperoxic period (days 3, 7 and 14 post-surgery).Results. Activation of the urea incretion from the operated liver to the bloodstream under hyperoxic conditions was accompanied by increased urea concentration in the arterial blood and excretion from the body with urine that was facilitated by the elimination of a stimulating effect of PHE on the reabsorption of urea in the kidneys by the HBO procedure. At the same time, the production of urea in the renal tissue was activated and further released to the circulation through the renal vein. Stimulation of the liver and intestinal urea circulation by HBO was accompanied by the preservation of the stimulating effect of PHE on its accumulation in duodenum and colon tissues. In the thyroid gland, spleen, heart, and lungs of operated rats, HBO activated the transition of the «arterial» urea from the free form to the bound one. Termination of HBO normalized the urea concentration in the arterial blood by the 11th day of the posthyperoxic period whereas urea continued to be accumulated the heart, spleen, lungs, and intestine. A preserved increased release of urea from the operated liver into the bloodstream after HBO was accompanied by partial retention in the hepatocytes of urea delivered via bloodstreem through the portal vein. On the 11th day after HBO, the repeated hyperoxic activation of the liver and intestinal urea circulation occured, as well as HBO-restored stimulation of its production in the kidneys resulted in urea release into the renal vein and excretion with urea.Conclusion. HBO provides a correcting effect on alteration of circulating urea caused by PHE.

Nitrogen handling in the elasmobranch gut: a role for microbial urease.

Ureotelic elasmobranchs require nitrogen for both protein growth and urea-based osmoregulation, and therefore are probably nitrogen-limited in nature. Mechanisms exist for retaining and/or scavenging nitrogen in the gills, kidney, rectal gland and gut, but as yet, the latter are not well characterized. Intestinal sac preparations of the Pacific spiny dogfish shark (Squalus acanthias suckleyi) incubated in vitro strongly reabsorbed urea from the lumen after feeding, but mucosal fluid ammonia concentrations increased with incubation time. Phloretin (0.25 mmol l-1, which blocked urea reabsorption) greatly increased the rate of ammonia accumulation in the lumen. A sensitive [14C]urea-based assay was developed to examine the potential role of microbial urease in this ammonia production. Urease activity was detected in chyme/intestinal fluid and intestinal epithelial tissue of both fed and fasted sharks. Urease was not present in gall-bladder bile. Urease activities were highly variable among animals, but generally greater in chyme than in epithelia, and greater in fed than in fasted sharks. Comparable urease activities were found in chyme and epithelia of the Pacific spotted ratfish (Hydrolagus colliei), a ureotelic holocephalan, but were much lower in ammonotelic teleosts. Urease activity in dogfish chyme was inhibited by acetohydroxamic acid (1 mmol l-1) and by boiling. Treatment of dogfish gut sac preparations with acetohydroxamic acid blocked ammonia production, changing net ammonia accumulation into net ammonia absorption. We propose that microbial urease plays an important role in nitrogen handling in the elasmobranch intestine, allowing some urea-N to be converted to ammonia, which is then reabsorbed for amino acid synthesis or reconversion to urea.

Modulation of kidney urea transporter UT-A3 activity by alpha2,6-sialylation.

Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.

The Kinetics of Urea in the Body after Liver Resection in the Experiment

Purpose. To study urea kinetics in the body after liver resection in the experiment. Material and Methods. Experiments were carried out on 45 white female rats weighing between 180 g and 220 g. Liver resection (LR) was performed under ester anesthesia, wherein 15—20% of the organ weight was removed. Urea content was studied in biological fluids (arterial blood, venous — v.porta, v.hepatica, v.renalis — blood, choledochal bile, urine), and tissues of visceral organs (the thyroid gland, lungs, heart, liver, kidneys, spleen, stomach, intestine) on days 3, 7, and 14 after LR. Results. LR, while reducing the urea content in the v. hepatica blood, does not lead to similar changes in the arterial blood. This is accompanied by increased urea reabsorption in kidneys and higher v.porta blood urea content, which, depending on the postoperative time, results either from reduced urea excretion into the small intestine lumen or from its greater production by enterocytes followed by metabolite intake into the portal blood flow. The urea intake from hepatocytes into the hepatic bile ducts did not change on day 3 after LR; however, it increased on day 7 and slowed down on day 14. LR caused no changes in the gastric tissues urea content; never theless, it led to its increased content in the duodenal and colonic tissues. Without affecting the cardiac muscleurea content, LR entailed its increase in the lungs and thyroid gland on postoperative days 3, 7, and 14. At the background of absence of similar changes in the arterial blood data indicates promotion of urea production by the cells of these organs or metabolite retention therein. Conclusions. LR not only changes urea kinetics in the portal system organs, but also activates extrahepatic mechanisms aimed at preventing development of the arterial blood urea deficit because of its abnormal intake from the resected organ into the central blood flow.

Morphological and molecular investigations of the holocephalan elephant fish nephron: the existence of a countercurrent-like configuration and two separate diluting segments in the distal tubule.

In marine cartilaginous fish, reabsorption of filtered urea by the kidney is essential for retaining a large amount of urea in their body. However, the mechanism for urea reabsorption is poorly understood due to the complexity of the kidney. To address this problem, we focused on elephant fish (Callorhinchus milii) for which a genome database is available, and conducted molecular mapping of membrane transporters along the different segments of the nephron. Basically, the nephron architecture of elephant fish was similar to that described for elasmobranch nephrons, but some unique features were observed. The late distal tubule (LDT), which corresponded to the fourth loop of the nephron, ran straight near the renal corpuscle, while it was convoluted around the tip of the loop. The ascending and descending limbs of the straight portion were closely apposed to each other and were arranged in a countercurrent fashion. The convoluted portion of LDT was tightly packed and enveloped by the larger convolution of the second loop that originated from the same renal corpuscle. In situ hybridization analysis demonstrated that co-localization of Na(+),K(+),2Cl(-) cotransporter 2 and Na(+)/K(+)-ATPase α1 subunit was observed in the early distal tubule and the posterior part of LDT, indicating the existence of two separate diluting segments. The diluting segments most likely facilitate NaCl absorption and thereby water reabsorption to elevate urea concentration in the filtrate, and subsequently contribute to efficient urea reabsorption in the final segment of the nephron, the collecting tubule, where urea transporter-1 was intensely localized.

Cloning and characterization of aquaglyceroporin genes from rainbow smelt (Osmerus mordax) and transcript expression in response to cold temperature

Aquaglyceroporins (GLPs) are integral membrane proteins that facilitate passive movement of water, glycerol and urea across cellular membranes. In this study, GLP-encoding genes were characterized in rainbow smelt (Osmerus mordax mordax), an anadromous teleost that accumulates high glycerol and modest urea levels in plasma and tissues as an adaptive cryoprotectant mechanism in sub-zero temperatures. We report the gene and promoter sequences for two aqp10b paralogs (aqp10ba, aqp10bb) that are 82% identical at the predicted amino acid level, and aqp9b. Aqp10bb and aqp9b have the 6 exon structure common to vertebrate GLPs. Aqp10ba has 8 exons; there are two additional exons at the 5' end, and the promoter sequence is different from aqp10bb. Molecular phylogenetic analysis suggests that the aqp10b paralogs arose from a gene duplication event specific to the smelt lineage. Smelt GLP transcripts are ubiquitously expressed; however, aqp10ba transcripts were highest in kidney, aqp10bb transcripts were highest in kidney, intestine, pyloric caeca and brain, and aqp9b transcripts were highest in spleen, liver, red blood cells and kidney. In cold-temperature challenge experiments, plasma glycerol and urea levels were significantly higher in cold- compared to warm-acclimated smelt; however, GLP transcript levels were generally either significantly lower or remained constant. The exception was significantly higher aqp10ba transcript levels in kidney. High aqp10ba transcripts in smelt kidney that increase significantly in response to cold temperature in congruence with plasma urea suggest that this gene duplicate may have evolved to allow the re-absorption of urea to concomitantly conserve nitrogen and prevent freezing.

IMPACT OF HYPERBARIC OXYGENATION ON RENAL AMMONIA EXCRETION DURING EXPERIMENTAL LIVER RESECTION

Objective: to study the impact of hyperbaric oxygenation (HBO) on renal ammonia excretion during liver resection (LR). Material and methods. Experiments were carried out on 138 pubescent rats (females). Three HBO sessions were performed at 3 ATA lasting 50 min once daily after LR (15—20% of the liver weight). The kidney, blood (aorta, v. renalis), and urine were examined. The levels of ammonia, glutamine, and urea were measured. Results. By eliminating postoperative arterial hyperammonemia, HBO stimulates urinary ammonia ion excretion. This is achieved through elimination of the inhibitory effect of LR under HBO on ammonia secretion into the renal tubules and through activation of intracellular ammoniagenesis in nephrocytes, including that uncoupled with arterial glutamine deamidation. HBO potentiates simultaneously the activating effect of LR on the glutamine cycle in the kidneys mainly through the hyperproduction of renal glutamine and its influx from them into the bloodstream. HPO enhances the stimulatory effect of LR on urea reabsorption in the kidneys, but, unlike non-oxygenated rats, there was no reduction in urine urea excretion due to increased arterial blood urea concentrations. Termination of the body's exposure to hyperoxia restores urea reabsorption in the kidneys with the persisting stimulatory impact of HBO on the formation of urea by nephrocytes and its incretion from the latter into the bloodstream.

Blood Urea Nitrogen/Creatinine Ratio and Response to Tolvaptan in Patients with Decompensated Heart Failure: A Retrospective Analysis.

Arginine vasopressin-stimulated reabsorption of urea occurs in the collecting duct via increased expression of the urea transporter. The aim of this study was to evaluate whether the blood urea nitrogen/creatinine (BUN/Cr) ratio is useful for predicting tolvaptan response in patients with decompensated heart failure (HF). Among 71 consecutive patients with HF who received oral tolvaptan between 2010 and 2014, we retrospectively studied 33 patients with decompensated HF without any mechanical circulatory assistance or inotropic support who had already been treated with loop diuretics. A responder to tolvaptan was defined as an individual who experienced a ≥30% increase in their respective 24-h urine volume. Among the 33 patients, 21 met the criteria of a responder. The area under the receiver operating characteristic curves of BUN/Cr and BUN were 0.790 and 0.714, respectively, and the respective cut-off values for responders to tolvaptan were 23.8 and 49.0. BUN/Cr and BUN retained their significant relationships with the responder status (odds ratio for BUN/Cr >23.8: 20.9; 95% confidence interval [CI] 2.7-531.1; p=0.002; odds ratio for BUN ≥49: 7.7; 95% CI 1.4-65.8; p=0.02). Our results suggest that high BUN/Cr may be a predictor of response to tolvaptan in decompensated HF patients. A prospective study with a large sample size is required to confirm this preliminary finding.

Abstract 19099: The Fractional Excretion of Urea is Substantially Altered by Diuretics in Heart Failure Patients: Implications for its Use in the Evaluation of Worsening Renal Function

Background: In the setting of acute decompensated heart failure (ADHF), significant renal dysfunction (RD) is a common both at presentation and during treatment. The fractional excretion of urea (FeUrea) is often recommended to differentiate “pre-renal” from intrinsic RD in patients receiving diuretics. However, the intra-renal physiology regulating urea reabsorption theoretically should be affected by high dose loop diuretics which are commonly utilized in ADHF. Methods: Heart failure patients presenting to the Yale Transition Care Clinic who received treatment with loop diuretics were enrolled (n=64). Urine was collected at baseline and 60-90 minutes following administration of loop diuretics. Possible AKI was defined as a presenting serum creatinine ≥0.3 mg/dl above the lowest outpatient level in the prior 12 months. Results: Overall, 75.9% of the cohort had an estimated glomerular filtration rate &lt; 60 ml/min/1.73m^2 and 66.7% met criteria for possible AKI. As would be expected in a sodium avid HF population, the mean pre-diuretic FeUrea was 31.9 ± 10.2%. This was below the “pre-renal” FeUrea cutoff (&lt;35%) in 62.5% of patients. Following administration of 160 mg (50 to 280) of IV furosemide equivalents, FeUrea increased by an average of 7.3 ±8.5% (p&lt;0.0001). After diuretics, the percentage of patients with a “pre-renal” FeUrea decreased to 37.7% which translated into 42.5% of initially “pre-renal” patients no longer being classified as “pre-renal.” The change in FeUrea was unrelated to the diuretic induced urine output (r=0.19 p=0.14) or the dose of diuretic (r=0.17, p=0.20). There was significant individual variability in the change in FeUrea induced by loop diuretics (Figure) such that a loop diuretic “correction factor” would not be appropriate. Conclusion: Contrary to traditional wisdom, FeUrea does not appear to be a reliable tool to workup changes in renal function in ADHF patients receiving treatment with high dose loop diuretics.

Morphological and functional characteristics of the kidney of cartilaginous fishes: with special reference to urea reabsorption.

For adaptation to high-salinity marine environments, cartilaginous fishes (sharks, skates, rays, and chimaeras) adopt a unique urea-based osmoregulation strategy. Their kidneys reabsorb nearly all filtered urea from the primary urine, and this is an essential component of urea retention in their body fluid. Anatomical investigations have revealed the extraordinarily elaborate nephron system in the kidney of cartilaginous fishes, e.g., the four-loop configuration of each nephron, the occurrence of distinct sinus and bundle zones, and the sac-like peritubular sheath in the bundle zone, in which the nephron segments are arranged in a countercurrent fashion. These anatomical and morphological characteristics have been considered to be important for urea reabsorption; however, a mechanism for urea reabsorption is still largely unknown. This review focuses on recent progress in the identification and mapping of various pumps, channels, and transporters on the nephron segments in the kidney of cartilaginous fishes. The molecules include urea transporters, Na(+)/K(+)-ATPase, Na(+)-K(+)-Cl(-) cotransporters, and aquaporins, which most probably all contribute to the urea reabsorption process. Although research is still in progress, a possible model for urea reabsorption in the kidney of cartilaginous fishes is discussed based on the anatomical features of nephron segments and vascular systems and on the results of molecular mapping. The molecular anatomical approach thus provides a powerful tool for understanding the physiological processes that take place in the highly elaborate kidney of cartilaginous fishes.