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.

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.

Identification and Expression of a Putative Facilitative Urea Transporter in Three Species of True Frogs (Ranidae): Implications for Terrestrial Adaptation

Urea transporters (UTs) help mediate the transmembrane movement of urea and therefore are likely important in amphibian osmoregulation. Although UTs contribute to urea reabsorption in anuran excretory organs, little is known about the protein’s distribution and functions in other tissues, and their importance in the evolutionary adaptation of amphibians to their environment remains unclear. To address these questions, we obtained a partial sequence of a putative UT and examined relative abundance of this protein in tissues of the wood frog (Rana sylvatica), leopard frog (R. pipiens), and mink frog (R. septentrionalis), closely related species that are adapted to different habitats. Using immunoblotting techniques, we found the protein to be abundant in the osmoregulatory organs but also present in visceral organs, suggesting that UTs play both osmoregulatory and nonosmoregulatory roles in amphibians. UT abundance seems to relate to the species’ habitat preference, as levels of the protein were higher in the terrestrial R. sylvatica, intermediate in the semiaquatic R. pipiens, and quite low in the aquatic R. septentrionalis. These findings suggest that, in amphibians, UTs are involved in various physiological processes, including solute and water dynamics, and that they have played a role in adaptation to the osmotic challenges of terrestrial environments.

Glycoforms of UT-A3 urea transporter with poly-N-acetyllactosamine glycosylation have enhanced transport activity

Urea transporters UT-A1 and UT-A3 are both expressed in the kidney inner medulla. However, the function of UT-A3 remains unclear. Here, we found that UT-A3, which comprises only the NH2-terminal half of UT-A1, has a higher urea transport activity than UT-A1 in the oocyte and that this difference was associated with differences in N-glycosylation. Heterologously expressed UT-A3 is fully glycosylated with two glycoforms of 65 and 45 kDa. By contrast, UT-A1 expressed in HEK293 cells and oocytes exhibits only a 97-kDa glycosylation form. We further found that N-glycans of UT-A3 contain a large amount of poly- N-acetyllactosamine. This highly glycosylated UT-A3 is more stable and is enriched in lipid raft domains on the cell membrane. Kifunensine, an inhibitor of α-mannosidase that inhibits N-glycan processing beyond high-mannose-type N-glycans, significantly reduced UT-A3 urea transport activity. We then examined the native UT-A1 and UT-A3 glycosylation states from kidney inner medulla and found the ratio of 65 to 45 kDa in UT-A3 is higher than that of 117 to 97 kDa in UT-A1. The highly stable expression of highly glycosylated UT-A3 on the cell membrane in kidney inner medulla suggests that UT-A3 may have an important function in urea reabsorption.

Cytokine-mediated regulation of urea transporters during experimental endotoxemia

Acute renal failure (ARF) is a frequent complication of sepsis and has a high mortality. Sepsis-induced ARF is known to be associated with significant impairment of tubular capacity. However, the pathogenesis of endotoxemic tubular dysfunction with failure of urine concentration is poorly understood. Urea plays an important role in the urinary concentrating mechanism and expression of the urea transporters UT-A1, UT-A2, UT-A3, UT-A4, and UT-B is essential for tubular urea reabsorption. The present study attempts to assess the regulation of renal urea transporters during severe inflammation in vivo. Lipopolysaccharide-(LPS)-injected mice presented with reduced glomerular filtration rate, fractional urea excretion, and inner medulla osmolality associated with a marked decrease in expression of all renal urea transporters. Similar alterations were observed after application of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, or IL-6. LPS-induced downregulation of urea transporters was not affected in knockout mice with deficient TNF-α, IL-receptor-1, IFN-γ, or IL-6. Glucocorticoid treatment inhibited LPS-induced increases of tissue TNF-α, IL-1β, IFN-γ, or IL-6 concentration, diminished LPS-induced renal dysfunction, and attenuated the downregulation of renal urea transporters. Renal ischemia induced by renal artery clipping did not influence the expression of urea transporters. Our data demonstrate that renal urea transporters are downregulated by severe inflammation, which likely accounts for tubular dysfunction. Furthermore, they suggest that the downregulation of renal urea transporters during LPS-induced ARF is mediated by proinflammatory cytokines and is independent from renal ischemia because of sepsis-induced hypotension.

High urea and creatinine concentrations and urea transporter B in mammalian urinary tract tissues

Although the mammalian urinary tract is generally held to be solely a transit and storage vehicle for urine made by the kidney, in vivo data suggest reabsorption of urea and other urine constituents across urinary tract epithelia. To determine whether urinary tract tissue concentrations are increased as a result of such reabsorption, we measured urea nitrogen and creatinine concentrations and determined whether urea transporter B (UT-B) was present in bladder, ureter, and other tissues from dogs and rats. Mean urea nitrogen and creatinine concentrations in dogs and rats were three- to sevenfold higher in urinary tract tissues than in serum and were comparable to those in renal cortex. In water-restricted or water-loaded rats, urea nitrogen concentrations in bladder tissues fell inversely with the state of hydration, were proportional to urine urea nitrogen concentrations, and were greater than the corresponding serum urea nitrogen concentration in every animal. Immunoblots of rat and dog urinary tract tissues demonstrated the presence of UT-B in homogenates of bladder and ureter, and immunocytochemical analysis localized UT-B to epithelial cell membranes. These findings are consistent with the notion that urea and creatinine are continuously reabsorbed from the urine across the urothelium, urea in part via UT-B, and that urine is thus altered in its passage through the urinary tract. Urea reabsorption across urinary tract epithelia may be important during conditions requiring nitrogen conservation and may contribute to pathophysiological states characterized by high blood urea nitrogen, such as prerenal azotemia and obstructive uropathy.

Changes in the renal handling of urea in sheep on a low protein diet exposed to saline drinking water

Previous trials have demonstrated that sheep on a low protein diet and free access to water, and sheep dosed with boluses of NaCl intraruminally also with free access to water, showed decreases in urea loss via the urine compared to control animals. We monitored urea excretion in sheep on a relatively poor protein diet when they were exposed to saline drinking water, i.e. they were unable to vary their intake of NaCl:water. Sheep on isotonic saline drinking water (phase 3) excreted significantly more urea via the urine (284 mM/day) compared to phase 1 when they were on non-saline drinking water (urea excretion = 230 mM/day) and phase 2 when they were on half isotonic saline drinking water (urea excretion = 244 mM/day).This finding was explained by the high glomerular filtration rate (GFR) 91.9 ℓ/day, compared to 82.4 ℓ/day (phase 1) and 77.9 ℓ/day (phase 2), together with a significantly raised fractional excretion of urea (FEurea) (51.1 %) during this phase, and was in spite of the significantly lower plasma concentrations of urea in phase 3 compared to phase 1. The FEurea probably results from the osmotic diuresis caused by the salt. There were indications of a raised plasma antidiuretic hormone (ADH) concentration and this would have opposed urea loss, as ADH promotes urea reabsorption. However, this ADH effect was probably counteracted to some extent by a low plasma angiotensin II concentration, for which again there were indications, inhibiting urea reabsorption during the phases of salt loading. As atrial natriuretic peptide both increases GFR and decrease sodium reabsorption from the tubule, it was probably instrumental in causing the increase in GFR and the increase in the fractional excretion of sodium (FENa).

Molecular and functional characterization of a urea transporter from the kidney of the Atlantic stingray

In general, marine elasmobranch fishes (sharks, skates, and rays) maintain body fluid osmolality above seawater, principally by retaining large amounts of urea. Maintenance of the high urea concentration is due in large part to efficient renal urea reabsorption. Regulation of renal urea reabsorption also appears to play a role in maintenance of fluid homeostasis of elasmobranchs that move between habitats of different salinities. We identified and cloned a novel 2.7-kb cDNA from the kidney of the euryhaline Atlantic stingray Dasyatis sabina (GenBank accession no. AF443781 ). This cDNA putatively encoded a 431-amino acid protein (strUT-1) that had a high degree of sequence identity (71%) to the shark kidney facilitated urea transporter (UT). However, the predicted COOH-terminal region of strUT-1 appears to contain an additional sequence that is unique among cloned renal UTs. Injection of strUT-1 cRNA into Xenopusoocytes induced a 33-fold increase in [14C]urea uptake that was inhibited by phloretin. Four mRNA bands were detected in kidney by Northern blot: a transcript at 2.8 kb corresponding to the expected size of strUT-1 mRNA and bands at 3.8, 4.5, and 5.5 kb. Identification of a facilitated UT in the kidney of the Atlantic stingray provides further support for the proposal that passive mechanisms contribute to urea reabsorption by elasmobranch kidney.

Regulation of Renal Urea Transporters

Urea is important for the conservation of body water due to its role in the production of concentrated urine in the renal inner medulla. Physiologic data demonstrate that urea is transported by facilitated and by active urea transporter proteins. The facilitated urea transporter (UT-A) in the terminal inner medullary collecting duct (IMCD) permits very high rates of transepithelial urea transport and results in the delivery of large amounts of urea into the deepest portions of the inner medulla where it is needed to maintain a high interstitial osmolality for concentrating the urine maximally. Four isoforms of the UT-A urea transporter family have been cloned to date. The facilitated urea transporter (UT-B) in erythrocytes permits these cells to lose urea rapidly as they traverse the ascending vasa recta, thereby preventing loss of urea from the medulla and decreasing urine-concentrating ability by decreasing the efficiency of countercurrent exchange, as occurs in Jk null individuals (who lack Kidd antigen). In addition to these facilitated urea transporters, three sodium-dependent, secondary active urea transport mechanisms have been characterized functionally in IMCD subsegments: (1) active urea reabsorption in the apical membrane of initial IMCD from low-protein fed or hypercalcemic rats; (2) active urea reabsorption in the basolateral membrane of initial IMCD from furosemide-treated rats; and (3) active urea secretion in the apical membrane of terminal IMCD from untreated rats. This review focuses on the physiologic, biophysical, and molecular evidence for facilitated and active urea transporters, and integrative studies of their acute and long-term regulation in rats with reduced urine-concentrating ability.