Pathology and proposed pathophysiology of diclofenac poisoning in free-living and experimentally exposed oriental white-backed vultures (Gyps bengalensis)

Oriental white-backed vultures (Gyps bengalensis; OWBVs) died of renal failure when they ingested diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), in tissues of domestic livestock. Acute necrosis of proximal convoluted tubules in these vultures was severe. Glomeruli, distal convoluted tubules, and collecting tubules were relatively spared in the vultures that had early lesions. In most vultures, however, lesions became extensive with large urate aggregates obscuring renal architecture. Inflammation was minimal. Extensive urate precipitation on the surface and within organ parenchyma (visceral gout) was consistently found in vultures with renal failure. Very little is known about the physiologic effect of NSAIDs in birds. Research in mammals has shown that diclofenac inhibits formation of prostaglandins. We propose that the mechanism by which diclofenac induces renal failure in the OWBV is through the inhibition of the modulating effect of prostaglandin on angiotensin II-mediated adrenergic stimulation. Renal portal valves open in response to adrenergic stimulation, redirecting portal blood to the caudal vena cava and bypassing the kidney. If diclofenac removes a modulating effect of prostaglandins on the renal portal valves, indiscriminant activation of these valves would redirect the primary nutrient blood supply away from the renal cortex. Resulting ischemic necrosis of the cortical proximal convoluted tubules would be consistent with our histologic findings in these OWBVs.     

In situ hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys

Recent evidence suggests that a local reninangiotensin system is operational in the kidney and that it mediates some of the actions of angiotensin II on renal tubules. In this study the ontogeny and renal distribution of the unique precursor to angiotensin II formation, angiotensinogen, was investigated in rats by use of immunohistochemistry, immuno-electron microscopy and non-isotopic hybridization histochemistry. At the light-microscopic level, intense staining for angiotensinogen was found in the proximal convoluted tubules of the cortex, with lighter staining in the straight proximal tubules of the outer stripe. The strongest immunostaining was found in the kidneys of neonatal rats, where glomerular mesangial cells and medullary vascular bundles were also immunopositive. The angiotensinogen content of the kidneys in late gestation embryos and neonates showed the presence of angiotensinogen by day E18 and a peak content in the neonate. Non-isotopic hybridization histochemistry with biotinylated oligodeoxynucleotide probes confirmed the presence of angiotensinogen mRNA expression in the proximal convoluted tubules of the renal cortex. Electron-microscopic immunohisto-chemistry showed staining of relatively few electron-dense structures close to the apical membrane of proximal convoluted tubule cells in the adult kidney. In the neonatal rat kidney, angiotensinogen immunostaining at the electron-microscopic level was found throughout the proximal tubule cells and was markedly stronger than that seen in adult kidney. The presence of angiotensinogen, from embryonic day 18, in the proximal tubules, mesangial cells and vasculature of the kidney suggests multiple potential sites of intrarenal angiotensin II generation with an ontogeny in late gestation.     

Angiotensin i-converting enzyme in the nephron.

Goat antibody to swine kidney angiotensin I-converting enzyme was coupled to fluorescein isothiocyanate and was used to react with the converting enzyme in slices of swine kidney. Converting enzyme was present throughout the nephron and was concentrated in the convoluted tubules, especially in the proximal tubule. This finding is supported by high converting enzyme activity in the homogenate of Morris MK2 renal tumor, which is a transplanted adenocarcinoma of the proximal tubules of the rat kidney.     

Effects of subacute 3-monochloropropane-1,2-diol treatment on the kidney of male albino rats

3-Monochloropropane-1,2-diol (3-MCPD) is a well-known food contaminant. Although the kidney is thought to be a target organ for 3-MCPD toxicity, nephrotoxic structural changes are relatively unstudied. We investigated the renal alterations caused by 3-MCPD in male albino rats. 3-MCPD was administered orally, at a dose of 60 mg/kg for 7 days. 3-MCPD caused significant elevation of serum creatinine and urea levels together with hydropic degeneration, necrosis and shedding of the cells of the proximal convoluted tubules, urinary casts in the distal convoluted tubules and interstitial inflammatory cell infiltration. Administration of 3-MCPD for a period as short as 7 days causes acute renal failure in male albino rats.     

Histochemical studies on the uptake of horseradish peroxidase by rat kidney slices

When rat kidney slices were incubated in the presence of horseradish peroxidase, there was an energy-dependent uptake of the protein by the cells of the kidney tubules. The uptake was greatest in the proximal convoluted tubules and in the thick ascending limbs of the loops of Henle; it was abolished by cold, anoxia, 2,4-dinitrophenol, and fluoroacetate, and was more readily depressed by unfavorable metabolic conditions in the proximal convoluted tubules than in the thick ascending limbs. Protein uptake was inhibited when the kidney slices were incubated in electrolyte-free media. In sodium chloride solutions, uptake was reduced as sodium was progressively replaced by choline, and ouabain inhibited uptake in the proximal convoluted tubules, but not in the thick ascending limbs. To a limited extent, lithium could replace sodium in the incubation medium with no depression of peroxidase uptake. These results suggest that a sodium-stimulated, ouabain-sensitive ATPase may be involved in the uptake of protein by cells of the kidney tubule. The intracellular transport of peroxidase in cells of the proximal convoluted tubules was abolished by cold, anoxia, and 2,4-dinitrophenol, but it was not affected by concentrations of ouabain which inhibited the uptake of the protein.     

小熊猫肾脏和输尿管的组织学研究

小熊猫的肾脏呈蚕豆形,表面光滑不分叶,只有1个肾锥体和1个肾盏,无肾盂。肾脏皮质内可见皮质迷路和髓放线。皮质迷路内有近曲小管、远曲小管和肾小体等结构。髓放线内有近端小管直部和远端小管直部。髓质可分为外髓和内髓两个区域。外髓有较多的集合管断面,少量的远端小管直部和细段,较多的直小血管束。内髓部位有大量的细段和乳头管。各种泌尿小管之间有少量的疏松结缔组织构成的间质,间质内有丰富的毛细血管。输尿管横切面呈圆形或卵圆形,管腔呈不规则的裂隙状。管壁由粘膜、肌肉层和外膜组成。并与大熊猫肾脏和输尿管的组织结构作了比较研究。     

Expression of the membrane-associated carbonic anhydrase isozyme XII in the human kidney and renal tumors.

Carbonic anhydrase isozyme XII (CA XII) is a novel membrane-associated protein with a potential role in von Hippel-Lindau carcinogenesis. Although Northern blotting has revealed positive signal for CA XII in normal human kidney, this is the first study to demonstrate its cellular and subcellular localization along the human nephron and collecting duct. Immunohistochemistry with a polyclonal antibody (PAb) raised against truncated CA XII revealed distinct staining in the basolateral plasma membrane of the epithelial cells in the thick ascending limb of Henle and distal convoluted tubules, and in the principal cells of the collecting ducts. A weak basolateral signal was also detected in the epithelium of the proximal convoluted tubules. In addition to the normal kidney specimens, this immunohistochemical study included 31 renal tumors. CA XII showed moderate or strong plasma membrane-associated expression in most oncocytomas and clear-cell carcinomas. The segmental, cellular, and subcellular distribution of CA XII along the human nephron and collecting duct suggests that it may be one of the key enzymes involved in normal renal physiology, particularly in the regulation of water homeostasis. High expression of CA XII in some renal carcinomas may contribute to its role in von Hippel-Lindau carcinogenesis.     

Metabolic adaptation of the renal carbohydrate metabolism. II

Summary The effects of a high carbohydrate diet on the renal gluconeogenic and glycolytic capacities and on the activities of the main enzymes of the carbohydrate metabolism, fructose 1,6-bisphosphatase, phosphofructokinase and pyruvate kinase have been studied. These parameters have been analysed in two separate and isolated fractions of the renal tubule, the proximal convoluted (PCT) and the distal convoluted (DCT) zones. The results presented in this study show a rapid adaptation capacity of the kidney in response to the high amount of dietary carbohydrate, which are characterized by a decrease in the glucose production and fructose 1,6-bisphosphatase activity in the proximal tubules, and an increase in the glycolytic flux and phosphofructokinase and pyruvate kinase activities in the distal tubules. The changes in these enzyme activities took place only at subsaturating substrate concentrations and not at maximum velocity which suggest that they are probably due to an allosteric and/or covalent modifications and so, they are independent of variations in the cellular levels of the enzymes.     

Renal metallothionein responds rapidly and site specifically to zinc repletion in growing rats

Metallothionein (MT) is important for heavy metals and free radical protection in the kidney. MT is responsive to zinc and primarily localized within the renal cortex. However, site-specific renal responses to dietary zinc repletion are understudied. The objective of this study was to examine the effects of dietary zinc deficiency and repletion on renal MT concentration and immunolocalization in rats. Weanling male Sprague Dawley rats were randomly assigned to either a zinc-deficient, zinc control, or pair-fed to zinc-deficient group. Half of the zinc-deficient and pair-fed rats were repleted with the control diet ad libitum for an additional 24 h. Renal tissue samples were assessed for total zinc, MT concentrations and MT immunostaining. Dietary zinc deficiency reduced renal zinc and MT concentrations, and attenuated intensity and localization of MT. Dietary zinc repletion for 24 h restored renal zinc and MT concentrations, the latter primarily in the proximal convoluted tubules of the cortex. Concentrations of renal MT, but not zinc, were elevated by diet restriction and MT (μg/mg protein) and partially normalized by 24 h diet repletion. In conclusion, renal MT modification due to zinc deficiency or diet restriction can be rapidly normalized in a site-specific manner with normal dietary zinc intake. The results support a role for MT in kidney homeostasis, in particular at the level of the proximal tubules in the cortex. The speed of MT repletion may have clinical implications for dietary zinc in the treatment of acute and chronic renal pathology due to toxins and free radicals.     

Localization of aquaporin-7 in rat and mouse kidney using RT-PCR, immunoblotting, and immunocytochemistry

To establish the segmental, cellular, and subcellular localization of AQP7 in rat and mouse kidney, we used RT-PCR, immunocytochemical, and immunoblotting approaches. RT-PCR of rat and mouse kidney zones revealed AQP7 mRNA in cortex and outer stripe of the outer medulla. RT-PCR on microdissected nephron segments revealed AQP7 mRNA in proximal convoluted and straight tubules. Immunoblotting using peptide-derived rabbit antibodies to either rat or mouse AQP7 revealed a 28-kDa band in kidney and testes from rat and mouse, respectively. Immunocytochemistry revealed strong AQP7 labeling of segment 3 proximal tubules and weaker labeling of proximal convoluted tubules in both rat and mouse kidneys. The labeling was almost exclusively confined to the brush border with no basolateral labeling. No labeling was observed of thin descending limbs or collecting duct. Immunolabeling controls were negative. The presence of AQP7 in the proximal tubule brush border indicates a role of AQP7 in proximal tubule water reabsorption.     

Expression and function of arginine-producing and consuming-enzymes in the kidney

The kidney plays a key role in arginine metabolism. Arginine production is controlled by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) which metabolize citrulline and aspartate to arginine and fumarate whereas arginine consumption is dependent on arginine:glycine amidinotransferase (GAT), which mediates creatine and ornithine synthesis. Histological and biochemical techniques have been used to study the distribution and activity of these enzymes in anatomically dissected segments, in isolated fragments of tubules and in whole tissues. ASS and ASL mRNAs and proteins are expressed in the proximal tubule. Within this nephron segment, the proximal convoluted tubule has a higher arginine synthesis capacity than the proximal straight tubules. Furthermore, this arginine-synthesizing portion of the nephron matches perfectly with the site of citrulline reabsorption from the glomerular filtrate. The kidney itself can produce citrulline from methylated arginine, but this capacity is limited. Therefore, intestinal citrulline synthesis is required for renal arginine production. Although the proximal convoluted tubule also expresses a significant amount of GAT, only 10% of renal arginine synthesis is metabolized to guanidinoacetic acid, possibly because GAT has a mitochondrial localization. Kidney arginase (AII) is expressed in the cortical and outer medullary proximal straight tubules and does not degrade significant amounts of newly synthesized arginine. The data presented in this review identify the proximal convoluted tubule as the main site of endogenous arginine biosynthesis.     

Localization of thyroxine 5'-monodeiodinase activity in the renal proximal tubules in rabbits and rats

To determine the localization of T4 5'-monodeiodinase activity in rabbit and rat nephron segments, the formation of tri-iodothyronine (T3) from thyroxine (T4) was measured in kidney homogenate and in isolated nephron segments obtained by the microdissection method. In order of decreasing activity, homogenates of rabbit renal cortex, outer medulla and inner medulla were capable of converting T4 to T3. In the isolated nephron segments of the rabbit cortex, the activities were noted in both proximal convoluted and proximal straight tubules. On the other hand, the activities were not detected in segments including the cortical thick ascending limb of Henle's loop, the distal convoluted tubule, the connecting tubule, and the cortical collecting tubule. It is concluded that both the convoluted and the straight tubules are the sites of T3 production in the kidney.     

Differences in the incorporation of L- and DL-Amino acids into renal tubular cells. An autoradiographic study.

The cytoplasmic uptake of 3H-L-leucine and 3H-L-proline by hepatocytes and cells of the proximal and distal convoluted and of the collecting tubules of the kidney was compared with that of 3H-DL-leucine and 3H-DL-proline in an autoradiographic study. 34 male white Sprague-Dawley rats were killed 1, 2, 6, and 24 hours after the intraperitoneal injection of these amino acids. The rate of incorporation of 3H-L-leucine in the liver and in the renal tubules, as judged by the number of silver grains counted, was about twice that of 3H-L-proline. In the tubules of the kidney the intensity of labelling progressively declined from the proximal convoluted to the collecting tubules. When the two 3H-DL-amino acids were used, almost identical rates of incorporation were found in the liver as well as in the kidney. The only exception was the pars recta of the proximal tubule: Here there could be found an unusually high uptake of 3H-DL-proline. The values were not only higher than those found for the uptake of 3DL-leucine in this particular segment, but they also surpassed those due to 3H-DL-proline and 3DL-leucine in the other parts of the renal tubules, as well as in the liver. The conspicuously high labelling seen in the pars recta after the injection of 3H-DL-proline suggests that there is present in the cells of this segment a d-amino acid oxidase, which may be relatively specific for D-proline. The possibility is considered that this enzyme may participate in a detoxifying function of the pars recta.     

Membrane traffic after inhibition of endocytosis in renal proximal tubules

This study was performed to examine quantitatively the cellular organelles involved in membrane recycling after inhibition of luminal endocytosis in renal proximal tubules. Paraffin oil was microinfused into rat renal proximal convoluted tubules to prevent luminal endocytosis. After 1-2 hr the kidneys were fixed by perfusion and prepared for electron microscopy. Segment 1 proximal tubules infused with paraffin oil and control tubules from the same kidney were studied. In addition we examined proximal tubules from kidneys fixed by immersion 30 sec after removal of the kidney. In the oil-infused tubules the large endocytic vacuoles (greater than 0.5 micron) disappeared, the amount of small endocytic vacuoles (less than 0.5 micron) was reduced to about 10%, and the amount of dense apical tubules was significantly increased. The dense apical tubules were very seldom seen connected to the apical plasma membrane in controls but this was occasionally observed in tubules fixed by immersion and relatively often in oil-infused tubules. An ultrastructural morphometric analysis substantiated and extended the qualitative observations and provided quantitative estimates of volumes and surface areas for large endocytic vacuoles, lysosomes, mitochondria, small endocytic vacuoles, and dense apical tubules in control and experimental tubules. The results strongly support the suggestion that the dense apical tubules located in the apical cytoplasm represent the vehicle for the recycling of membrane from endocytic vacuoles back to the plasma membrane, and show that in renal proximal tubule cells small and large endocytic vacuoles are transformed into dense apical tubules when endocytosis is stopped.     

Comparison of the distribution of tissue kallikrein and esterase A, a kallikrein-like enzyme, in rat kidney using specific monoclonal antibodies

Tissue kallikrein (E.C. 3.4.21.35) and arginine esterase A, another closely related, kinin-generating serine protease, have been localized by immunocytochemistry in rat kidney, using monoclonal antibodies that do not crossreact with other kallikrein-related enzymes or with tonin. Kallikrein was present primarily in the apical cytoplasm of the connecting tubule and the cortical collecting duct. Esterase A, on the other hand, was present primarily in the basolateral region of both proximal and distal straight tubules in the outer medulla and medullary rays. In addition, esterase A was demonstrable in distal convoluted tubules and, to a lesser extent, in proximal convoluted tubules. The presence of different kinin-generating enzymes at these sites would permit the formation of kinins from appropriate substrates on both the vascular and luminal poles of separate segments of the kidney tubule.     

Mitochondrial and Metabolic Dysfunction in Renal Convoluted Tubules of Obese Mice: Protective Role of Melatonin

Obesity is a common and complex health problem, which impacts crucial organs; it is also considered an independent risk factor for chronic kidney disease. Few studies have analyzed the consequence of obesity in the renal proximal convoluted tubules, which are the major tubules involved in reabsorptive processes. For optimal performance of the kidney, energy is primarily provided by mitochondria. Melatonin, an indoleamine and antioxidant, has been identified in mitochondria, and there is considerable evidence regarding its essential role in the prevention of oxidative mitochondrial damage. In this study we evaluated the mechanism(s) of mitochondrial alterations in an animal model of obesity (ob/ob mice) and describe the beneficial effects of melatonin treatment on mitochondrial morphology and dynamics as influenced by mitofusin-2 and the intrinsic apoptotic cascade. Melatonin dissolved in 1% ethanol was added to the drinking water from postnatal week 5–13; the calculated dose of melatonin intake was 100 mg/kg body weight/day. Compared to control mice, obesity-related morphological alterations were apparent in the proximal tubules which contained round mitochondria with irregular, short cristae and cells with elevated apoptotic index. Melatonin supplementation in obese mice changed mitochondria shape and cristae organization of proximal tubules, enhanced mitofusin-2 expression, which in turn modulated the progression of the mitochondria-driven intrinsic apoptotic pathway. These changes possibly aid in reducing renal failure. The melatonin-mediated changes indicate its potential protective use against renal morphological damage and dysfunction associated with obesity and metabolic disease.     

Smad2/3蛋白及其活化形式在人肾脏中的表达和定位

目的研究Smad2和Smad3蛋白及其活化形式P—Smad2、P—Smad3在人正常肾脏组织中的表达、定位及其意义。方法采用免疫组织化学技术（SP法）检测20例人肾脏中Smad2、Smad3蛋白及P—Smad2、P-Smad3的表达和定位。结果Smad2、Smad3在肾小管、肾小球和集合小管中广泛表达,主要定位于细胞质,其中远端小管曲部呈强阳性;P-Smad2、p-Smad3也在肾脏皮、髓质中广泛分布,主要定位于细胞核,远端小管更多见。结论Smad2、Smad3在正常肾脏中有着活跃的功能。     

鸡肾发生的组织学观察

采用组织学方法和电镜技术,对9个不同发育时期的鸡(Callus domestiaus)胚胎进行了观察.通过对鸡胚胎肾组织发生过程的观察,探讨鸡胚中肾的发生与退化,后肾的发生、分化规律和特点.结果表明,孵育到第16期在中肾前端附近出现一些中肾小泡.孵育到第18期形成中肾小管.孵育到第26期,中肾小管的盲端内陷,原始的肾小囊和肾血管球形成,中肾小管显著伸长并迂回曲折.孵育到第33～37期,体前后部中肾组织均已形成完整的肾单位.第37～46期体前部至后部的中肾组织依次退化.孵育到第26期从泄殖腔附近发出的输尿管芽向生后肾组织侵入生长,生后.肾组织产生许多生后肾小泡.第33期出现肾小囊和肾小管,肾小管伸长并发生折叠,出现集合小管、近端小管和远端小管的形态分化.第37～46期肾小体逐渐发育成熟,肾小管继续分化出现细段.鸡的中肾具有排泄功能.鸡后肾的发生与分化存在明显的时间差异.肾单位的分化中,同一胚龄肾组织内可存在不同发育阶段的肾小体,集合小管分化较早,诱导近端小管和远端小管分化,细段分化较迟.     

Eucalyptus foliar chemistry explains selective feeding by koalas

The koala is the quintessential specialist herbivore, feeding almost exclusively on Eucalyptus foliage. Consequently, the limitations imposed on the koala's diet by plant defences indicate the extent to which evolutionary adaptations allow mammalian herbivores to circumvent such defences. We tested whether a recently discovered group of plant secondary metabolites, the formylated phloroglucinol compounds (FPCs), deters koalas from feeding on some eucalypt foliage. We found that captive koalas ate less foliage in a single night from trees with high FPC concentrations. Individual trees also differ in the types of FPC they possess, but for a given eucalypt species, most FPCs were similarly effective deterrents. Two closely related and sympatric eucalypt species could be clearly separated by the amounts that koalas ate from each; however, this difference could not be explained by total FPC concentrations alone. We suggest, that in this case, the presence of a distinct type of FPC deters koala herbivory on the less palatable species, and may have facilitated the evolutionary divergence of these species. We conclude that plant defences probably play an important role in determining the distribution and abundance of koalas.     

Effect of phosphate deprivation on enzymes of the cyclic adenosine monophosphate metabolism in rat proximal convoluted and proximal straight tubules.

Phosphate deprivation causes a resistance to the phosphaturic effect of parathyroid hormone (PTH). The present study determined whether acute phosphate deprivation alters basal or stimulated activities of key enzymes of the cyclic adenosine monophosphate (cAMP) metabolism in microdissected proximal convoluted and proximal straight tubules, since blunted cAMP levels in these proximal subsegments might account for refractoriness to the effect of PTH on phosphate reabsorption in the proximal convoluted and proximal straight tubule segments. In the proximal convoluted tubules of rats fed a normal-phosphate diet (NPD), PTH increased the adenylate cyclase activity by tenfold. In the proximal convoluted tubule of rats fed a low-phosphate diet (LPD), PTH also increased the adenylate cyclase activity by tenfold. In addition, forskolin increased the adenylate cyclase activity to levels similar to PTH in the proximal convoluted tubule of rats fed NPD or LPD. In the proximal straight tubule of rats fed NPD, PTH resulted in an approximately fivefold increase in adenylate cyclase activity. In the proximal straight tubule of rats fed LPD, PTH resulted in a fourfold increase in adenylate cyclase activity. The forskolin-stimulated adenylate cyclase activity was markedly decreased (46%) in the proximal straight tubule of phosphate-deprived rats. The cAMP-phosphodiesterase activity in the proximal convoluted tubule was significantly increased by 26% in phosphate-deprived rats. The cAMP-phosphodiesterase activities in the proximal straight tubules from rats fed NPD or LPD were similar. We conclude that distinct differences in key enzymes of cAMP metabolism exist in the proximal convoluted and proximal straight tubule subsegments. Further, phosphate deprivation affects the cAMP-phosphodiesterase and adenylate cyclase activities differently in these nephron subsegments.(ABSTRACT TRUNCATED AT 250 WORDS)