Cell junctions in the renal tubule of a fresh-water teleost,Salmo gairdneri Rich.

Summary Cell junctions in the renal tubule of the fresh-water rainbow trout were studied with thin-section and freeze-fracture techniques. Gap junctions were restricted to the proximal tubule, which is consistent with other vertebrate classes. Segments I and II of the proximal tubule and the collecting tubule/collecting duct system exhibited a well-developed zonula occludens with anastomosing strands. The distal segment showed a narrow zonula occludens composed of few parallel strands. The structure of the occluding junctions along the renal tubule of this teleost displays several similarities with the pattern of the zonulae occludentes in the amphibian and the mammalian nephron. From these observations, in conjunction with available data from other vertebrate classes, it can be concluded that in the proximal tubule the development of a deep and complex zonula occludens is a general feature of cold-blooded vertebrates.     

Peptidases in the kidney of male rats following castration and testosterone substitution

The localization of aminopeptidase M (APM), dipeptidyl peptidase I (DAP I), II (DAP II) and IV (DAP IV) in the renal section was investigated histochemically, and their activities were determined fluorometrically in renal homogenate of normal, castrated and testosteron treated male rats.--After castration the activities of the lysosomal DAP II (pars convoluta of the proximal tubule), DAP I (distal and proximal tubule) and of the mainly membrane-bound DAP IV (glomeruli, brush border of the proximal tubule) increase in comparison to normal males, whereas the activities of the brush border-bound APM decrease. After testosteron treatment of castrated animals (0.1, 0.5 and 1.0 mg testosterone proprionate/100 g BW and day; 5-day treatment) the activities of DAP I, II and IV decrease again, so that after treatment with 0.1 mg testosterone proprionate, the activities of DAP I and II approach those in normal males.--The additionally determined urinary protein excretion shows that there is a significant decrease in proteinuria after castration, whereas testosterone treatment of castrated animals is accompanied by an increase of proteinuria.--Our results would suggest that the protein catabolism in the proximal tubule and the proteinuria are interrelated, and that testosterone influences (decreases) the protein catabolism in the proximal tubule. This means that high activities of lysosomal proteinases in the proximal tubule (castrates) are accompanied by a low proteinuria, and low activities of those proteinases (testosterone treated castrated or normal males) by a high proteinuria.     

Fungi isolated from cultured eggs, alevins and broodfish of brown trout in a hatchery affected by saprolegniosis

The aquatic fungi cultured from eggs, alevins and broodfish of brown trout Salmo trutta belonged to the genus Saprolegnia and were identified as S. diclina , S. australis , S. ferax , S. furcata , S. hypogyna , S. unispora and S. parasitica . The species obtained from infected eggs and alevins were different to those from infected fish. Several Saprolegnia species were isolated from eggs and alevins, whereas all the isolates obtained from broodfish were the pathogenic S. parasitica .     

Cloning of rainbow trout SLC26A1: involvement in renal sulfate secretion

The kidney plays an important role in ion regulation in both freshwater and seawater fish. However, ion transport mechanisms in the teleost kidney are poorly understood, especially at the molecular level. We have cloned a kidney-specific SLC26 sulfate/anion exchanger from rainbow trout (Oncorhynchus mykiss) that is homologous to the mammalian SLC26A1 (Sat-1). Excretion of excess plasma sulfate concentration after Na2SO4 injection corresponded to significantly higher expression of the cloned SLC26A1 mRNA. Detailed morphological observation of rainbow trout renal tubules was also performed by light microscopy and transmission electron microscopy. According to the structure of brush border and tubular system in the cytoplasm, renal tubules of rainbow trout were classified into proximal tubule first and second (PI and PII) segments and distal tubules. In situ hybridization revealed that SLC26A1 anion exchanger mRNA is specifically localized in the PI segment of kidneys from both seawater- and freshwater-adapted rainbow trout. With immunocytochemistry, Na+-K+-ATPase and vacuolar-type H+-ATPase were colocalized to the same cells and distributed in the basolateral and the apical membranes, respectively, of the cells where the SLC26A1 mRNA expressed. These findings suggest that the cloned kidney-specific SLC26A1 is located in kidney proximal tubules and is involved in excretion of excess plasma sulfate in rainbow trout.     

亲本死亡时间对近太湖新银鱼人工授精受精率的影响

银鱼捕起后易死,较难用活的亲本进行人工繁殖,在实际生产中,多采用死的亲本进行人工受精,亲本死亡的时间对卵的受精率影响较大。本文对徐家河水库近太湖新银鱼亲本死亡时间与卵受精率的关系进行了系列研究,亲本死亡后240分钟内,每隔15分钟进行一次人工受精,结果表明,卵的受精率随着亲本死亡时间的延长,其受精率越来越低,240分钟后,受精率趋于0。水温在15℃以下,亲本死亡105分钟后进行人工受精,平均受精率为43%,因此建议水温在15℃以下近太湖新银鱼的人工受精应在亲本死亡后105分钟之内进行。       

Reabsorption of fluorescein-isothiocyanate-labelled-ovalbumin in the kidney of normal and castrated male and female rats

Summary The reabsorption of ovalbumin double labelled with fluorescein isothiocyanate (FITC) in the kidneys of normal and castrated male and female rats was investigated using fluorometry and fluorescence microscopy. The animals received an intravenous injection of either 2 or 7 mg fluorescein-thiocarbamyl (FTC)-ovalbumin per kilogram bodyweight (bw) and were killed 4 or 8 min post-injection. Animals injected with unlabelled ovalbumin (7.0 mg/kg bw) served as controls. Fluorescence microscopy revealed that FTC-ovalbumin was reabsorbed exclusively in the renal proximal tubule, the highest level of reabsorption being observed in its first part. Four and eight minutes after the mjection, FTC-ovalbumin was only observed in apical reabsorption vacuoles, with lysosomes exhibiting no specific fluoreseence. Fluorometric determinations for the renal homogenate supernatant showed that the renal reabsorption of FTC-ovalbumin was up to 24% higher in normal females than in normal males. Castration resulted in a significant increase in renal reabsorption in male rats (up to 38%; significant), whereas a minor decrease was observed in castrated females. The renal uptake differences in normal and castrated animals are discussed in the light of the sex-hormone-dependent catabolism of lysosomal proteins in the renal proximal tubule of rats.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Reabsorption of fluorescein-isothiocyanate-labelled-ovalbumin in the kidney of normal and castrated male and female rats

The reabsorption of ovalbumin double labelled with fluorescein isothiocyanate (FITC) in the kidneys of normal and castrated male and female rats was investigated using fluorometry and fluorescence microscopy. The animals received an intravenous injection of either 2 or 7 mg fluorescein-thiocarbamyl (FTC)-ovalbumin per kilogram bodyweight (bw) and were killed 4 or 8 min post-injection. Animals injected with unlabelled ovalbumin (7.0 mg/kg bw) served as controls. Fluorescence microscopy revealed that FTC-ovalbumin was reabsorbed exclusively in the renal proximal tubule, the highest level of reabsorption being observed in its first part. Four and eight minutes after the injection, FTC-ovalbumin was only observed in apical reabsorption vacuoles, with lysosomes exhibiting no specific fluorescence. Fluorometric determinations for the renal homogenate supernatant showed that the renal reabsorption of FTC-ovalbumin was up to 24% higher in normal females than in normal males. Castration resulted in a significant increase in renal reabsorption in male rats (up to 38%; significant), whereas a minor decrease was observed in castrated females. The renal uptake differences in normal and castrated animals are discussed in the light of the sex-hormone-dependent catabolism of lysosomal proteins in the renal proximal tubule of rats.     

Hybridization dynamics between sympatric species of trout: loss of reproductive isolation

Although reinforcement should enhance reproductive barriers in sympatric species, sympatric trout species do hybridize. Using mitochondrial and nuclear species markers, we investigated hybridization directionality, hybrid mating biases, and selection against hybrids in 13 sympatric cut-throat and rainbow trout populations on Vancouver Island, Canada. Approximately 50% of the genotyped fish were hybrid (F1 or higher-order) and populations ranged from very recent (all F1 hybrids) to extremely advanced higher-order hybridization. Overall, interbreeding was reciprocal, although some populations showed directional hybridization. Pronounced cytonuclear disequilibrium in post-F1 hybrids indicated a remarkable mating bias not previously reported, which is most likely because of behavioural reproductive preferences. Selection against hybrids was observed in only two populations, indicative of extrinsic selection. Two populations were 'hybrid swarms', with a complete loss of reproductive isolation. The complex hybridization dynamics in this system represent a valuable natural experiment of the genetic and evolutionary implications of recent and on-going interspecific hybridization.     

Acute renal venous obstruction is more detrimental to the kidney than arterial occlusion: implication for murine models of acute kidney injury

In this study, we compared the traditional murine model with renal pedicle clamp with models that clamped the renal artery or vein alone as well as to a whole body ischemia-reperfusion injury (WBIRI) model. Male C57BL/6J mice underwent either clamping of the renal artery, vein, or both (whole pedicle) for 30 or 45 min followed by reperfusion, or 10 min of cardiac arrest followed by resuscitation up to 24 h. After 30 min of ischemia, the mice with renal vein clamping showed the mostly increased serum creatinine and the most severe renal tubule injury. After 45 min of ischemia, all mice with renal vasculature clamping had a comparable increase in serum creatinine but the renal tubule injury was most severe in renal artery-clamped mice. Renal arterial blood flow was most decreased in mice with a renal vein clamp compared with a renal artery or pedicle clamp. A 30-or 45-min renal ischemia time led to a significant increase in the protein level of interleukin-6, keratinocyte-derived chemokine (KC), and granular colony-stimulating factor in the ischemic kidney, but the KC was the highest in the renal pedicle-clamped kidney and the lowest in the renal vein-clamped kidney. Of note, 10 min of WBIRI led to kidney dysfunction and structural injury, although less than longer time clamping of isolated renal vasculature. Our data demonstrate important differences in ischemic AKI models. Understanding these differences is important in designing future experimental studies in mice as well as clinical trials in humans.     

Peptidases in the kidney and urine of rats after castration

Summary The localization of various peptidases in the renal section of the rat was investigated histochemically, and their activities were determined fluorometrically in renal homogenate. The membrane-bound peptidases aminopeptidase A (APA), aminopeptidase M (APM), -glutamyl-transferase (-GT), dipeptidylpeptidase IV (DAP IV), and the lysosomal dipeptidyl peptidases I (DAP I) and II (DAP II) were investigated in male and female (estrus) rats both before and 30 days after castration. In addition, protein excretion and APA, APM, DAP I and DAP IV activities were measured in the urine of these animals. Histochemically, the membrane-bound peptidases are demonstrable mainly in the brush borders of the proximal tubules. In addition, APA and DAP IV are found in the glomeruli, -GT and DAP IV in the thin descending limbs of the loops of Henle, and -GT in the basal labyrinth of the S₂ and S₃ segments. The lysosomal peptidases are most concentrated in the S₁ and S₂ segments of the proximal tubule, in the distal tubule, and in certain cells of the connecting tubule and collecting duct, where they are contained in lysosomes of varying size. Sex differences and castration effects are demonstrable both histochemically and biochemically for the investigated peptidases. Histochemically these effects are most pronounced in the S₃ segments for the membrane-bound peptidases, and in the lysosomes of the proximal tubule for the lysosomal peptidases. Biochemical tests in controls show significantly higher lysosomal peptidase activities in the renal homogenate of females than of males. After castration the lysosomal peptidase activities in males increase, approaching those of females. This appears to have bearing on the sex-dependent proteinuria in rats, for lysosomal peptidases and proteinases are particularly important in the degradation of filtered proteins that are reabsorbed in the proximal tubule. In females high lysosomal peptidase activities correlate with a low proteinuria, while males demonstrate lower lysosomal peptidase activities and a significantly higher proteinuria than females. After castration, the lysosomal peptidase activities and proteinuria in males approach those in females. Renal peptidases are also excreted in the urine, again with sex differences, and so these excreted peptidases contribute to the proteinuria in rats.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Proximal tubule function in chronic serum sickness glomerulonephritis of rats

Fatal immune complex glomerulonephritis can be induced in rats by chronic intravenous administration of bovine serum albumin. There are three distinct stages, mild, moderate, and severe, in the development of renal immunopathology and pathophysiology in this model of chronic serum sickness. The work described here was undertaken to evaluate aspects of proximal tubule function in those different stages. Tissue water distribution, oxidative metabolism, and transport of representative organic anions and cations were measured in renal cortical slices. In mild chronic serum sickness all functions were normal except the transport of p-aminohippurate (PAH, organic anion), which was significantly decreased. This decrease appeared to be attributable to immunization with Freund's adjuvant. In the moderate stage of chronic serum sickness, proximal tubule functions and morphology appeared essentially normal. Only Na-K-ATPase activity was somewhat lower than in controls. However, proximal tubule dysfunction was a feature of severe chronic serum sickness. A significant inhibition of anion and cation transport was observed. Reduction in transport functions occurred together with impaired oxidative metabolism and severe reduction in Na-K-ATPase activity. Abnormalities of mitochondrial structure, a decrease in number of mitochondria, and a significant increase in intracellular H2O content provided additional evidence of degenerative changes in proximal tubule cells during the severe stage of chronic serum sickness. It was concluded that decreased transport of organic ions by the basolateral membrane in proximal tubules of rats with severe chronic serum sickness resulted from a breakdown in the metabolic machinery of the tubule epithelium rather than a specific injury to organic ion transport systems.     

Aromatase expression in testes of XY, YY, and XX rainbow trout (Oncorhynchus mykiss)

The main goal of the present study was to show whether testicular cells of rainbow trout (Oncorhynchus mykiss Walbaum) either hormonally manipulated (XX males) or produced by using gamma irradiation and pressure shock (YY males, "supermales") are able to aromatize androgens into estrogens compared with the control (XY males). The expression of aromatase gene at the level of the protein and its presence in testicular tissue was investigated by means of immunohistochemistry and Western blot analysis, respectively. The positive staining for aromatase was detected in testicular cells of all trout and in efferent duct cells of XY and YY males. However, the staining intensity varied among particular trout, being strong in YY males, moderate in XY males, and weak in XX trout. It was confirmed by quantitative image analysis in which the staining intensity was expressed as relative optical density (ROD) of diaminobenzidine deposits. Significant differences were found between XY and YY trout ((**)p<0.01) and XY and XX trout ((*)p<0.05). Such differences could reflect various levels of estrogens, possibly dependent on the genetic background of the trout studied. It seems likely that differential expression of the enzyme, especially that of weak or strong intensity, causes some alterations in testicular morphology of homogametic trout. Additionally, the results indicate that an imbalance in sex hormone biosynthesis may provoke the functional alterations in testes of YY males, and, in consequence, negatively affect the fertility of "supermales".     

Aromatase expression in testes of XY, YY, and XX rainbow trout (Oncorhynchus mykiss).

The main goal of the present study was to show whether testicular cells of rainbow trout (Oncorhynchus mykiss Walbaum) either hormonally manipulated (XX males) or produced by using gamma irradiation and pressure shock (YY males, "supermales") are able to aromatize androgens into estrogens compared with the control (XY males). The expression of aromatase gene at the level of the protein and its presence in testicular tissue was investigated by means of immunohistochemistry and Western blot analysis, respectively. The positive staining for aromatase was detected in testicular cells of all trout and in efferent duct cells of XY and YY males. However, the staining intensity varied among particular trout, being strong in YY males, moderate in XY males, and weak in XX trout. It was confirmed by quantitative image analysis in which the staining intensity was expressed as relative optical density (ROD) of diaminobenzidine deposits. Significant differences were found between XY and YY trout ((**)p<0.01) and XY and XX trout ((*)p<0.05). Such differences could reflect various levels of estrogens, possibly dependent on the genetic background of the trout studied. It seems likely that differential expression of the enzyme, especially that of weak or strong intensity, causes some alterations in testicular morphology of homogametic trout. Additionally, the results indicate that an imbalance in sex hormone biosynthesis may provoke the functional alterations in testes of YY males, and, in consequence, negatively affect the fertility of "supermales".     

Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells

The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.     

Electron microscopic study of the innervation of the renal tubules and urinary bladder epithelium in Rana catesbeiana and Necturus maculosus

The fine structure of the kidney and the bladder of the bullfrog (Rana catesbeiana), the bullfrog tadpole, and the mudpuppy (Necturus maculosus) were studied with special attention to the innervation of renal tubule cells and bladder epithelial cells. In the bullfrog kidney, nerve terminals and varicosities were frequently associated with the tubule cells, apparently in an increasing order from the proximal tubule to the connecting tubule. Although these terminals and varicosities did not directly contact the tubular cell membrane, an aggregation of synaptic vesicles on the side facing the tubule was considered as morphological evidence that neurotransmitter can be released here and can affect the transport activity of the tubule cells. The association of nerve varicosities with canaliculi cells in the connecting tubule was also demonstrated. In the bullfrog tadpoles, renal tubule cells were occasionally innervated. In the mudpuppy, renal tubule cells were only poorly innervated. The epithelium of the bullfrog bladder was commonly innervated. Nerve terminals with synaptic vesicles were located very near basal cells and even contacted them directly on rare occasions. In the mudpuppy, the innervation of the bladder epithelium was observed infrequently. The bullfrog tadpoles did not possess an apparent bladder. In all materials studied, renal arterioles and bladder smooth muscle cells were innervated.     

Alteration of surfactant protein A expression in renal cell carcinoma

Surfactant protein-A (SP-A) belongs to a family of collagen-containing C-type lectins called collectins. SP-A is expressed by renal tubule epithelial cells. We investigated the distribution of SP-A in renal cell carcinomas (RCC) using immunohistochemical techniques and western blotting. We used 35 formalin fixed, paraffin embedded (FFPE) RCC tissue samples. We compared results with clinico-pathological parameters of RCC including age, sex, Fuhrman grade, tumor volume, tumor node metastasis (TNM) and clinical stage. SP-A was localized in the glomerulus and renal tubule epithelium in nontumor tissue and strong SP-A immunoreactivity was observed in tumor tissue. SP-A was expressed in the RCC tumor cells (64%) and nontumor cells (34%) in males and RCC tumor cells (90%) and nontumor cells (30%) in females. There was a significant correlation between SP-A immunoreactivity in tumor cells and gender, age, tumor diameter, Fuhrman grade and tumor diameter. Western blot analysis supported the immunohistochemical findings. We present evidence for involvement of SP-A in RCC and suggest that increased SP-A expression in RCC is associated with favorable prognosis.     

A1 adenosine receptor allosteric enhancer PD-81723 protects against renal ischemia-reperfusion injury

Activation of A(1) adenosine receptors (ARs) protects against renal ischemia-reperfusion (I/R) injury by reducing necrosis, apoptosis, and inflammation. However, extrarenal side effects (bradycardia, hypotension, and sedation) may limit A(1)AR agonist therapy for ischemic acute kidney injury. Here, we hypothesized that an allosteric enhancer for A(1)AR (PD-81723) protects against renal I/R injury without the undesirable side effects of systemic A(1)AR activation by potentiating the cytoprotective effects of renal adenosine generated locally by ischemia. Pretreatment with PD-81723 produced dose-dependent protection against renal I/R injury in A(1)AR wild-type mice but not in A(1)AR-deficient mice. Significant reductions in renal tubular necrosis, neutrophil infiltration, and inflammation as well as tubular apoptosis were observed in A(1)AR wild-type mice treated with PD-81723. Furthermore, PD-81723 decreased apoptotic cell death in human proximal tubule (HK-2) cells in culture, which was attenuated by a specific A(1)AR antagonist (8-cyclopentyl-1,3-dipropylxanthine). Mechanistically, PD-81723 induced sphingosine kinase (SK)1 mRNA and protein expression in HK-2 cells and in the mouse kidney. Supporting a critical role of SK1 in A(1)AR allosteric enhancer-mediated renal protection against renal I/R injury, PD-81723 failed to protect SK1-deficient mice against renal I/R injury. Finally, proximal tubule sphingosine-1-phosphate type 1 receptors (S1P(1)Rs) are critical for PD-81723-induced renal protection, as mice selectively deficient in renal proximal tubule S1P(1)Rs (S1P(1)R(flox/flox) PEPCK(Cre/-) mice) were not protected against renal I/R injury with PD-81723 treatment. Taken together, our experiments demonstrate potent renal protection with PD-81723 against I/R injury by reducing necrosis, inflammation, and apoptosis through the induction of renal tubular SK1 and activation of proximal tubule S1P(1)Rs. Our findings imply that selectively enhancing A(1)AR activation by locally produced renal adenosine may be a clinically useful therapeutic option to attenuate ischemic acute kidney injury without systemic side effects.     

Differential handling of urea and its analogues suggests carrier-mediated urea excretion in freshwater rainbow trout

The possible presence of urea transport mechanisms in the gill and kidney of the freshwater rainbow trout (Oncorhynchus mykiss) was investigated in vivo by comparing the branchial and renal handling of analogues acetamide and thiourea with the handling of urea. Trout were fitted with indwelling dorsal aortic catheters and urinary catheters and injected with an isosmotic dose of [(14)C]-labeled urea analogue (acetamide or thiourea) calculated to bring plasma analogue concentrations close to plasma urea concentrations. Urea and analogue concentrations were significantly greater in the urine than in the plasma. Branchial clearance rate of acetamide was only 48% of urea clearance, whereas the clearance of thiourea was only 22%, a pattern that was also observed in branchial uptake of these substances and was similar to our previous observations in toadfish and midshipmen. The renal secretion clearance rates of urea and acetamide were similar, and on average, both substances were secreted on a net basis, although reabsorption did occur in some cases. In contrast, thiourea was neither reabsorbed nor secreted by the kidney tubule. The secretion clearance rates of both acetamide and urea were well correlated with the secretion clearance rates of Na(+), Cl(-), and water, whereas there was no relationship between thiourea and these substances. The pattern of acetamide, thiourea, and urea handling by the gill of the trout is similar to that found in the gills of the midshipman and the gulf toadfish and strongly suggests the presence of a UT-type facilitated diffusion urea transport mechanism. The pattern of differential handling in the kidney is unlike that in the gill and also unlike that in the kidney of the midshipman and the gulf toadfish, suggesting a different mechanism. In addition, renal urea secretion occurs against a concentration gradient, suggesting the involvement of an active transport mechanism.     

Differential effects of 3,4,5,3',4',5' -hexachlorobiphenyl (hcb) on interrenal steroidogenesis in male and female rainbow trout Oncorhynchus mykiss

1. The conversion of progesterone to 17α-hydroxyprogesterone (17α-OHP), 11-deoxycortisol (11-DOCR) and deoxycorticosterone (DOC) was significantly higher in female rainbow trout than in male trout; in contrast, the interrenal production of cortisol (CR) plus cortisone (CN) was higher in males than in females.2. Following treatment with 1 mg/kg of HCB, the interrenal conversion of progesterone to 17α-OHP and 11-DOCR was significantly increased in male and female trout but at 20 mg/kg of HCB, the production of these metabolites was increased in males and decreased in females; CR + CN production was unchanged after HCB treatment in both sexes.