Ochratoxin A-induced mycotoxic porcine nephropathy: alterations in enzyme activity in tubular cells.

Mycotoxic porcine nephropathy was induced by p.o. administration of crystalline ochratoxin A for periods of 5 days, 3 months and 2 years. Enzyme activities of the renal tissue were studied histochemically. These were NADH-tetrazolium reductase, NADPH-tetrazolium reductase, lactate dehydrogenase, isocitrate dehydrogenase, succinate dehydrogenase, glucose-6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, unspecific acid phosphatase and unspecific alkaline phosphatase. The activity of NADH-tetrazolium reductase and succinate dehydrogenase was reduced in the proximal tubule of all nephrons after 5 days ochratoxin A exposure and remained reduced after 3 months and 2 years exposure. The effect of ochratoxin A on these enzymes would appear to cause the impairment of proximal tubular function and the morphological changes observed in the proximal tubule in ochratoxin A-induced mycotoxic porcine nephropathy. The localization of alterations in enzyme activity corresponds to the localization of ochratoxin A previously demonstrated in the kidney. The activities of NADPH-tetrazolium reductase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and unspecific alkaline phosphatase were reduced focally corresponding to the areas with focal tubular atrophy and the degree of reduction was roughly parallel to the degree of atrophy.     

Comparative responses to mode of oral administration and dose of ochratoxin A or nephrotoxic extract of Penicillium polonicum in rats

Administration of Penicillium polonicum extract to male Sprague-Dawley rats (200 g), either mixed in feed or given daily by gavage, for 5 days, had no clinical effects. However, at necropsy on day 6 marked histopathological changes occurred in renal tubule epithelia, including mitotic figures, karyomegalic nuclei, and frequent apoptosis identified specifically by TUNEL methodology and confocal microscopy. Ochratoxin A given similarly to rats (daily, 1 mg or 0.2 mg) was also clinically asymptomatic except for the 1 mg dose given by gavage; rats in this group lost weight. Marked renal tubular necrosis, though even without any significant accompanying apoptosis, was evident only at this higher dose by gavage; it was associated also with the highest incidence of renal DNA adducts and a disproportionately high concentration of ochratoxin A in plasma on day 6. Significantly fewer renal DNA adducts were detected in rats given 1 mg ochratoxin A in feed. The study demonstrates the potential for exaggerated toxicological responses to ochratoxin A administered by gavage through predicted consequential surges in the circulating concentration of the mycotoxin.     

Fumonisin B1 and ochratoxin A nephrotoxicity in Japanese quail: an ultrastructural assessment

In the present study, fumonisin B₁ (200 ppm) and ochratoxin A (2 ppm) were fed alone and in combination to day old Japanese quail chicks for 21 days to evaluate renal ultrastructural changes. The severity and intensity of renal ultrastructural changes varied with the type of treatment, and predominant and consistent lesions were found in the proximal convoluted tubule (PCT) lining cells. Distortion and dilatation of cisternae of endoplasmic reticulum leading to the formation of vesicular and tubular structures was a consistent finding noticed in the fumonisin B₁-fed group. Ochratoxin A treatment revealed characteristic changes in the epithelial brush border of PCTs with blunting and variable loss of microvilli. Reduced number of mitochondria with mitochondrial pleomorphism (round, oval, curved, elongated, dumbbell, banana, pear, and tennis racket) was almost a consistent finding noticed in the ochratoxin A treatment group. Ultrastructural changes in the combination fed group were almost similar to those noticed in the individual treatment groups but were of a higher intensity. Or in other words, we can say that on simultaneous exposure, fumonisin B₁ potentiated the toxic effects of OTA on renal ultrastructure.     

Human exposure to ochratoxin A in areas of Yugoslavia with endemic nephropathy

Ochratoxin A is a mycotoxin with pronounced nephrotoxic potency in all species of single-stomach animals studied; it is a major disease determinant of porcine nephropathy and a disease occurring endemically in several countries. This disease is comparable with Balkan (endemic) nephropathy, suggesting a common causal relationship. Ochratoxin A has been found in foodstuffs in many countries, but the highest frequency of ochratoxin A contamination in foods (10.3% of 1,553 samples of foodstuffs) was encountered in an area of Yugoslavia, where Balkan (endemic) nephropathy is prevalent. Detection of ochratoxin A in human blood samples confirmed the prevalent exposure to this food contaminant. Relative risk calculations indicated a tendency to an association between this mycotoxin and Balkan (endemic) nephropathy, supporting the hypothesis of a causal role of ochratoxin A in this disease.     

Effects of two metabolites of ochratoxin A, (4R)-4-hydroxyochratoxin A and ochratoxin alpha, on immune response in mice.

The metabolites of ochratoxin A, (4R)-4-hydroxyochratoxin A and ochratoxin alpha, were investigated for immunosuppressive properties in BALB/c mice. The standard plaque-counting technique for the estimation of antibody-producing spleen lymphocytes was used. (4R)-4-hydroxyochratoxin A was found to be an immunosuppressor almost as highly effective as ochratoxin A. Doses of 1 microgram of (4R)-4-hydroxyochratoxin A per kg administered to mice caused an 80% reduction in the number of cells producing immunoglobulin M (90% with ochratoxin A) and a 93% reduction in cells synthesizing immunoglobulin G (92% with ochratoxin A). Ochratoxin alpha, however, was ineffective. A possible mode of action is discussed.     

A micropuncture study of the renal handling of lithium

Although clearance studies in man and experimental animals indicate that filtered lithium is reabsorbed primarily in the proximal tubule, it is unclear whether lithium is also reabsorbed in distal portions of the nephron. Micropuncture studies were, therefore, performed to determine the nephron sites involved in lithium transport during free flow. A method was established to estimate the concentration of lithium in nanoliter samples, using the Helium Glow photometer, which permitted the accurate measurement of lithium in tubular fluid samples over a range from 0.5–30.0 mM.Approximately 56% of filtered lithium and tubular fluid was reabsorbed at the end of the proximal convolution, while at the early distal tubule 75% of filtered lithium and water was reabsorbed. There was no change in net transepithelial movement of lithium beyond the loop of Henle.These data suggest that lithium transport is localized to the proximal tubule, including the pars recta. Lithium reabsorption does not occur in distal tubule or collecting duct. Beyond the early distal tubule net movement of lithium and sodium is dissociated.     

Analysis of wheat and kidney samples for ochratoxin a using immunoaffinity columns in conjunction with HPLC

Ochratoxin A is a highly nephrotoxic mycotoxin commonly associated with cereals and cereal products. Residues of the toxin are also frequently found in animal tissues, e.g. pig kidney, when animals are fed with contaminated feed. The analysis of foodstuffs for ochratoxin A has been simplified by the introduction of immunoaffinity methods such as the Biocode ochratoxin EASI EXTRACT column. These columns contain monoclonal antibodies raised against ochratoxin A and allow purification and concentration of the toxin from a foodstuff. Analysis involves a solvent extraction stage prior to application to the ochratoxin EASI EXTRACT column, with detection of the toxin by high‐pressure liquid chromatogra‐phy. Several solvent extraction systems have been evaluated for both wheat and kidney. Percentage recoveries from spiked material ranged between approximately 35 and 80% for kidney samples and between approximately 65 and 80% for wheat samples depending on the extraction solvent used. Immunoaffinity analysis for ochratoxin A appears to be the method of choice for accurate quantification.     

Ochratoxin A levels in human serum and foods from nephropathy patients in Tunisia: Where are you now?

Ochratoxin A is a natural mycotoxin with nephrotoxic properties that can contaminate food products. It has been detected in high amount in human serum collected from nephropathy patients, especially those categorized as having a chronic interstitial nephropathy of unknown etiology. In the present study, ochratoxin A levels were measured in commonly consumed food items and in serum samples from nephropathy and healthy subjects in Tunisia. To assess ochratoxin A, a high performance liquid chromatography method was optimized. The ochratoxin A assay showed very different scales of ochratoxin A serum and food contamination from 0.12 to 1.5 ng/mL and 0.11 to 6.1 ng/g respectively, and in healthy subjects and 0.11 to 33.8 ng/g for food and 0.12 to 3.8 ng/mL for serum in nephropathy patients suffering from chronic interstitial nephropathy of unknown etiology. The disease seems related to ochratoxin A serum levels and food contaminations, since the healthy group was significantly different from the nephropathy group (P < 0.001) for both food and serum ochratoxin A contamination. Those results combined with data published already, emphasize the likely endemic aspect of ochratoxin A-related nephropathy occurring in Tunisia.     

The distribution and function of aquaporins in the kidney: resolved and unresolved questions

The membrane water channel aquaporin (AQP) family is composed of 13 isoforms in mammals, eight of which are reportedly expressed in the kidney: AQP1, 2, 3, 4, 6, 7, 8, and 11. These isoforms are differentially expressed along the renal tubules and collecting ducts. AQP1 and 7 are distributed in the proximal tubules, whereas AQP2, 3, and 4 occur in the collecting duct system. They play important roles in the reabsorption of water and some solutes across the plasma membrane. In contrast to other aquaporins found in the kidney, AQP6, 8, and 11 are localized to the cytoplasm rather than to the apical or basolateral membranes. It is therefore doubtful that these isoforms are directly involved in water or solute reabsorption. AQP6 is localized in acid-secreting type A intercalated cells of the collecting duct. AQP8 has been found in the proximal tubule but its cellular location has not yet been defined by immunohistochemistry. AQP11 seems to be localized in the endoplasmic reticulum (ER) of proximal tubule cells. Interestingly, polycystic kidneys develop in AQP11-null mice. Many vacuole-like structures are seen in proximal tubule cells in kidneys of newborn AQP11-null mice. Subsequently, cysts are generated, and most of the mice die within a month due to severe renal failure. Although ER stress and impairment of polycystin-1, the product of the gene mutated in autosomal-dominant polycystic kidney disease, are possible causes of cystogenesis in AQP11-null mice, the exact mechanism of pathogenesis and the physiological function of AQP11 are yet to be resolved.     

Comparison of the local effects of amiloride hydrochloride on the isotonic fluid absorption in the distal and proximal convoluted tubule

The isotonic fluid absorption (Jv) was measured under standard conditions in the proximal and distal convolution of the rat kidney. The peritubular blood capillaries were perfused simultaneously. Amiloride was applied either intraluminally or peritubularly. When applied intraluminally, amiloride strongly inhibited Jv in the distal tubule at concentrations up to 10(-6) M. In the proximal tubule similar effects were obtained only after intraluminal application of one thousand-fold greater concentrations of amiloride. In contrast to amphibian epithelia, amiloride also inhibits Jv in the distal tubule when applied peritubularly, but at higher concentrations and less completely than after intraluminal application. Amiloride was found to be generally more effective in the distal tubule than furosemide and mefruside, although in the proximal tubule it was less effective than these diuretics. That amiloride is most effective after intraluminal application in the distal tubule would suggest a dominant action at the luminal membrane of the distal tubule cell, while not excluding a concomitant effect at the peritubular membrane.     

Urinary cystatin C as a biomarker for acute kidney injury and its immunohistochemical localization in kidney in the CDDP-treated rats

Cystatin C, a cysteine protease inhibitor, is a novel biomarker of renal damage. In the present study, we examined the urinary and plasma levels of cystatin C and how useful they are for the early detection of acute kidney injury (AKI) in CDDP-treated rats in comparison with other biomarkers (β2-microglobulin, calbindin, clusterin, EGF, GST-α, GST-μ, KIM-1, NGAL, osteopontin, TIMP-1, and VEGF). The urinary levels of cystatin C, GST-α, KIM-1, and EGF changed prior to proximal tubule damage and increases in plasma urea nitrogen and creatinine levels, suggesting their usefulness for predicting AKI. On the other hand, the plasma cystatin C level hardly changed. We also investigated the localization of cystatin C in the kidney according to the progression of renal damage. Cystatin C was predominantly localized in the proximal tubule of the cortex, and its immunohistochemical expression was not affected by CDDP treatment. In addition, cystatin C was observed in the lumen of the renal tubule in the cortex, cortico-medullary junction, and medulla during the progression of renal damage, although its immunoreactive area ratio was very low.In conclusion, urinary cystatin C measurements can detect CDDP-induced AKI as early as KIM-1, GST-α, and EGF in rats, although the change ratio of the cystatin C was smaller than others. Immunohistochemical cystatin C expression in the proximal tubule of the kidney was hardly changed by the CDDP treatment, but it was newly observed in the renal tubule lumen after CDDP treatment.     

Loss of antigens associated with the apical endocytotic pathway in proximal tubules from rats with heymann nephritis.

In addition to the glomerular lesions associated with Heymann nephritis, a rat model of human membranous nephritis, proximal tubule damage, and a perturbation of proximal tubule function also have been reported to occur in this disease. The aim of the present study was to examine in more detail the nature of the apical plasma membrane damage in proximal tubules using specific antibodies directed against clathrin, gp330, and a proton-pumping adenosine triphosphatase, all of which are components of the apical endocytotic apparatus of these epithelial cells. Immunocytochemical studies revealed a marked reduction in staining for all three antigens in proximal tubules from rats with active Heymann nephritis. Furthermore endocytotic uptake of intravenously injected FITC-dextran was considerably lower in diseased animals than in normal rats. Gp330 and rat IgG were identified as components of the luminal debris that accumulated during the course of Heymann nephritis. These results show that perturbation of proximal tubule endocytosis occurs in Heymann nephritis together with a loss of three apical antigens that are normally localized on membrane domains associated with the apical endocytotic pathway in these cells. The results also suggest that antibody-antigen complexes may be shed from the plasma membrane in both the glomerulus and the proximal tubule in this disease.     

The changes of calretinin immunoreactivity in paraquat-induced nephrotoxic rats

Calcium-binding proteins are present in the kidneys: calbindin D-28k in the distal tubules and calretinin in the proximal tubules. Since paraquat causes degeneration in the brush border-bearing proximal tubule cells in rat kidneys, we investigated the changes of calretinin immunoreactivity in the proximal tubule cells of paraquat-induced nephrotoxicity in experimental male Sprague-Dawley rats following chitosan oligosaccharide pretreatment to investigate its protective properties. Paraquat (60mg/kg) was administered intraperitoneally with or without chitosan oligosaccharide (500mg/kg, p.o.) pretreatment. The changes on calretinin were compared with those of calbindin D-28k by immunohistochemistry and Western Blot analysis. Calretinin was immunolocalized on the apical surface of proximal tubule cells in the deeper cortex of normal kidney, and disappeared after paraquat administration with minor changes of calbindin D-28k immunoreactivity in the distal tubules and collecting ducts. Chitosan oligosaccharide pretreatment caused increased expression of calretinin and calbindin D-28k before paraquat injection and helped preserve proximal tubules after paraquat treatment. However, Western blot analysis on calretinin and calbindin D-28k could not explain the degeneration of the proximal tubule cells in paraquat-induced nephrotoxicity. These findings suggested that calretinin is a possible and more useful histopathological marker for proximal tubule cells in paraquat-induced nephrotoxic rats.     

Effect of volume expansion on renal glucose transport in normal and uremic dogs.

Proximal and distal tubule micropuncture studies were performed in normal and uremic remnant-kidney dogs to examine the tubule mechanism of glucose reabsorption before and after 10% extracellular volume expansion. In normal dogs volume expansion markedly inhibited glucose reabsorption in the proximal convoluted tubule, but the ensuing increase in further distal glucose delivery was nearly completely reabsorbed in the intermediate segment (between late proximal tubule and distal tubule). In the uremic, remnant-kidney dogs, glomerulotubular balance for glucose was well maintained in the proximal convoluted tubule despite an adaptive increase in nephron filtration rate. Volume expansion markedly increased glucose delivery out of the proximal convoluted tubule and an incomplete glucose reabsorption in the intermediate segment led to glycosuria. When glucose delivery to the intermediate segment was increased to a comparable degree by subthreshold glucose loading in hydropenic normal dogs, glucose reabsorption in this segment was virtually complete, suggesting that in the volume-expanded uremic dogs glucose reabsorptive capacity in the intermediate segment was reduced. Thus, the intermediate segment appears to play a significant role in the fine regulation of urinary glucose excretion.     

Atubular glomeruli in patients with chronic pyelonephritis

In an animal model of chronic nephropathy a large proportion of the apparently normal glomeruli have been shown to be small and without connection to a proximal tubule. The present study examines the degree to which atubular glomeruli are also present in human renal disease. Eleven patients with chronic pyelonephritis (CP) and seven controls were investigated. The number of glomeruli connected to a normal proximal tubule was determined in serial sections and the volumes of individual glomeruli estimated with stereological methods. Only glomeruli with little or no sclerosis were investigated. The volume fractions of proximal tubules and interstitial tissue were estimated using point counting. The results showed that 50% of glomeruli in the CP group were connected to a normal proximal tubule, whereas 35% of the glomeruli were without any recognizable connection to a proximal tubule (atubular glomeruli). The remaining 15% were connected to an atrophic tubule. The mean volume of the glomeruli without a connection to a normal proximal tubule was only half that of glomeruli with a normal proximal tubule. No significant difference was found between the mean glomerular volume in the two groups, but the intraindividual variation of glomerular volumes was larger in the CP group. A significant negative correlation was found in the CP group between the percentage of glomeruli without connection to a normal proximal tubule and the volume fraction of proximal tubules. A significant positive correlation was found between the percentage of glomeruli that were not connected to a normal proximal tubule and the volume fraction of the interstitial tissue. This study shows that atubular glomeruli, which only can be identified in serial sections, constitute a large proportion of glomeruli in chronic pyelonephritis. Their existence could be a major reason for the irreversibility of nonglomerular chronic renal diseases.     

Tissue distribution of an inducible cystatin in isoproterenol-treated rats

A well-characterized, monospecific rabbit antiserum directed to an isoproterenol-inducible type 2 salivary cystatin was used for immunocytochemical localization of this cystatin in rat salivary glands, as well as in other organs of normal and isoproterenol-treated rats. Immunocytochemical analysis revealed a moderate staining of secretory granules within the acinar cells of submandibular glands, which was more pronounced in tissues obtained from female rats. In addition, the inducible cystatin was readily detected within granular convoluted tubule cells and striated duct cells of submandibular glands of both male and female rats, although not all such structures were stained. Cystatin was also localized in the proximal convoluted tubule cells of the kidney in isoproterenol-treated female rats. Western blotting and Ouchterlony double diffusion analysis showed that the cystatin from submandibular gland and kidney extracts was immunologically identical.     

Site of renal phosphate reabsorption

Using modified microinfusion and free flow micropuncture techniques in the same intact and acutely thyroparathyroidectomized (TPTX) Munich-Wistar rats the nephron sites for phosphate reabsorption were reinvestigated. In intact animals, 62% of filtered phosphate was reabsorbed in the proximal tubule but none in the loop of Henle, here defined as the nephron segment between the last accessible proximal and the first distal convolution. Delivery of phosphate to the superficial distal tubule significantly exceeded urinary phosphate excretion but no phosphate reabsorption could be detected in the terminal nephron by distal microinfusions of radioactively labelled phosphate (32P). In TPTX rats, proximal phosphate reabsorption was enhanced and there was marked phosphate reabsorption in the loop of Henle. Similarly, 32P microinfused in the late proximal tubule was almost completely reabsorbed. Again, no phosphate tracer outflux was detected after distal microinfusion. It is concluded that phosphate reabsorption is confined to the proximal tubule and the loop of Henle.     

Spontaneous toxic nephropathy in poultry associated with ochratoxin A.

At a poultry slaughterhouse 14 birds with macroscopic renal changes were collected, and the kidneys were examined histologically and the muscular tissue was analysed for ochratoxin A residues. Out of 14 birds 5 birds had ochratoxin A residues ranging from 4.3 to 29.2 mug/kg. In 4 of these birds a toxic nephropathy was found characterised by atrophy and degeneration of proximal and distal tubules and interstitial fibrosis. The possibility of birds with ochratoxin A residues being presented for human consumption is discussed.     

Autometallographic localization of inorganic mercury in the kidneys of rats: effect of unilateral nephrectomy and compensatory renal growth

The histochemical technique of autometallography was used in the present study to demonstrate the zonal and tubular localization of inorganic mercury in the kidneys of unilaterally nephrectomized (NPX) and sham-operated (SO) rats given either a nontoxic 0.5 mumol/kg or a toxic 2.5 mumol/kg dose of mercuric chloride 10 days after surgery. Deposits were found in the cortex and outer stripe of the outer medulla in both groups of rats given either dose of mercuric chloride. The deposits were localized exclusively in the convoluted and straight portion of the proximal tubule. Forty eight hours after the administration of the 0.5 mumol/kg dose of mercuric chloride, there were significantly more deposits in the renal outer stripe of the NPX rats than in the renal outer stripe of the SO rats. The number of deposits in the renal outer stripe of the NPX and SO rats given the 2.5 mumol/kg dose of mercuric chloride was similar after 24 hr, but was greater than the corresponding rats given the nontoxic dose. These findings suggest that the proximal tubule (particularly the pars recta) is the primary site for the accumulation of inorganic mercury in the kidney. They also suggest that, in the rat, there is enhanced accumulation of inorganic mercury in the pars recta of proximal tubules in the outer stripe of the renal outer medulla when a nontoxic dose of inorganic mercury is given after unilateral nephrectomy or when a toxic dose of mercuric chloride is administered.     

Free flow electrophoresis. Application to the separation of 2 populations of proximal tubule cells from the rabbit kidney

Two cell populations from the proximal tubule of the rabbit kidney were separated by free flow electrophoresis from a pure suspension of proximal tubular cells obtained by a combination of a Ca-binding agent, gentle mechanical forces and differential sifting. Before the electrophoretic separation, distal and proximal enzyme activities were measured on the cortical homogenates, on the proximal tubule suspensions and on the isolated cell samples in order to assess the purity of the cell preparation. The isolated cells were very poor in distal tubule marker activities and were enriched in proximal tubule marker enzymes. Cell oxygen consumption was measured before and after the electrophoretic run were similar and reflected high cell metabolic capacity. The cells in the slow-moving electrophoresis fractions had a high gamma-glutamyl transpeptidase activity and the fast moving cells showed a high glucose-6-phosphatase activity. These results point out a separation of viable cells from straight and convoluted portion of the proximal tubule from the rabbit kidney. These two cell populations can be suitable for further use in biochemical and physiological studies.