Carol M. Herak-Kramberger

Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organs

Metallothioneins are cysteine-rich, small metal-binding proteins present in various mammalian tissues. Of the four common metallothioneins, MT-1 and MT-2 (MTs) are expressed in most tissues, MT-3 is predominantly present in brain, whereas MT-4 is restricted to the squamous epithelia. The expression of MT-1 and MT-2 in some organs exhibits sex, age, and strain differences, and inducibility with a variety of stimuli. In adult mammals, MTs have been localized largely in the cell cytoplasm, but also in lysosomes, mitochondria and nuclei. The major physiological functions of MTs include homeostasis of essential metals Zn and Cu, protection against cytotoxicity of Cd and other toxic metals, and scavenging free radicals generated in oxidative stress. The role of MTs in Cd-induced acute and chronic toxicity, particularly in liver and kidneys, is reviewed in more details. In acute toxicity, liver is the primary target, whereas in chronic toxicity, kidneys are major targets of Cd. The intracellular MTs bind Cd ions and form CdMT. In chronic intoxication, Cd stimulates de novo synthesis of MTs; it is assumed that toxicity in the cells starts when loading with Cd ions exceeds the buffering capacity of intracellular MTs. CdMT, released from the Cd-injured organs, or when applied parenterally for experimental purposes, reaches the kidneys via circulation, where it is filtered, endocytosed in the proximal tubule cells, and degraded in lysosomes. Liberated Cd can immediately affect the cell structures and functions. The resulting proteinuria and CdMT in the urine can be used as biomarkers of tubular injury.

Expression of Aquaporin 9 in the Adult Rat Epididymal Epithelium Is Modulated by Androgens

Abstract Reabsorption of fluid and solutes across the epithelium lining the male excurrent duct is important for adequate sperm maturation, concentration, and storage. Water channels contribute to water movement across epithelia in many tissues. Aquaporin 9 (AQP9) is abundantly expressed in the apical membrane of principal cells that line the epididymis, and in reabsorptive and secretory epithelial cells of the male reproductive tract. In this study we show that the nonsteroidal antiandrogen flutamide, given to adult rats at a dose of 50 mg kg−1 day−1 for 2 wk via osmotic minipumps significantly decreased the amount of AQP9 in the epididymis. This down-regulation was observed by immunofluorescence of cryostat tissue sections and by Western blotting of epididymal brush border membrane preparations. In addition, castrated adult rats showed lower levels of epididymal AQP9 compared with adult controls, whereas systemic testosterone treatment of castrated adult rats induced a recovery of the expression of AQP9 to control levels. These data indicate that the expression of AQP9, a likely candidate for apical transepithelial fluid and solute transport in several regions of the male reproductive tract, is modulated by androgens in the adult rat epididymis.

Distribution of the Vacuolar H+ATPase Along the Rat and Human Male Reproductive Tract

Abstract Luminal acidification in parts of the male reproductive tract generates an appropriate pH environment in which spermatozoa mature and are stored. The cellular mechanisms of proton (H+) secretion in the epididymis and the proximal vas deferens involve the activity of an apical vacuolar H+ATPase in specialized cell types, as well as an apical Na+/H+ exchanger in some tubule segments. In this study we used Western blotting and immunocytochemistry to localize the H+ATPase in various segments of the male reproductive tract in rat and man as a first step toward a more complete understanding of luminal acidification processes in this complex system of tissues. Immunoblotting of isolated total cell membranes indicated a variable amount of H+ATPase in various segments of the rat reproductive tract. In addition to its known expression in distinct cell types in the epididymis and vas deferens, the H+ATPase was also localized at the apical pole and in the cytoplasm of epithelial cells in the efferent duct (nonciliated cells), the ampulla of the vas deferens and the ventral prostate (scattered individual cells), the dorsal and lateral prostate, the ampullary gland, the coagulating gland, and all epithelial cells of the prostatic and penile urethra. Both apical and basolateral localization of the protein were found in epithelial cells of the prostatic ducts in the lateral prostate and in periurethral tissue. Only cytoplasmic, mostly perinuclear localization of the H+ATPase was found in all epithelial cells of the seminal vesicles and in most cells of the ventral prostate and coagulating gland. No staining was detected in the seminiferous tubules, rete testis, and bulbourethral gland. In human tissue, H+ATPase-rich cells were detected in the epididymis, prostate, and prostatic urethra. We conclude that the vacuolar H+ATPase is highly expressed in epithelial cells of most segments of the male reproductive tract in rat and man, where it may be involved in H+ secretion and/or intracellular processing of the material endocytosed from the luminal fluid or destined to be secreted by exocytosis.

Subchronic cadmium treatment affects the abundance and arrangement of cytoskeletal proteins in rat renal proximal tubule cells

Disfunction of proximal tubules (PT) in cadmium (Cd) nephrotoxicity in mammals results from the diminished functional capacity of brush-border membrane (BBM) caused by (a) direct inhibition of BBM transporters by Cd, (b) shortening and loss of microvilli, and (c) loss of specific BBM transporters. The loss of transporters may partially result from impaired intracellular vesicle recycling due to loss or/and inhibition of vacuolar H+-ATPase in the PT cell organelles. Cytoskeleton plays an important role in vesicle-mediated recycling and processing of BBM transporters in PT cells. Experiments in vitro have indicated that Cd may affect the state of polymerization of some cytoskeletal proteins. In this work we studied the in vivo effect of CdCl2-treatment in rats (2 mg Cd/kg b. m., s.c., daily for 14 days) upon abundance and arrangement of actin filaments, actin-bundling protein villin, and microtubules (MT) in PT cells. Cd-treatment elicited a dramatic accumulation of Cd in the kidney cortex (200 microg/g tissue wet mass after 14 days) and a strongly increased abundance of metallothionein in PT cells. As revealed by immunocytochemistry in tissue cryosections, the staining intensity of actin and villin in PT cells of Cd-treated rats was generally decreased, without a marked change in their intracellular distribution, whereas MT became largely irregular, diminished in most cells, and lost in many cells. However, the immunoblots revealed an increased content of villin and alpha-tubulin in cortical tissue homogenates from Cd-treated rats, thus indicating an impaired bundling of actin and greatly depolymerized MT in cells intoxicated with Cd. The partial loss of apical actin and villin in PT cells of Cd-treated rats may reflect (or cause) shortening and loss of microvilli, whereas derangement and depolymerization of MT may contribute to the impairment of intracellular recycling of BBM proteins, and lead to the loss of BBM transporters.

Renal type II Na/Pi-cotransporter is strongly impaired whereas the Na/sulphate-cotransporter and aquaporin 1 are unchanged in cadmium-treated rats

The cellular mechanisms of cadmium (Cd) nephrotoxicity are poorly understood. In this study we investigated the cellular causes of the Cd-induced phosphaturia in the rat. Compared to controls, Cd-treated rats (2 mg Cd/kg body weight, s.c. for 14 days) showed a marked polyuria, proteinuria and phosphaturia. As studied by the rapid filtration technique in isolated cortical brush-border membrane vesicles (BBMV), Na+-gradient-driven uptake of phosphate ([32Pi]) and of [3H] glucose were markedly decreased in Cd-treated rats, whereas uptake of sulphate ([35S]) remained unchanged. By Western blotting of BBMV proteins and by indirect immunocytochemistry in 4-μm thick frozen fixed kidney sections, using an antibody against the type II Na/Pi-cotransporter (NaPi-2), we found a diminished expression of this protein in the brush-border membrane from Cd-treated rats. However, the expression of the water channel aquaporin 1, estimated from the specific antibody staining in brush-border membranes, remained unchanged by Cd. Northern blot analysis showed a strong reduction of 2.7 kb NaPi-2-related mRNA in Cd-affected kidneys. Our data indicate that: (1) Cd may reduce reabsorption of Pi in proximal tubules by affecting the expression of the functional Na/Pi-cotransporters in the luminal membrane, and (2) Cd effects on brush-border transporters are selective.

The integrity of renal cortical brush-border and basolateral membrane vesicles is damaged in vitro by nephrotoxic heavy metals

Poisoning of experimental animals with cadmium (Cd), mercury (Hg), lead (Pb) or cis-diamminedichloroplatinum (cis-Pt) causes shortening and focal loss of microvilli in proximal tubule (PT) cells, thus indicating that the reduced reabsorptive surface due to damaged integrity of brush-border membrane (BBM) may contribute to the reabsorptive and secretory defects in these toxic states. In addition, in in vitro studies with isolated renal cortical BBM vesicles (BBMV), heavy metals (HM) inhibit transport of various compounds, and these data were interpreted as being a result of a direct inhibition of the respective membrane transporters. In this work we used a DeltapH-driven acridine orange fluorescence quench assay to test if various divalent cations affect in vitro the integrity of BBMV and basolateral membrane vesicles (BLMV) isolated from the rat renal cortex. In Cd-treated BBMV we found that: (a) the integrity of vesicles decreased with increasing concentrations of Cd; and (b) the loss of sealed vesicles was high at 37 degrees C, intermediate at 25 degrees C, and very low at 0 degrees C. The loss of sealed BBMV was caused also by Hg, Cu, Pb and Zn (Hg>>>Cu=Cd>Pb=Zn). Cis-Pt, Al, Fe, Ba, Mg and Mn had no effect. BLMV were damaged by HM with an efficiency Hg>>>Cd=Pb=Cu, whereas other divalent cations, including Zn, were ineffective. An SH-group protector, dithiothreitol, prevented the loss of sealed vesicles in some (Hg, Pb, Cu) but not in all (Cd, Zn) cases. We conclude that the nephrotoxic HM directly damage the integrity of PT cell plasma membranes; this may cause shortening and loss of microvilli and basolateral invaginations in HM-treated experimental animals in vivo. The data also indicate that caution should be taken when effects of HM on various transports are studied in isolated membrane vesicles in vitro; an impaired transport may result from the loss of vesicle integrity, and not necessarily from the direct inhibition of a transporter.

Cadmium inhibits vacuolar H+ATPase-mediated acidification in the rat epididymis

Abstract In rats, an acidic luminal pH maintains sperm quiescence during storage in the epididymis. We recently showed that vacuolar H+ATPase-rich cells in the epididymis and vas deferens are involved in the acidification of these segments. Treatment of rats with cadmium (Cd) leads to alkalinization of this fluid by an unknown mechanism. Because Cd may affect H+ATPase function, we examined 1) the in vivo effect of Cd poisoning on H+ATPase-rich cell morphology and on the abundance and distribution of the 31-kDa H+ATPase subunit in cells along the rat epididymis, and 2) the in vitro effect of Cd on H+ATPase activity and function in the isolated vas deferens. Immunofluorescence and immunoblotting data from rats treated with Cd for 14–15 days (2 mg Cd/kg body mass/day) showed that 1) H+ATPase-positive cells regressed to a prepubertal phenotype, and 2) H+ATPase was lost from the apical pole of the cell and was redistributed into an intracellular compartment. In experiments in vitro, Cd inhibited bafilomycin-sensitive ATPase activity in isolated total cell membranes and, as measured using a proton-selective extracellular microelectrode, inhibited proton secretion in isolated vas deferens. We conclude that alkalinization of the tubule fluid in the epididymis and vas deferens of Cd-treated rats may result from the loss of functional H+ATPase enzyme in the cell apical domain as well as from a direct inhibition of H+ATPase function by Cd.

Immunocytochemical characterization of the incubated rat renal cortical slices

The use of renal cortical slices in vitro and the data obtained in these studies have been subjects of controversy, largely due to uncertain viability, e.g., structural and functional integrity of the proximal and other tubules. However, detailed studies of tubule integrity have not been reported. To correlate functional and structural viability of the hand-cut rat renal cortical slices, incubated in optimally conditioned media for up to 25xa0h, we studied the time course of p-aminohippurate (PAH) uptake, the immunocytochemical distribution of several proteins that reside in the proximal tubule basolateral [Na/K-ATPase, organic anion transporters (OAT)1 and OAT3], or brush border [megalin, sodium-proton exchanger (NHE)3] membrane, as well as the general integrity of the tubule epithelium and its cytoskeleton (actin filaments, microtubules). PAH uptake in slices was proportional to time within 1xa0h of incubation and gradually declined thereafter. The immunostaining experiments indicated a fast, time-dependent loss of basolateral transporters, at a rate of OAT1 > Na/K-ATPase > OAT3. In the brush border membrane, the loss of megalin was faster than that of NHE3, and a partial redistribution of NHE3 into the basolateral domain indicated the loss of cell polarity. The loss of intracellular actin and tubulin cytoskeleton in the proximal tubule was already visible after 15xa0min of incubation and gradually increased with time, whereas a partial redistribution of actin to the basolateral domain indicated a compromised polarity of the cells. The data also revealed very early (after 15xa0min) necrotic events in the proximal tubule epithelium, with sloughing of brush border and cell debris into the tubule lumen, detachment of cells from the basal membrane, and opening and widening of the tubule lumen. We conclude that the loss of cellular structure, cytoskeleton, and cell membrane transporters in the nephron epithelium is a very early event in the incubated rat renal cortical slices.

Serotonin transporter in rat platelets. Level of protein expression underlies inherited differences in uptake kinetics.

By breeding selection for the extreme values of platelet serotonin level (PSL), two sublines of Wistar-derived rats, with constitutionally high or low PSL and platelet serotonin uptake (PSU), have been developed. Searching for the basis of these differences, we performed quantitative western blot analysis of serotonin transporter (5HTt) in platelet membranes isolated from both rat sublines. A polyclonal anti-5HTt antibody labeled a single, 5HTt-related 94 kDa protein band in platelet membranes, with significantly stronger intensity in membranes from rats that exhibited a high PSL. We conclude that the inherited differences in PSL and PSU in rats, following breeding selection, are determined by the level of 5HTt expression in platelet membranes.

Sex-dependent expression of water channel AQP1 along the rat nephron

In the mammalian kidney, nonglycosylated and glycosylated forms of aquaporin protein 1 (AQP1) coexist in the luminal and basolateral plasma membranes of proximal tubule and descending thin limb. Factors that influence AQP1 expression in (patho)physiological conditions are poorly known. Thus far, only angiotensin II and hypertonicity were found to upregulate AQP1 expression in rat proximal tubule in vivo and in vitro (Bouley R, Palomino Z, Tang SS, Nunes P, Kobori H, Lu HA, Shum WW, Sabolic I, Brown D, Ingelfinger JR, Jung FF. Am J Physiol Renal Physiol 297: F1575-F1586, 2009), a phenomenon that may be relevant for higher blood pressure observed in men and male experimental animals. Here we investigated the sex-dependent AQP1 protein and mRNA expression in the rat kidney by immunochemical methods and qRT-PCR in tissue samples from prepubertal and intact gonadectomized animals and sex hormone-treated gonadectomized adult male and female animals. In adult rats, the overall renal AQP1 protein and mRNA expression was ∼80% and ∼40% higher, respectively, in males than in females, downregulated by gonadectomy in both sexes and upregulated strongly by testosterone and moderately by progesterone treatment; estradiol treatment had no effect. In prepubertal rats, the AQP1 protein expression was low compared with adults and slightly higher in females, whereas the AQP1 mRNA expression was low and similar in both sexes. The observed differences in AQP1 protein expression in various experiments mainly reflect changes in the glycosylated form. The male-dominant expression of renal AQP1 in rats, which develops after puberty largely in the glycosylated form of the protein, may contribute to enhanced fluid reabsorption following the androgen- or progesterone-stimulated activities of sodium-reabsorptive mechanisms in proximal tubules.