Expression and immunolocalization of metallothioneins MT1, MT2 and MT3 in rat nephron.

Rodent kidneys exhibit three isoforms of metallothioneins (MTs), MT1, MT2 and MT3, with poorly characterized localization along the nephron. Here we studied in adult male Wistar rats the renal expression of MTs mRNA by end-point RT-PCR and MT proteins by immunochemical methods The expression pattern of MT1 mRNA was cortex (CO)>outer stripe (OS)=inner stripe (IS)=inner medulla (IM), of MT2 mRNA was IM>CO>IS=OS, and of MT3 mRNA was IM>CO=OS=IM. MT1/2-antibody stained with heterogeneous intensity the cell cytoplasm and nuclei in proximal tubule (PT) and thin ascending limb, whereas MT3-antibody stained weakly the cell cytoplasm in various cortical tubules and strongly the nuclei in all nephron segments. However, the isolated nuclei exhibited an absence of MT1/2 and presence of MT3 protein. In MT1/2-positive PT cells, the intracellular staining appeared diffuse or bipolar, but the isolated brush-border, basolateral and endosomal membranes were devoid of MT1/2 proteins. In the lumen of some PT profiles, the heterogeneously sized MT1/2-rich vesicles were observed, with the limiting membrane positive for NHE3, but negative for V-ATPase, CAIV, and megalin, whereas their interior was positive for CAII and negative for cytoskeleton. They seem to be pinched off from the luminal membrane of MT1/2-rich cells, as also indicated by transmission electron microscopy. We conclude that in male rats, MTs are heterogeneously abundant in the cell cytoplasm and/or nuclei along the nephron. The MT1/2-rich vesicles in the tubule lumen may represent a source of urine MT and membranous material, whereas MT3 in nuclei may handle zink and locally-produced reactive oxygen species.

Sex-independent expression of chloride/formate exchanger Cfex (Slc26a6) in rat pancreas, small intestine, and liver, and male-dominant expression in kidneys.

Chloride/formate exchanger (CFEX; SLC26A6) mediates oxalate transport in various mammalian organs. Studies in Cfex knockout mice indicated its possible role in development of male-dominant hyperoxaluria and oxalate urolithiasis. Rats provide an important model for studying this pathophysiological condition, but data on Cfex (rCfex) localisation and regulation in their organs are limited. Here we applied the RT-PCR and immunochemical methods to investigate rCfex mRNA and protein expression and regulation by sex hormones in the pancreas, small intestine, liver, and kidneys from intact prepubertal and adult as well as gonadectomised adult rats treated with sex hormones. rCfex cDNA-transfected HEK293 cells were used to confirm the specificity of the commercial anti-CFEX antibody. Various biochemical parameters were measured in 24-h urine collected in metabolic cages. rCfex mRNA and related protein expression varied in all tested organs. Sex-independent expression of the rCfex protein was detected in pancreatic intercalated ducts (apical domain), small intestinal enterocytes (brush-border membrane; duodenum > jejunum > ileum), and hepatocytes (canalicular membrane). In kidneys, the rCfex protein was immunolocalised to the proximal tubule brush-border with segment-specific pattern (S1=S2

Expression of basolateral organic anion and cation transporters in experimental cadmium nephrotoxicity in rat kidney.

Cadmium (Cd)-intoxicated experimental animals exhibit impaired renal secretion of organic anions (OA) and cations (OC), indicating their transporters (Oats and Octs) in the proximal tubule (PT) basolateral membrane as possible targets of Cd. To correlate transport data from the literature with the expression of relevant transporters, we performed immunochemical and RT-PCR studies of renal Oats and Octs in the subchronic (treatment with CdCl2; 2 mg Cd/kg b.m./day, for 2 weeks) and acute (treatment with Cd-metallothionein (CdMT); 0.4 mg Cd/kg b.m., 6 or 12 h before killing) models of Cd nephrotoxicity. In the subchronic model, PT exhibited a minor loss of basolateral invaginations and overall unchanged expression of Na(+)/K(+)-ATPase and GAPDH proteins and mRNAs, while the expression of Oat and Oct proteins and their mRNAs was strongly downregulated. In the acute model, a time-related redistribution of basolateral transporters to the intracellular vesicular compartment was a major finding. However, 6 h following CdMT treatment, the total abundance of Oat and Oct proteins in the renal tissue remained unchanged, the expression of mRNAs decreased only for Oats, while a limited Oat1 and Na(+)/K(+)-ATPase immunoreactivity in the PT apical membrane indicated loss of cell polarity. As tested in rats treated with colchicine, the observed loss/redistribution of basolateral transporters in both models may be independent on microtubules. Therefore, the diminished renal secretion of OA and OC via PT in Cd nephrotoxicity may result from (a) limited loss of secretory surface (basolateral invaginations), (b) selective loss of Oats and Octs, and

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.

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.

Loss of basolateral invaginations in proximal tubules of cadmium-intoxicated rats is independent of microtubules and clathrin.

Experimental cadmium (Cd) nephrotoxicity after treating rats with CdCl(2) (2mg Cd/kg b.m./day) for 2 weeks (subchronic nephrotoxicity) or with Cd-metallothionein (CdMT, a single dose of 0.4 mg Cd/kg b.m.) for a few hours (acute nephrotoxicity) is characterized by significant damage to cortical proximal tubules (PT) that results in reabsorptive and secretory defects. Most of the damage, studied so far, has been reported at the PT cell apical domain. This includes the loss of apical transporters and brush-border microvilli, and is considered to be the main cause of the kidney malfunction seen in this condition. However, in some studies the loss of basolateral (BL) invaginations and the activity of Na/K-ATPase in PT cells was also observed, but this "basolateral" aspect of intracellular Cd toxicity has been poorly investigated. In this report we induced subchronic and acute Cd nephrotoxicity in rats, and we studied the expression and intracellular distribution of microtubules and clathrin, and the abundance of Na/K-ATPase associated with BL invaginations in renal cortical tubules. Methods used were immunofluorescence microscopy, transmission and immunogold microscopy and immunoblotting of tissue homogenates and isolated total cell membranes. In both experimental models, in the cortical PT we demonstrated: (a) significantly damaged morphology of the cells, (b) fragmentation and depolymerization of microtubules, (c) loss of clathrin in the subapical domain and its relocation into vesicles scattered throughout the cytoplasm and (d) loss of BL invaginations and the associated Na/K-ATPase immunostaining. A similar loss of microtubules and redistribution of clathrin in the cortical PT was observed in rats treated with microtubule depolymerizing agent colchicine, but without any detectable loss of BL invaginations. We conclude that the loss of BL invaginations and the associated Na/K-ATPase in the cortical PT of Cd-intoxicated rats may contribute to the loss of PT function that characterizes Cd nephrotoxicity. This loss is accompanied by, but it is not dependent on perturbation of microtubule organization and loss of membrane-associated clathrin.

Rat renal glucose transporter SGLT1 exhibits zonal distribution and androgen-dependent gender differences.

SGLT1 (SLC5A1) mediates a part of glucose and galactose reabsorption in the mammalian proximal tubule (PT), but the detailed localization of the transporter along the tubule is still disputable. Here, we used several methods to localize rat SGLT1 (rSGLT1) in the kidneys of intact and variously treated male (M) and female (F) rats. In immunoblots of isolated cortical (C) and outer stripe (OS) brush-border membranes (BBM), a peptide-specific polyclonal antibody for rSGLT1 labeled a sharp inzone-, and gender-dependent approximately 40-kDa protein and a broad approximately 75-kDa band that exhibited strong zonal (OS > C) and gender differences (F > M). In tissue cryosections, the antibody strongly stained BBM of the S3 PT segments in the OS and medullary rays (F > M) and smooth muscles of the blood vessels and renal capsule (F approximately M) and weakly stained the apical domain of other PT segments in the C (F approximately M). The phlorizin-sensitive uptake of d-[(3)H]galactose in BBM vesicles, as well as the tissue abundance of rSGLT1-specific mRNA, matched the immunoblotting data related to the 75-kDa protein and the immunostaining in S3, proving zonal and gender differences in the functional transporter. Ovariectomy had no effect, castration upregulated, whereas treatment of castrated rats with testosterone, but not with estradiol or progesterone, downregulated the 75-kDa protein and the immunostaining in S3. We conclude that in the rat kidney, the expression of SGLT1 is represented by a 75-kDa protein localized largely in the PT S3 segments, where it exhibits gender differences (F > M) at both the protein and mRNA levels that are caused by androgen inhibition.

EPR study of lipid phase in renal cortical membrane organelles from intact and cadmium-intoxicated rats.

Numerous studies have demonstrated various structure/function correlations at the level of transport proteins in the kidney cell membranes and various intracellular organelles. However, characterization of the lipid phase of these membranes is rare. Here, we report the differences in lipid organization and dynamics of the brush-border membranes (BBM), basolateral membranes (BLM) and endocytotic vesicles (EV), isolated from the kidney cortex of intact rats, studied with the EPR spectroscopy of the spin-labeled membrane lipids. The EPR spectra were analyzed by comparing experimentally observed line shapes with the line shapes calculated according to the theoretical model developed for liquid crystals. In the fitting procedure, three different lipid domains were assumed, which revealed clear differences in the lipid ordering and rotational correlation times, as well as in the lipid partition of these domains in each of the three types of membranes. A similar approach, used to compare the spectroscopic characteristics of BBM from control and cadmium-intoxicated rats, showed significantly changed ordering and increased molecular mobility in the lipid phase of BBM from Cd-treated animals. As tested by an established fluorescence assay, the Cd-induced changes in the lipid mobility co localized with approximately 5-fold higher conductance of BBM for potassium, with unchanged conductance for protons.

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 25 h, 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 1 h 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 15 min 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 15 min) 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.

Rat renal cortical OAT1 and OAT3 exhibit gender differences determined by both androgen stimulation and estrogen inhibition.

In rats, the secretion of p-aminohippurate (PAH) by the kidney is higher in males (M) than in females (F). The role of the major renal PAH transporters, OAT1 and OAT3, in the generation of these gender differences, as well as the responsible hormones and mechanisms, has not been clarified. Here we used various immunocytochemical methods to study effects of gender, gonadectomy, and treatment with sex hormones on localization and abundance of OAT1 and OAT3 along the rat nephron. Both transporters were localized to the basolateral membrane: OAT1 was strong in proximal tubule S2 and weak in the S3 segments, whereas OAT3 was stained in proximal tubule S1 and S2 segments, thick ascending limb, distal tubule, and in principal cells along the collecting duct. Gender differences in the expression of both transporters in adult rats (M > F) were observed only in the cortical tubules. OAT1 in the cortex was strongly reduced by castration in adult M, whereas the treatment of castrated M with testosterone, estradiol, or progesterone resulted in its complete restitution, further depression, or partial restitution, respectively. In adult F, ovariectomy weakly increased, whereas estradiol treatment of ovariectomized F strongly decreased, the expression of OAT1. The expression of OAT3 in the M and F cortex largely followed a similar pattern, except that ovariectomy and progesterone treatment showed no effect, whereas in other tissue zones gender differences were not observed. In prepubertal rats, the expression of OAT1 and OAT3 in the kidney cortex was low and showed no gender differences. Our data indicate that gender differences in the rat renal cortical OAT1 and OAT3 (M > F) appear after puberty and are determined by both a stimulatory effect of androgens (and progesterone in the case of OAT1) and an inhibitory effect of estrogens.

Cd-MT causes endocytosis of brush-border transporters in rat renal proximal tubules.

Nephrotoxicity in humans and experimental animals due to chronic exposure to cadmium (Cd) is manifested by defects in the reabsorptive and secretory functions of proximal tubules (PT). The main symptoms of Cd nephrotoxicity, including polyuria, phosphaturia, aminoaciduria, glucosuria, and proteinuria, suggest that various brush-border membrane (BBM) transporters are the main targets of Cd. Specific transporters may be either directly inhibited by Cd or lost from the BBM after Cd treatment, or both. We have recently proposed that Cd may impair the vesicle-dependent recycling of BBM transporters by inhibiting vacuolar H+-ATPase (V-ATPase) activity and endocytosis in PT cells (Herak-Kramberger CM, Sabolic I, and Brown D. Kidney Int 53: 1713-1726, 1998). The mechanism underlying the Cd effect was further explored in an in vivo model of experimental Cd nephrotoxicity induced by Cd-metallothionein (Cd-MT; 0.4 mg Cd/kg body mass; a single dose sc) in rats. The time-dependent redistribution of various BBM transporters was examined in this model by fluorescence and gold-labeling immunocytochemistry on tissue sections and by immunoblotting of isolated renal cortical BBM. In PT cells of Cd-MT-treated rats, we observed 1) shortening and loss of microvilli; 2) time-dependent loss of megalin, V-ATPase, aquaporin-1 (AQP1), and type 3 Na+/H+ exchanger (NHE3) from the BBM; 3) redistribution of these transporters into vesicles that were randomly scattered throughout the cell cytoplasm; and 4) redistribution of NHE3, but not megalin, into the basolateral plasma membrane. The internalization of BBM transporters was accompanied by fragmentation and loss of microtubules and by an increased abundance of alpha-tubulin monomers in PT cells. Transporter redistribution was detectable as early as 1 h after Cd-MT treatment and increased in magnitude over the next 12 h. We conclude that the early mechanism of Cd toxicity in PT cells may include a colchicine-like depolymerization of microtubules and impaired vesicle-dependent recycling of various BBM proteins. These processes may lead to a time-dependent loss of cell membrane components, resulting in reabsorptive and secretory defects that occur in Cd-induced nephrotoxicity.