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Cadmium (Cd)-intoxicated experimental animals exhibit impaired renal secretion of organic anions (OA) and cations (OC), indicating relevant transporters (Oat1/Slc22a6, Oat3/Slc22a8, Oct1/Slc22a1, Oct2/Slc22a2) in the proximal tubule (PT) basolateral membrane (BLM) as possible targets of Cd. To correlate previous transport data with the expression of relevant transporters in experimental Cd- nephrotoxicity, we performed the immunochemical and RT-PCR studies of renal Oats and Octs in rats treated with CdCl2 for two weeks (subchronic model) or Cd-metallothionein (CdMT) for 6-12 hours (acute model). In both models, PT exhibited loss of basolateral invaginatios. In subchronic model, the expression of Oat1, Oat3, Oct1, and Oct2 proteins and their mRNAs was strongly downregulated. In acute model, a progressive loss of transporters from the BLM and their internalization and accumulation in intracellular organelles was observed. However, as estimated at 6 hours following CdMT- treatment, the total abundance of Oat and Oct proteins in the tissue remained unchanged, whereas the expression of their mRNAs was heterogeneously diminished (Oats >Octs). In some PT cells, finding of a limited Oat1 immunoreactivity in the brush-border membrane indicated loss of cell polarity. As tested in rats treated with colchicine, redistribution of Oats and Octs in intracellular organelles in acute Cd-nephrotoxicity was independent on microtubules. Therefore, the diminished renal secretion of OA and OC in Cd-nephrotoxicity may result from: a) loss of BLM invaginations, b) loss of BLM transporters due to inhibition of intracellular vesicle trafficking, c) diminished mRNA-dependent synthesis of the transporter proteins, and d) loss of the PT cell polarity.

P-glycoprotein (P-gp ; ABCB1 in humans/Abcb1 in rodents) is an ATP-dependent multidrug efflux transporter in the cell membrane, also known as the multidrug resistant protein 1 (MDR1 in humans/Mdr1 in animals), constitutively expressed in the a) bile canaliculi of hepatocytes, b) brush-border membrane of the renal proximal tubule cells, c)luminal membrane of the intestinal enterocytes, d) blood-brain, blood-testis, and mother-foetus barriers, and e) hematopoietic cells. P-gp mediates the transport of some endogenous compounds and various xenobiotics (drugs, toxins, environmental organic compounds, and their metabolites) out of the cells, thus protecting the cell’s interior from the potentially toxic effects of these compounds. Although P-gp is a highly studied transporter in a variety of(patho)physiological conditions in human and animal organs, its age-dependent expression is still a controversial issue. Here, we investigated the ontogenic pattern of P-gp protein expression in rat liver and kidneys to provide insight into the drug transport capacity in these organs at different ages. P-gp protein expression was studied by immunocytochemistry and Western blotting in organs from neonatal (age, 1 day), prepubertal (age, 3 weeks), adult(age, 3 months), and old (age, 2 years) male and female Wistar rats. In both sexes, the liver P-gp expression pattern was: neonatal (highest) > prepubertal > adult < old. In the kidneys, the P-gp expression exhibited the pattern: neonatal < prepubertal < adult (highest) > old. Better knowledge of the ontogeny of P-gp expression may improve experimental design and the interpretation of results of toxicity studies in juvenile animals as well as the understanding of drug toxicity in different age groups in translational studies.

CFEX (chloride/formate exchanger ; Slc26a6) is an important anion exchanger of chloride, bicarbonate, oxalate (OX), formate, and hydroxyl ions in kidneys, liver, and the small intestine. Studies on CFEX-knockout mice indicated a possible role of CFEX in the development of hyperoxaluria and OX urolithiasis/nephrolithiasis, which in humans is more frequent in men. Here we studied the expression of the CFEX protein and mRNA in the organs of male and female rats, and employed a rat model of ethylene glycol (EG)-induced OX urolithiasis in order to correlate the expression of CFEX with sex-related hyperoxaluria. Rats drank EG in water (0.75 % vol/vol) or water (control) for 30 days. Tissue expressions of the CFEX protein and mRNA were analysed by immunochemical methods and qRT-PCR, respectively. The specificity of an anti-CFEX antibody, used in immunochemical studies, was confirmed in HEK293 cells transiently transfected with CFEX cDNA. In kidneys, the CFEX protein was immunolocalized to the proximal tubule brush-border membrane (BBM) with segmental (S3>>S1~S2) and sex (male>female) differences. Sex-unrelated expression was detected in the BBM of enterocytes (duodenum>jejunum) and in the hepatocyte canalicular membrane. In immunoblots, the CFEX protein band of ~120 kDa in various organs showed an expression pattern comparable to that in immunocytochemistry ; however, renal CFEX mRNA expression was not sex-dependent. Compared to controls and EG-treated females, the EG-treated male rats exhibited hyperoxalemia, hyperoxaluria and OX crystaluria, but the expression of CFEX mRNA and protein remained unaffected in the organs of both sexes. Thus, basic CFEX expression in both rat sexes was sufficient for OX handling even upon EG-treatment, indicating that in rats, CFEX plays no major role in generating EG-induced hyperoxaluria and nephrolithiasis.

Reabsorption of glucose in the renal proximal tubule is of pivotal physiological importance in health and disease. The reabsorption of glucose from ultrafiltrate prevents loss of metabolic energy and is important for glucose homeostasis and sodium and water balance [1]. For almost 2 decades, there is a huge interest in therapies to treat diabetic patients, and one is to control blood glucose by inhibiting Sglt2 in the kidney [2]. Sglt2 (slc5a2) is responsible for the majority of glucose reabsorption in the mammalian renal proximal tubule (PT). In our previous studies [3] in rat kidneys we confirmed the Sglt2 protein (rSglt2) expression in the brush border membrane (BBM) of cortical PT S1 and S2 segments with the female (F)-dominant sex differences (Figure 1.). In order to test the presence and determine localization of Sglt2 protein in mouse kidneys (mSglt2), two different polyclonal antibodies, rSglt2-Ab (cross-reacts with mSglt2-Ab) and mSglt2-Ab, were used in immunochemical studies. Usefulness and specificity of the antibodies for mSglt2 was proven by a) Western blotting (WB) of isolated whole kidney total cell membranes (TCM) and brush-border membranes (BBM) ; the antibodies labeled a single protein band of ~75 kDa in the membranes from adult wild-type (WT) mouse kidneys, b) immunofluorescence cytochemistry (IC) in tissue cryosections of the formalin-fixed kidneys ; the antibodies stained the brush-border of cortical PT S1 and S2 segments in WT animals, and c) absence of the labeled protein band and IC staining when using the immunizing peptide-blocked antibodies or membrane and tissue samples from the mSglt2 knockout (KO) mice (Figure 2.). In adult WT mice, the IC studies revealed the male (M)-dominant staining intensity of the renal Sglt2. Accordingly, in WB of the renal TCM and BBM, the density of mSglt2 protein band was significantly stronger in M then in F, whereas the band density of α-actin was similar in membranes from both sexes (Figure 3.).We conclude that in mice the renal Sglt2 protein expression exhibits sex differences (M > F) which are opposite from those in rats (M < F). The presence of sex- and species-related differences in renal transporters, such as shown here for Sglt2 in rats and mice, leaves doubts whether these animals are good experimental models for physiological and pharmacological studies in humans.

Water channel 1 (AQP1), a small integral membrane protein expressed in the basolateral (BLM) and apical (AM) cell membranes of proximal tubule and thin descending limb of Henle, mediates ≥ 65% of water reabsorption in the mammalian kidneys. The expression of AQP1 has been extensively studied in various rat and human organs, while the corresponding data in pig are mostly restricted to the reproductive tract. The aim of this work was to determine whether the expression of AQP1 in pig kidneys is influenced by sex hormones. Swedish landrace pigs (10 of each sex) were either sham operated (5 males (M) and 5 female (F)) or gonadectomized (5 M and 5 F) at the age of 4 weeks and sacrificed at the age of 10 months. Kidney tissue samples were collected and tested for AQP1 protein expression by Western blotting of isolated membranes and by immunostaining of tissue cryosections using specific polyclonal antibodies. With both methods, the expression of AQP1 protein along the nephron was strongly gender-depend, being in sham operated F˃M. Castration had no effect, while ovariectomy downregulated the expression approximately to the levels in sham operated M. These data indicate that the expression of AQP1 in pig kidneys is stimulated by estrogens. This is in contrast to the situation in rats, where the renal AQP expression is male dominant due to androgen stimulation. Opposite gender- and sex hormone-driven AQP1 protein expression in rat and pig kidneys proves the existence of species differences regarding this water channel.

Chloride-formate exchanger (CFEX), a member of the Solute carrier family 26 (Slc26a6), in various mammalian organs mediates transport of chloride, bicarbonate, oxalate, formate and hydroxyl ions. Its cellular localization in rat organs/tissues is poorly documented. Here we used a commercial polyclonal anti-CFEX antibody (CFEX-Ab) to investigate the CFEX protein expression in the kidneys, intestine, liver and pancreas of Wistar rats by immunofluorescence cytochemistry (IFC) in tissue cryosections and by Western blotting (WB) of total cell membranes (TCM) isolated from the respective organs. To determine sex-dependent renal expression, the CFEX protein abundance was studied in the kidneys of prepubertal, adult intact and gonadectomized rats of both sexes, and of sex hormone-treated castrates. By IFC, the CFEX-Ab stained the brush-border membrane (BBM) of proximal tubules with heterogeneous intensity (S1~S2>S3), other nephron segments being CFEX-negative. The renal CFEX protein expression was a) male-dominant, b) downregulated by castration, c) not affected by ovariectomy, d) upregulated by androgen treatment, and e) low and sex-independent in prepubertal animals. In the intestine, CFEX was localized in the BBM of duodenal and jejunal enterocytes (duodenumjejunum) ; no staining was seen in the ileum, caecum and colon. By WB of TCM, the CFEX-Ab labeled a single protein band of 120 kDa whose density matched the IFC findings. The protein was also immunolocalized to the hepatocyte canalicular membrane and apical domain of pancreatic ducts. Therefore, the CFEX protein is expressed in various rat organs/tissues, whereas in the kidneys, its expression is male-dominant due to post-pubertal androgen stimulation.

The chloride/formate exchanger CFEX, a member of the solute carrier family SLC26A6 in humans/Slc26a6 in rodents, in various mammalian organs mediates transport of anions including chloride, bicarbonate, oxalate, formate and hydroxyl ion. Except in mice, its distribution and expression in the kidneys of other species are poorly documented. Here we used a commercial polyclonal anti-peptide antibody (CFEX-Ab) to study distribution of protein (rCFEX) along the nephron, and its possible sex-related expression in adult male (M) and female (F) Wistar rats. In order to validate specificity of the CFEX-Ab, we transiently transfected the HEK293 cells with the rCFEX cDNA ; total RNA was isolated from the rat kidney, cDNA was synthesized and amplified by PCR using specific primers, a full-length rCFEX cDNA was inserted into the pcDNA3.1/His C and used for transformation of the competent DH5α E. coli, and the HEK293 cells were transfected with this vector using polyethyleneimine. Specificity of the CFEX-Ab was verified by immunofluorescence cytochemistry (IFC) ; the antibody strongly stained the plasma membrane of rCFEX-transfected cells, whereas the staining was not observed in mock-transfected cells and in the rCFEX-transfected cells incubated with the peptide-blocked antibody. The CFEX-Ab was further characterized by IFC on tissue cryosections and by Western blotting (WB) of isolated total cell (TCM) or brush-border (BBM) membranes. By IFC, the CFEX-Ab stained the BBM of proximal tubules (PT) with heterogeneous intensity (S1 ~ S2 > S3). By WB of TCM or BBM, the CFEX-Ab labeled a single protein band of 120 kDa. The rCFEX-related staining of PT BBM and labeling of the protein band were abolished with the immunizing peptide-blocked antibody. The intensity of rCFEX-related staining in PT BBM, as well as the density of rCFEX-related protein band exhibited strong sex differences, M > F. The observed expression of rCFEX protein was downregulated by castration and unaffected by ovariectomy, whereas in the sex hormone-treated gonadectomized males, testosterone upregulated, while estrogen and progesterone had no effect on the renal expression of protein. Collectively, in the rat kidneys the rCFEX protein is localized to the PT BBM showing zonal differences (cortex  outer stripe) and M-dominant expression due to androgen stimulation.

Cadmium nephrotoxicity (Cd-NTX) manifests itself through impaired proximal tubule (PT) reabsorptive and secretory functions. Urine symptoms (phosphaturia, proteinuria, aminoaciduria, glucosuria, increased excretion of inorganic and organic anions and cations, polyuria) indicate that Cd affects various transporters in the PT brush-border (BBM) and basolateral (BLM) membranes. In order to investigate their expression in Cd-NTX, we exploited experimental models of subchronic (treatment with CdCl2 for 14 days) and acute (treatment with Cd-metallothionein 6 h to 12 h before sacrifice) Cd-NTX in rats. Immunocytochemistry, Western blotting, transmission electron microscopy, and end-point RT-PCR were applied to screen transporters located in the PT BBM (NaPi2, V-ATPase, NHE3, Sglt1, Sglt2), BLM (Na/K-ATPase, Oat1, Oat3, Oct1, Oct2), or in both membranes (AQP1). In both models, PT exhibited a loss of BBM and BLM. In the subchronic model, the expression of various transporters was strongly downregulated at both the protein and mRNA level. In the acute model we observed a time-dependent loss of BBM and BLM transporters and their redistribution in intracellular organelles, translocation of certain transporters (NHE3) from the BBM to the BLM, and minimal changes in the expression of mRNAs. The data indicate that functional defects of PT in Cd-NTX result from the loss of 1) absorptive and secretory surface, 2) transporting proteins in BBM and BLM, and 3) cell polarity. In subchronic Cd-NTX, the loss of membrane transporters is mRNA-related, whereas in acute Cd-NTX, their loss may result from the disrupted intracellular vesicle sorting and trafficking.

P-gp is an ATP-dependent transmembrane efflux transporter constitutively expressed in the liver bile canaliculi (BC), renal proximal tubules (PT), intestinal epithelium, blood-tissue barriers, maternal-fetal barriers, and hematopoietic cells. In the BC, the P-gp expression exhibits sex differences (males (M)M) in the P-gp expression in BC ; gonadectomy in M, but not in F increased this expression. In the kidneys, the antibody stained the PT brush-border membrane ; gonadectomy in M, but not in F, decreased this expression in the cortex and increased in the outer stripe. In the intestine, the P-gp expression increased from proximal to distal segments, but sex differences or effects of gonadectomy were not observed. The data indicate that in rats, androgens inhibit the expression of P-gp in BC and PT S3 segments, and stimulate in the PT S2 segments.

The physiological function of aquaporin 1 (AQP1) has been thoroughly characterized in the mammalian kidneys, where it is responsible for constitutive water reabsorption. The AQP1 protein is found in the apical and basolateral plasma membranes of the proximal tubules and descending thin limbs. In the membrane, it takes two forms: non-glycosylated (NG, ~28 kDa) and glycosylated (G, 40 kDa to 50 kDa). The factors influencing renal AQP1 expression in (patho)physiological conditions are poorly understood. The aim of our work was to investigate the possible effects of sex hormones on renal AQP1 expression in rats, using immunocytochemistry, Western blotting, and real time RT-PCR. The effects were extensively studied in prepubertal and adult, gonadectomised and sex hormone-treated gonadectomised rats. The AQP1 protein expression in various kidney zones was higher in males than in females and observed sex differences were present in both forms of AQP1 (NG and G) in adult rats. The AQP1 mRNA expression was in concordance with the protein expression. Castration reduced and ovariectomy increased the abundance of AQP1 protein in isolated renal total cell membranes. Furthermore, the treatment of castrated animals with testosterone upregulated, whereas treatment with estradiol and progesterone had no significant effect on AQP1 protein expression. The abundance of AQP1 protein and mRNA in the kidneys of prepubertal rats was similar in both sexes. We conclude that sex differences exist in the expression of AQP1 along the nephron of adult rats ; AQP1 is more abundant in males due to upregulating effect of androgens.

AQP1 is assumed to be a constitutive water channel responsible for bulk water reabsorption in the mammalian kidneys. The protein is located in the cell membrane of proximal tubule and descending thin limb epithelium, where it exhibits non-glycosylated (~28 kDa) and glycosylated (40 kDa-50 kDa) forms. Possible factors influencing renal AQP1 expression in (patho)physiological conditions are largely unknown. Here we investigated the effects of sex hormones on renal AQP1 expression by immunofluorescence cytochemistry in tissue cryosection, by Western blotting in isolated membranes, and by real time RT-PCR in prepubertal and variously treated adult rats. In adult rats, the AQP1 protein expression was higher in males than in females ; sex differences were observed in both protein forms. The AQP1 mRNA expression matched the protein expression. Castration reduced and ovariectomy slightly increased, while the treatment of castrated animals with testosterone increased the abundance of AQP1 protein in the kidneys. The treatment with estradiol and progesterone had no effect. In prepubertal rats, the expression of both AQP1 protein and mRNA in the kidneys was similar in both sexes. Therefore, in the kidneys of adult rats, sex differences exist in the expression of AQP1 due to stimulating effect of androgens.

P-gp is an ATP-dependent transmembrane efflux transporter constitutively expressed in the membrane of hepatocyte bile canaliculi (BC), brush border membrane (BBM) of the renal proximal tubule (PT) and intestinal epithelium, blood-tissue barriers, maternal-fetal barriers, and hematopoietic cells. Sex steroids regulate P-gp expression and function in the rat liver, exhibiting female (F) dominant sex differences. However, the hormone(s) responsible for these differences in the rat liver, and possibly in other P-gp expressing organs, have not been resolved. Here we used the commercial monoclonal antibody (clone C219) to study effects of sex and gonadectomy on the localization and abundance of P-gp by immunocytochemistry (IC) and Western blotting (WB) in the liver, kidneys, and gastrointestinal tract of adult male (M) and F Wistar rats. We confirmed sex differences (M < F) in the P-gp expression in BC, and found that gonadectomy in M increases this expression. In the kidneys, the antibody stained BBM of the PT S2 segments in the M cortex (CTX) and S3 segments in medullary rays and outer stripe (OS) in both M and F. Gonadectomy in M, but not in F, increased the P-gp expression in OS and decreased in CTX. In the small intestine, the P-gp abundance increased from proximal to distal segments, but possible sex differences or effects of gonadectomy could not be detected due to high variability of the P-gp expression. The data indicate that in rats, androgens inhibit the expression of P-gp in BC and PT S3 segmenst, and stimulate it in the PT S2 segments.

CFEX in various mammalian organs exchanges anions, such as chloride, bicarbonate, oxalate, formate, and hydroxyl ion. The tissue expression and localization of CFEX protein has been studied in mice, but its presence in rat organs is poorly documented. Here we used an anti-CFEX antibody (CFEX-Ab) to study the expression of this transporter in the kidneys, liver, gastrointestinal tract, and pancreas of adult Wistar rats of both sexes. In the kidneys, possible sex-dependent expression of this protein was studied in more detail. The experiments were performed by immunofluorescence cytochemistry (IC) in tissue cryosections, and by Western blotting (WB) of total cell membranes (TCM) isolated from the tissues. By IC, the CFEX-Ab stained with heterogeneous intensity the brush border membrane of renal proximal tubules (PT ; S1~S2>S3) and intestinal enterocytes (duodenumjejunum), the canalicular membrane of hepatocytes, and the apical cell membrane of pancreatic ducts. By WB of TCM, the antibody labeled a protein band of 100 kDa. In PT, the expression of CFEX protein was stronger in males, downregulated by castration, and unaffected by ovariectomy. In conclusion, the CFEX protein was detected in all tested rat organs. In the kidneys, the male-dominant expression of this transporter is caused by androgen stimulation.

Antineoplastic cis-diaminodichloroplatinum (cisplatin, CDDP) is a highly nephrotoxic drug that causes hypoosmotic polyuria and decreased renal secretion of organic anions (OA) and cations (OC). The aim of this work was to investigate the expression of membrane proteins responsible for the transport of water (water channels/aquaporins, AQP1 and AQP2), OA (Oat1 and Oat3), and OC (Oct1 and Oct2) along the nephron in a rat model of cisplatin nephrotoxicity. Adult male Wistar rats were injected with a single dose of cisplatin (5 mg kg-1 b.m., i.p.) or 0.9 % NaCl (control), and tested for five consecutive days for urine excretion and standard biochemical parameters in urine. The animals were then sacrificed, the kidneys were sampled, and the platinum tissue content was determined by atomic absorption spectrometry. The expression of various transporters was studied using protein-specific polyclonal antibodies by Western blotting (WB) in isolated membranes and by immunofluorescence cytochemistry in tissue cryosections. In comparison with control animals, the cisplatin- treated rats exhibited hypoosmolar polyuria and a high accumulation of platinum in the kidney tissue. The abundance of AQP1, AQP2, Oat1, Oat3, Oct1, and Oct2 proteins in isolated renal membranes from the cisplatin-treated animals was strongly diminished. These WB findings were fully supported by immunocytochemical data. We conclude that the increased excretion of water (urine) OA and OC in cisplatin nephrotoxicity is caused by the downregulation of respective membrane transporters in epithelial cells along the nephron.

In mice, the chloride-formate exchanger (CFEX, Slc26a6), also known as the putative anion transporter-1 (PAT-1), has been localized to the brush-border membrane (BBM) of renal proximal tubule (PT) and small intestine in mice. Its localization and protein expression in rat organs is unknown. Here, an anti-CFEX polyclonal antibody (CFEX-Ab) was used to investigate in Wistar rats the cell localization and abundance of CFEX protein in 1) kidneys, liver, gastrointestinal tract, and pancreas of adult intact males, 2) kidneys of adult male and female rats, and 3) kidneys of gonadectomised rats of both sexes. The studies were performed by immunocytochemistry in tissue cryosections and by Western blotting (WB) in isolated total cell membrane. In the kidneys, CFEX-Ab exclusively stained the BBM of PT with heterogeneous intensity (S1~S2>S3). In the liver, stained was the hepatocyte canalicular membrane, while the bile ducts were CFEX-negative. In the gastrointestinal tract, the antibody strongly stained the enterocyte BBM in duodenum and jejunum (duodenumjejunum) ; ileum, cecum, and colon remained unstained. In pancreas, CFEX protein was localized to the pancreatic duct apical membrane. A CFEX-related protein band of 100 kDa was detected in the membranes isolated from the above-mentioned organs, exhibiting zonal differences in the kidneys (cortexouter stripe) and gastrointestinal tract (duodenum>jejunum). Renal expression of CFEX protein was male- dominant, downregulated by castration, and unaffected by ovariectomy. We conclude that in rats, CFEX protein is expressed in the kidneys, liver, pancreas, and intestine, but only in the kidneys its expression exhibits stimulation by androgens.

Ageing is characterized by an impaired functional and structural integrity of mammalian kidneys. A diminished handling of organic compounds, drugs, and water, as observed in the kidneys of older humans and experimental animals, indicates that ageing may affect the expression and/or activity of the renal transporters that mediate excretion and/or reabsorption of organic anions and cations, glucose, and water. We compared the expression of relevant transporters in the kidneys of 3-month- old and 24-month-old male Wistar rats, including Na/K-ATPase, organic anion transporters Oat1 (Slc22a6), Oat2 (Slc22a7), Oat3 (Slc22a8), and Oat5 (Slc22a19), organic cation transporters Oct1 (Slc22a1) and Oct2 (Slc22a2), glucose transporters Sglt1 (Slc5a1) and Sglt2 (Slc5a2), and water channels AQP1 and AQP2. These transporters/channels reside in specific cell membrane domains along the nephron. Protein expression was studied by immunofluorescence cytochemistry (IC) in tissue cryosections and Western blotting (WB) in isolated membranes, whereas expression of mRNA in the tissue was tested by RT-PCR. The protein and mRNA expression of Oat1, Oat3, Oct1, Oct2, and AQP1 was significantly weaker in senescent animals. However, the expression of Na/K-ATPase, Oat2, Oat5, Sglt1, Sglt2, and AQP2 at both protein and/or mRNA levels was not affected by age. Our findings imply that the cellular uptake and excretion of endogenous and/or exogenous compounds in the kidneys can be compromised due to the ageing-related downregulation of certain renal transporters. This can lead to an impaired ability to maintain the homeostasis of specific compounds and an increased susceptibility to drug interaction and toxicity in kidneys and other organs.

Aging is an inevitable physiological process characterized by a gradual decline in the functional capabilities regarding personal and environmental needs. In the absence of underlying diseases that accelerate aging (diabetes, cancer, etc…), kidneys are crucial organs for „healthy aging“. Aging kidneys exhibit functional, structural, and genomic losses. The impaired secretory and reabsorptive functions in handling water, organic compounds, drugs, toxins, and the increased tendency to drug-drug interactions, drug-induced adverse reactions, and drug (nephro)toxicity indicate that aging affects various renal transporters, but relevant studies on their activity and/or expression are missing. To initiate such studies, we compared various biochemical, immunochemical, and mRNA parameters in young (Y ; 3 months old) and old (O ; 2 years old) male rats. In comparison with Y, the O rats had higher body mass, lower blood testosterone, lower urine flow and excretion of inorganic ions, and unchanged excretion of glucose. The O rats exhibited lower expression of organic anion (Oat1, Oat3) and cation (Oct1, Oct2) transporters, Na+-phosphate cotransporter NaPi2, and water channel AQP1. The expression of Na/K-ATPase, Oat2, Oat5, Na+-glucose cotransporters (Sglt1, Sglt2), and AQP2 was similar in Y and O rats. We concluded that aging in rats selectively downregulates the renal transporters that are androgen-dependent.

SGLT1 is a high affinity/low capacity transporter of glucose (G) in the mammalian small intestine and kidneys. In the small intestine, SGLT1 is responsible for the entire G absorption, whereas in kidneys, it contributes to ~10% of G reabsorption, the bulk being handled by the low affinity/high capacity SGLT2. In our recent studies in rat kidneys (Am J Physiol Renal Physiol 290:F913, 2006 & Am J Physiol Cell Physiol 295:C475, 2008), the SGLT1 protein was characterized with the rat-specific polyclonal antibody, and immunolocalized to the proximal tubule (PT) brush border membrane (BBM) and intracellular organelles, exhibiting segmental (S1males (M)) differences in expression. Specific immunoreactivity was also observed in the luminal membrane of cortical thick ascending limb of Henle (TALH) and macula densa. However, previous studies in mice did not reveal clear expression and localization of SGLT1 in their kidneys and other organs due to lack of specific antibody. In order to characterize the SGLT1 protein in mouse organs, we have generated a novel polyclonal antibody against the mouse SGLT1 (mSGLT1-ab). Specificity of the antibody was confirmed by Western blotting (WB) of BBM isolated from the mouse small intestine and kidneys, and by immunostaining of tissue cryosections using wild type (WT) and Sglt1 knockout (KO) mice. In WT mice, mSGLT1-ab labeled the ~75 kDa protein band in the BBM from the small intestine and kidneys, and stained the brush-border of epithelial cells in both organs, whereas in KO mice, both the protein band and immunostaining were absent. In the kidneys of WT mice, the antibody strongly stained the BBM of PT S2 and S3 segments (S1 was negative), exhibiting segmental (S2>S3) and zonal (cortex>outer stripe) differences in staining intensity, similar in both sexes. The SGLT1-ab further stained the apical domain of TALH in the kidney cortex and outer stripe, liver bile ducts, and pancreatic ducts. In these organs, as well as in the small intestine, similar staining intensity in F and M was observed. The cells of macula densa remained unstained. Other tested extrarenal organs, such as brain, spleen, skeletal and heart muscles, and eyes were negative. Therefore, comparison of the data in rats and mice indicates the presence of species differences in renal expression and localization of SGLT1 protein.

In the mammalian kidneys, AQP1 is supposed to be a constitutive water channel located in the apical and basolateral domain of proximal tubule (PT) and descending thin limb (DTL) epithelium. In the membrane it exists in two, nonglycosylated (NG, ~28 kDa) and glycosylated (G, 40-50 kDa) forms, both being water permeable. Factors influencing renal AQP1 expression in (patho)physiological conditions are poorly known ; thus far only angiotensin II and hypertension were found to upregulate its protein and mRNA expression in the rat PT (Am J Physiol Renal Physiol 297:F1575, 2009). In order to investigate possible sex and species differences in the expression of renal AQP1, we used an anti-AQP1 polyclonal antibody and performed immunocytochemistry on tissue cryosections and Western blotting (WB) of cell membranes isolated from various kidney zones of adult male (M) and female (F) rats, mice, pigs and humans. Effects of sex hormones on AQP1 expression were studied more thoroughly in prepubertal and adult, gonadectomized and sex hormone-treated gonadectomized rats. In rats and mice, the AQP1-related immunostaining in various kidney zones in M was stronger than in F, whereas the expression (immunostaining intensity) of AQP1 in the pig and human kidneys of both sexes was similar. These results were confirmed by WB of total cell membranes (TCM), and brush-border and basolateral membranes isolated from the respective kidney zones. The observed sex differences in expression were comparable for both NG and G forms of AQP1. In the adult rats, castration had no effect, while ovariectomy increased the abundance of AQP1 in the renal TCM. Furthermore, treatment of castrated animals with testosterone upregulated, whereas treatment with estradiol and progesterone had no significant effect on NG and G forms of AQP1. Strong, but sex-independent AQP1 expression was detected in red blood cell membranes isolated from adult rats, whereas in TCM isolated from the kidneys of prepubertal rats, the AQP1 expression was weak and similar in both sexes. We conclude that a) sex differences exist in the expression of AQP1 along the nephron of adult rats and mice (M > F), which result from both upregulating effects of androgens in M and downregulating effects of estrogens in F after the puberty, and b) similar sex differences are absent in the pig and human kidneys, thus indicating the presence of species differences in the expression of renal AQP1.

Background and Aims. The symptoms of cadmium (Cd) nephrotoxicity (Cd-NTX) in humans and experimental animals manifest in the defects of reabsorptive and secretory functions of proximal tubules (PT), and include phosphaturia, aminoaciduria, glucosuria, proteinuria, increased excretion of organic anions and cations, and polyuria. These symptoms indicate that Cd targets various transporters in the PT brush-border (BBM) and basolateral (BLM) membrane. The aim of this study is to characterize the expression of these transporters in experimental subchronic and acute model of Cd-NTX in rats. Methods. Cd-NTX was induced by treating rats s.c. with CdCl2 (2 mg Cd/kg b.m./day for 14 days ; subchronic model) or Cd-metallothionein (CdMT ; a single dose of 0.4 mg Cd/kg b.m. 6 or 12 hours before sacrifice ; acute model). Control animals were vehicle-treated. Various methods (immunocytochemistry, Western blotting, transmission and immunogold microscopy, end-point RT-PCR) were applied to study the expression of transporters localized in the PT BBM (VATPase, NaPi2, megalin, NHE3, SGLT1, SGLT2), BLM (Na/K-ATPase, OAT1, OAT3, OCT1, OCT2), or in both membranes (AQP1). Results. In both models of Cd-NTX, PT exhibited loss of BBM and BLM. In subchronic model, the expression of specific transporters was strongly downregulated at the level of protein and mRNA. In acute model we observed: a) time-dependent loss of various BBM transporters and their accumulation in the randomly scattered intracellular vesicles, b) redistribution of NHE3 into the BLM, and c) time-dependent loss of various BLM transporters, and their redistribution in intracellular vesicles that accumulated in the cell subapical domain. Conclusions. The data indicate that the functional defects of PT in Cd-NTX result from: a) loss of absorptive and secretory surface, b) loss of transporting proteins in BBM and BLM, and c) loss of cell polarity. In subchronic Cd-NTX, loss of membrane transporters is largely mRNA-related, whereas in acute Cd-NTX, loss of membrane transporters due to derranged intracellular vesicle trafficking seems to be the major phenomenon.

In the mammalian kidneys, transport of various organic compounds (organic anions and cations, glucose) and water is mediated by specific proteins localized in the luminal and/or contraluminal cell membrane domains along the nephron, largely in proximal tubules. These transporters contribute to reabsorption and/or secretion of endogenous and xenobiotic organic compounds, including various anionic and cationic drugs that are used in human and veterinary medicine. Recent studies have shown that some of these transporters (“drug transporters”) contribute to development of drug resistance, drug-drug interactions, and drug-induced nephrotoxicity, whereas their malfunction due to truncated forms of proteins or point mutations in their genes can cause genetic diseases. In rodents (rats, mice, rabbits), many renal transporters exhibit sex differences in their protein and/or mRNA expression, whereas in pigs and humans, some transporters are absent, some exhibit localization in the cell membrane domains different from that in rodents, but the sex-related expression of thus far tested transporters could not been confirmed. For some transporters common to rodent, pig and human kidneys, species differences were observed in selectivity for substrates, distribution along the nephron, expression of mRNA and/or protein, sensitivity to inhibitors, and regulation of the activity. In addition, recent findings in this field in rodents revealed the species-related discrepancies in the expression of some transporters at the level of protein and their mRNA, e.g., sex differences in the transporter protein can exist with or without equivalent differences in the expression of its mRNA. Moreover, these two parameters can be in an opposite relationship. We, therefore, conclude the following: a) the data on renal transporters in one sex and species can not simply be regarded as relevant for other sex and species, b) posttranscriptional regulation may represent crucial mechanism in controlling the sex- and species-related protein expression of the renal transporters, c) various physiological, pharmacological, and toxicological findings related to the transporter-mediated handling of organic substances and water in the rodent kidneys do not reflect the situation in the pig and human kidneys, and d) the kidneys in pigs, not in rodents, may represent much better model for studying the human-related expression and functions of various renal transporters.

Prijenos različitih organskih spojeva (organski anioni i kationi, glukoza) i vode u bubrezima sisavaca posredovan je posebnim proteinima smještenim u luminalnoj i/ili kontraluminalnoj strani stanične membrane duž nefrona, glavninom u proksimalnim kanalićima. Ovi prijenosnici sudjeluju u reapsorpciji i/ili sekreciji endogenih i ksenobiotskih organskih spojeva, uključujući različite anionske i kationske lijekove koji se rabe u tretmanu bolesti u humanoj i veterinarskoj medicini. Nedavna istraživanja pokazala su da neki od dotičnih prijenosnika igraju ulogu u nastanku otpornosti na lijekove, međudjelovanju lijekova i lijekovima-izazvanu nefrotoksičnost, a njihova poremećena aktivnost zbog strukturne nesavršenosti ili točkastih mutacija može dovesti do genetske bolesti. U glodavaca (štakor, miš, kunić), neki bubrežni prijenosnici tvari iskazuju spolne razlike u ekspresiji proteina i/ili mRNA, a u bubrezima svinja i ljudi, neki prijenosnici su odsutni, neki su smješteni na staničnoj domeni suprotnoj od one u glodavaca, a spolna-ovisnost u njihovoj ekspresiji nije utvrđena. Za neke prijenosnike, koji su zajednički glodavcima i ljudima, primijećene su vrstne razlike u selektivnosti prema supstratima, lokalizaciji duž nefrona, razini ekspresije mRNA i/ili proteina, osjetljivosti na inhibitore, i regulaciji aktivnosti. Prema tome, a) podaci o bubrežnim prijenosnicima tvari u jednom spolu i vrsti na mogu se smatrati relevantnim za drugi spol i vrstu i b) različiti fiziološki, farmakološki i toksikološki rezultati, koji se odnose na prijenosnicima-posredovan tok organskih tvari i vode u bubrezima glodavaca, ne odražava stanje u humanim bubrezima.

Introduction. SGLT1 mediates a part of glucose and galactose reabsorption in the mammalian proximal tubule (PT). Previous transport studies in isolated brush-border membranes (BBM) from various kidney regions, and hybridization studies in the kidney tissue from rats and rabbits, localized this transporter largely to the PT S3 segments in the outer stripe (OS) and medullary rays (MR). However, due to lack of good antibodies, the detailed localization of SGLT1 along the mammalian nephron has not been resolved. Material and Methods. In this work we used a peptide-specific polyclonal antibody for rat SGLT1 (rSGLT1) and studied the transporter along the nephron of intact and gonadectomized male (M) and female (F) rats, and of sex hormone-treated castrated M rats, by immunoblotting of BBM isolated from the kidney cortex (C) and OS, and by immunocytochemistry in tissue cryosections. These findings were correlated with the phlorizin-sensitive, Na+-dependent uptake of 3H-D-galactose in the BBM vesicles, measured by filter technique, and with the abundance of rSGLT1-specific mRNA in the C and OS tissue, determined by Northern blotting. Results. In immunoblots of BBM from control animals, the antibody labeled a sharp ~40 kDa band, that was not clearly zone- and gender-dependent, and a broad ~75 kDa band, that in both sexes exhibited strong zonal (OS > C) and gender differences (F > M). By immunocytochemistry, the antibody stained strongly brush-border in S3 in the OS and MR and smooth muscles of the blood vessels and renal capsula, and weakly the apical domain of other PT segments in the C. Strong gender differences (F > M) in the staining intensity were observed in S3 in the OS and MR, but not in the blood vessels. The phlorizin-sensitive uptake of 3H-D-galactose in BBM vesicles completely matched the immunoblotting data related to the 75 kDa band and the immunocytochemical staining of brush-border, thus proving zonal and gender differences in the functional transporter. Relevant gender differences (F > M) were also found for the rSGLT1-specific mRNA in the OS. Ovariectomy had no effect, castration caused upregulation, whereas treatment of castrated rats with testosterone, but not with estradiol or progesterone, caused downregulation of the 75 kDa protein and the relevant immunostaining. Conclusions. The data indicate that in the rat kidney the expression of SGLT1 is represented by 75 kDa protein localized largely in the PT S3 segments, where it exhibits gender differences (F > M) at both protein and mRNA level. These gender differences are caused by androgen inhibition.

The gender differences (GD) in the renal secretion of organic anions may be due to different expression levels of the organic anion transporter 1 (OAT1) in the basolateral membrane (BLM) of the S2 proximal tubule segment. By using immunofluorescence cytochemistry in cryosections of fixed renal tissue and immunoblotting of isolated renal cortical BLM and total cell membranes, we studied localization and the abundance of OAT1 in adult and prepubertal male (M) and female (F) rats, as well as an effect of gonadectomy and steroid replacement therapy. The expression of OAT1 protein in the S2 segment was stronger in M than in F. In prepubertal rats, OAT1 abundance was low and exhibited no GD. In adult M, the abundance of OAT1 decreased by 45% two weeks after castration, whereas daily s.c. treatment of castrated M with testosterone, estradiol, or progesterone (2.5 mg/kg b.m. each) for 8 days resulted in a complete restitution, further (88%) depression, or overexpression (22%) of OAT1, respectively. In F rats, ovariectomy had no effect, but additional treatment with estradiol caused a strong decrease (73%) of OAT1. Immunoblotting data completely matched the findings by immunofluorescence. We conclude that GD in renal OAT1 expression are due to stimulation by androgens and progesterone, and inhibition by estrogens.

In the last fifteen years there were several feed-related outbreaks of morbidity and mortality in the Institute's breeding colony of Wistar rats. The last event took place in April 1999, one month after the use of a new supply of the usual standard rodent feed. Animals did not thrive and manifested generalised oedema, hypoalbuminaemia, elevated liver enzymes, and high mortality. The effect of feed was assessed first by feeding a group of sick females during 14 days with either suspected feed (A-March) or with the earlier supply of feed (A-January) of the same producer. Then a group of healthy male rats Y59 from another breeding colony was fed either suspected feed (A-March) or feed from another producer (feed B). Although neither chemical nor microbiological deviation in feed analysis had been detected, decreased consumption and slower body weight gain in all animals fed with feed A-March suggested an association between this batch of feed and the increased morbidity in those animals. Eventually, the entire rat colony was put down and replaced with a new breed which was given a new brand of feed.

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 towards 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 was 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.

AQP1 mediates a constitutive water permeability in the mammalian kidney. In the membrane, the functional protein exists in both nonglycosylated (NG, MM=28 kDa) and glycosylated (G, MM=40-60 kDa) forms. Factors that may influence the expression of AQP1 in physiological conditions are poorly known. To investigate possible sex differences, we compared the expression of AQP1 by immunocytochemistry in kidney tissue cryosections of adult male (M) and female (F) rats, and by immunoblotting in total cell membranes (TCM) isolated from the same tissue. Immunocytochemistry with serial dilutions of the polyclonal anti-AQP1 antibody revealed a higher intensity of the specific staining along the M nephron. By semiquantitative densitometry of the immunoblotting data (relative units+SEM ; n= 6 M and 6 F) in TCM from the kidney cortex (C), outer stripe (OS) and inner stripe&papilla (ISP) showed that the total abundance of AQP1 (NG+G) in M tissues (160+14.5, 167+11.1, and 162+12.7, respectively) was higher than in F tissues (123+5.4, 127+10.5, and 100+5.5, respectively ; vs. respective M data PF). These differences are not reflected in the ability of M and F kidney to excrete and concentrate the urine.

In most principal cells (PC) along the mammalian collecting duct, AQP2 is located in the apical membrane and intracellular recycling vesicles. Recent data by Coleman et al. (AJP 279:F874, 2000) showed that AQP2 may also be present basolaterally in PC of the rat renal CNT/CCD. The importance of this finding for (patho)physiology of water transport in the respective parts of the nephron is unclear. In order to define possible mechanisms of regulation and expression of the basolateral AQP2 in PC of the CNT/CCD, we performed immunofluorescence and immunogold labeling studies in cryosections of the kidney cortex from adult male (M) and female (F) rats. By immunocytochemistry, the AQP2 staining in PC of the CNT/CCD exhibited the following characteristics: a) in both sexes it was present in the apical and the basolateral domain, b) the basolateral staining colocalized with AQP3 and AQP4, and c) the intensity of basolateral staining in F was stronger than in M. As measured by a computer-guided imaging system (IP LabSpectrum, Scanalytics, VA, USA), the intensity of AQP2 staining at the PC apical domain was (average pixel intensity/cell membrane domain, +/-SEM) 1113+/-61 (n=6) and 1373+/-103 (n=6, N.S.) in M and F rats, respectively. The respective data for the basolateral cell domain were 372+/-36 (n=6) and 617+/-33 (n=6) (Pmale).

CisPt, a potent anticancer drug in humans and experimental animals, causes nephrotoxicity as its major side effect. Hypoosmotic polyuria is a marked symptom in cisPt nephrotoxicity. Polyuria develops in two stages: a vasopressin (V)-sensitive peak occurs 24 h following cisPt administration, and recedes the day after, whereas the late, V-insensitive stage starts at day 3, and steadily worsens thereafter. To study causes of polyuria and a role of water channels in these stages, we treated rats with a single dose of cisPt (5 mg/kg b.m., i.p.) and compared the distribution pattern of AQP1 and AQP2 by immunoblotting (WB) in total cell membranes (TCM) from various kidney regions and by immunocytochemistry in tissue cryosections at day 1 (D1) and day 5 (D5) following cisPt administration. In comparison with controls, at D1 the urine flow (UF) and osmolality (O) in cisPt-treated rats increased 3 fold and decreased 67%, respectively. However, similar density of protein bands in WB of TCM from the cortex (C), outer (OS) and inner stripe (IS), and papilla (PAP), and similar staining pattern in undamaged tubules indicated that the abundance and the intracellular distribution of AQP1 and AQP2 along the nephron were not affected at D1. At D5, however, the UF and O were 3.4-fold higher and 65% lower, respectively, than in control rats, and the animals exhibited glucosuria, proteinuria, and moderate phosphaturia. By WB, the abundance of AQP1 in TCM at D5 decreased between 10% (C) and 47% (OS), whereas the abundance of AQP2 decreased between 38% (PAP) and 74% (IS), the decrease in other regions being in between these extremes. By immunocytochemistry of D5 tissues we found: a) heavily denuded apical domain of the proximal tubule (PT) pars recta, b) decreased AQP1 and AQP2 staining in the respective nephron segments, and c) largely apical localization of the remaining AQP2 in the respective cells. We conclude that polyuria at D1 may result from the direct inhibition of water channels by cisPt, possibly via chelation of functionally important SH groups. At D5, polyuria is partially osmotic, and largely due to a loss of both reabsorptive surface along the PT and aquaporins in the respective nephron segments.