Novel expression of sodium/myo-inositol co-transporter in podocytes in puromycin aminonucleoside nephrosis
Yusuke Watanabe1,3,
Tatsuya Kobayashi1,3,
Eishin Yaoita1,
Hiroshi Kawachi2,
Atsushi Yamauchi4,
Tsutomu Inoue3,
Fujio Shimizu2,
Yutaka Yoshida1,
Adel G. A. El-shemi1,
Hirokazu Okada3,
Hiromichi Suzuki3 and
Tadashi Yamamoto1
1Department of Structural Pathology and 2Department of Cell Biology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, 3Department of Nephrology, Saitama Medical School, Saitama and 4Division of Nephrology, Department of Medicine, Osaka Rosai Hospital, Osaka, Japan
Correspondence and offprint requests to: Eishin Yaoita, Department of Structural Pathology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Niigata, 951-8510, Japan. Email: ren-path{at}med.niigata-u.ac.jp Y. Watanabe and T. Kobayashi contributed equally to this work.
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Abstract
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Background. How podocytes respond to injury is poorly understood, although podocyte injury in the glomerulus has been proposed as the crucial mechanism in the pathogenesis of proteinuria and focal segmental glomerulosclerosis. An increase in sodium/myo-inositol co-transporter (SMIT) transcripts, an osmoprotective gene, has been demonstrated in a variety of brain injury models. In the present study, we investigated SMIT expression in podocytes in experimental nephrosis.
Methods. Two types of nephrosis were induced in rats: puromycin aminonucleoside (PAN) nephrosis and monoclonal antibody (mAb) 5-1-6 nephropathy. Podocyte injury was morphologically distinct in the former type of nephrosis and limited to a minimum in the latter. SMIT expression in isolated glomeruli was estimated by ribonuclease protection assay. Localization of SMIT-expressing cells in glomeruli was examined by in situ hybridization.
Results. SMIT transcripts in glomeruli increased conspicuously in the nephrotic stage of PAN nephrosis, whereas the transcripts in cortices and medullae did not show significant changes. In situ hybridization revealed that podocytes were predominant cells expressing SMIT in the glomerulus. Significant increase of SMIT mRNA in the glomeruli was detected before the onset of massive proteinuria. In contrast, up-regulation of SMIT expression was not observed in mAb 5-1-6 nephropathy, whose urinary protein levels were comparable with those in the nephrotic stage of PAN nephrosis.
Conclusions. These findings suggest that SMIT expression in podocytes is not provoked by an effect of massive proteinuria but by extensive cellular injury.
Keywords: injury; monoclonal antibody 5-1-6; podocyte; puromycin aminonucleoside; sodium/myo-inositol co-transporter
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Introduction
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Podocytes are regarded as a terminally differentiated cell types such as neurons. They exhibit an elaborate morphology consisting of cell bodies, primary processes and foot processes. In addition to scarcity of their 3H-thymidine uptake, podocytes do not increase in number during postnatal and hypertrophic kidney growth [1]. Evidence has indicated that podocytes in adults are unable to undergo cell division. Regardless of the nature of the initial insult, once podocytes are injured and lost, they cannot be replaced by newly replicated cells. A considerable number of clinical and experimental studies have presented evidence that podocyte injury is the starting point of focal segmental glomerulosclerosis and eventual glomerular tuft destruction, which are common histological findings in the progression of chronic renal diseases [1]. Thus, podocyte depletion is a crucial hallmark of glomerulosclerosis and has been a central problem in the progression of renal diseases [1].
Puromycin aminonucleoside (PAN) nephrosis is widely used as a model of nephrotic syndrome. Glomerular injury induced by PAN has been demonstrated to progress to focal glomerulosclerosis [2]. Although the pathogenesis of proteinuria is not clearly explained, it has been reported that oxygen radicals that are produced during the metabolism of PAN, cause podocyte injury, resulting in nephrotic syndrome [3]. In recent in situ hybridization studies, we have discovered a striking change of expression of an osmoprotective gene, sodium/myo-inositol co-transporter (SMIT), in podocytes in PAN nephrosis. The abundance of SMIT mRNA and the transcription rate of the SMIT gene have been shown to increase when cells are cultured in hypertonic medium [4]. SMIT up-regulation results in intracellular accumulation of myo-inositol, which is one of the major organic osmolytes that does not perturb cellular enzymes and therefore allow cellular function unaltered in hypertonic environment. SMIT expression in the kidney has been found predominantly in the outer medulla, where myo-inositol reaches its highest concentration in the kidney [5]. Up-regulation of SMIT mRNA has been also demonstrated in a variety of brain injury models [68]. These recent studies suggest that SMIT induction depends not only on hypertonicity but also on unknown factors related to injury.
In the present study, we show that SMIT transcripts conspicuously increased in the glomerulus, especially podocytes, in the nephrotic stage of PAN nephrosis, which is not attributed to a podocyte response to hypertonic stress. We speculate that the SMIT expression may be regulated in podocytes in response to injury.
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Subjects and methods
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Induction of PAN nephrosis in WKY rats
A total of 50 WKY rats weighing
200 g were purchased from Charles River Japan (Atsugi, Japan) and used in this experiment. PAN nephrosis was induced in 25 rats by a single i.v. injection of PAN (Sigma, St Louis, MO, USA) at a dose of 5 mg/100 g of body weight in phosphate-buffered saline (PBS). Twenty-five control animals received the same volume of PBS. The rats were housed individually in metabolic cages and their 24-h urine specimens were collected before injection and 4, 6 and 10 days after injection of PAN. Protein concentration in the urine samples was measured by protein assay kit (Nippon Bio-Rad Laboratories, Tokyo, Japan). Ten rats of PAN-treated and control each, were killed under ether anaesthesia groups 3 days after PAN injection, and 15 rats each at day 10. The kidneys were removed and processed for ribonuclease protection assay or in situ hybridization. Renal cortices from four or six kidneys at each time point were mixed and used for isolation of glomeruli by a serial sieving method using stainless steel 60-mesh (pore size, 250 µm), 100-mesh (pore size, 150 µm) and 200-mesh (pore size, 75 µm) screens. We often found tubular fragments on the 200-mesh screen, which were larger or longer than 150 µm. To improve the purity of glomeruli, tissue fragments retained on 200-mesh were passed through a 100-mesh screen again to remove large tubular fragments.
Induction of monoclonal antibody (mAb) 5-1-6 induced nephropathy and PAN nephrosis in Wistar rats
A total of 16 Wistar rats (Charles River Japan) weighing
200 g were used in this experiment. Five rats were intravenously injected with 5 mg of mAb 5-1-6 [9]. Proteinuria excretion per 24 h was determined before and 1 and 3 days after the injection. At day 3, kidneys were removed and processed for total RNA preparation. Six rats were intravenously injected with PAN (8 mg/100 g body weight) and the remaining five rats were injected with PBS as normal control. Twenty-four-hour urine samples were collected before and 5 and 10 days after PAN injection for proteinuria estimation. At day 10, kidneys were removed and processed for total cellular RNA preparation. Each RNA sample was prepared from glomeruli isolated from two kidneys of a rat.
Ribonuclease protection assay
Ribonuclease protection assay was carried out as described previously [10]. Isolated glomeruli were homogenized in TRIzol (Gibco BRL, Grand Island, NY, USA) with a sonicator, and total cellular RNA was extracted from these samples. A fragment of rat SMIT cDNA (490 bp) was amplified by PCR with specific 5' and 3' primers for SMIT cDNA from MDCK cells corresponding to base pairs 12851774 (M85063), and subcloned in pT7 Blue (R) T-vector plasmid (Novagen, Madison, WI) [5]. Antisense cRNA probes for mRNA of SMIT and glyceraldehyde-3-phosphate dehydrogenase [GAPDH: 114 bp corresponding to base pairs 674787 (M17701) as a housekeeping gene] were 32P-labelled by in vitro transcription. Total RNA (10 µg each) from rat glomeruli, cortex and medulla were hybridized with the cRNA probes at 45°C overnight in hybridization buffer (80% formamide, 40 mM 1,4-piperazinediethanesulfonic acid, 0.4 M NaCl, 1 mM ethylenediamine-tetraacetic acid). Unhybridized probes were digested with ribonuclease A (4 µg/ml) and ribonuclease T1 (120 U/ml) for 60 min at 30°C, and then ribonucleases were digested with proteinase K (0.5 µg/ml) at 37°C for 30 min. After phenolchloroform extraction, the hybridized probes were precipitated with ethanol and heat-denatured for electrophoresis on 6% polyacrylamide gels. The dried gels were exposed to X-ray films (Fuji Photo Film, Kanagawa, Japan) for 16 h at 80°C, and the density of each protected band was quantified by a computerized image analysis system (NIH Image, National Institutes of Health, Bethesda, MD, USA). The results of ribonuclease protection assay were represented as the ratio of SMIT to GAPDH.
In situ hybridization
In situ hybridization was done by a modified procedure as described previously [10,11]. In brief, digoxigenin-labelled antisense and sense cRNA probes were synthesized by in vitro transcription with the same linearized templates that were used in the ribonuclease protection assay. Rat kidneys were fixed in 4% paraformaldehyde in PBS for 5 h at 4°C, immersed in 20% sucrose-PBS solution at 4°C for overnight, and frozen in n-hexan at 70°C. Frozen sections of kidneys on poly-L-lysine-coated glass slide were fixed again in 4% paraformaldehyde in PBS, treated with 0.5 µg/ml proteinase K for 15 min at room temperature, and hybridized with the digoxigenin-labelled probes overnight at 50°C. After washing in 3x standard saline citrate (SSC), 1 mM ethylenediamine-tetraacetic acid, and 0.05% Triton X-100, the sections were treated with 20 µg/ml ribonuclease A in 10 mM TrisHCl (pH 7.6) for 30 min at room temperature, followed by washing in 3x SSC, 1 mM EDTA, 0.05% Triton X-100. For immunodetection of digoxigenin-labelled probes, sections were incubated with alkaline phosphatase-conjugated sheep anti-digoxigenin antibody at a dilution of 1:500 for 2 h at room temperature. Visualization was performed using nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolylphosphate.
Statistics
All data were expressed as means±SD. MannWhitney U test was employed for comparison between control animals and PAN-treated animals, and the difference was considered statistically significant for P<0.05.
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Results
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Urinary protein in PAN nephrosis
Measurement of 24-h urinary protein levels revealed that a single i.v. injection of PAN induced massive proteinuria in WKY rats at day 6 (116.1±52.7 mg/day) and day 10 (201.3±39.3 mg/day) after the injection. No or slight increase in urinary protein was detected at day 4 (3.5±1.6 mg/day) in comparison with control rats (1.7±0.3 mg/day).
SMIT expression in the nephrotic stage of PAN nephrosis
The levels of SMIT transcripts in the normal kidney and PAN nephrosis at day10 were evaluated by ribonuclease protection assay (Figure 1). Significant signals for SMIT were found in every compartment of the kidney: glomerulus, total cortex and medulla, although SMIT was expressed predominantly in the medulla as shown previously [16]. SMIT expression changed distinctly in the glomerulus at day 10 of PAN nephrosis, whereas SMIT in the total cortex and medulla did not show significant changes. Densitometric analysis revealed an 8.9-fold increase of SMIT mRNA in the glomerulus (0.124±0.030 in five samples of nephrotic rats vs 0.014±0.017 in five samples of normal rats, P<0.01, each sample was made from glomeruli isolated from three rats).

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Fig. 1. Expression of SMIT mRNA in the normal kidney and PAN nephrosis at day 10. (A) Significant signals for SMIT transcripts are detected in total RNA samples (10 µg each) from isolated glomeruli (G), total cortices (C) and medullae (M) by ribonuclease protection assay, although dominant in medullae. A distinct increase of SMIT signals is seen only in glomeruli in PAN nephrosis. (B) SMIT expression levels in (A) are quantified by densitometry, and shown as the ratio of the signal intensity for SMIT to that for GAPDH. Data represent means ± SD (n = 5). n.s., not significant.
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To elucidate which cells in the glomerulus expressed SMIT, the distribution of SMIT mRNA was examined by in situ hybridization (Figure 2). In normal control rats, no signals could be detected in glomeruli, whereas SMIT mRNA was seen in distal tubules including macula densa. In PAN nephrosis, significant hybridization signals in glomeruli were observed predominantly in podocytes and parietal epithelial cells of Bowman's capsule. No significant changes were seen in tubular cells.

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Fig. 2. Localization of SMIT mRNA in glomeruli from normal kidney (A and B) and PAN nephrosis at day 10 (C and D) detected by in situ hybridization using digoxigenin-labelled cRNA antisense probe (A and C) and sense probe (B and D). No significant expression is observed in the normal glomerulus (A), whereas podocytes (arrows) show distinct expression of SMIT mRNA in addition to parietal epithelial cells of Bowman's capsule (arrowheads) (C). Bar, 50 µm.
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SMIT expression in the early stage of PAN nephrosis
The levels of SMIT mRNA in the glomerulus were evaluated 3 days after PAN injection, when massive proteinuria had not appeared, in order to examine whether SMIT up-regulation at day 10 of PAN nephrosis was related to a possible increase in osmotic pressure due to massive proteinuria. At day 3 of PAN nephrosis, 24-h urinary protein was 1.43±0.31 mg/day, which was at the same level of the normal control (1.21±0.26 mg/day). Nevertheless, ribonuclease protection assay revealed a 1.9-fold increase in SMIT mRNA in glomeruli of PAN-treated rats (0.044±0.008 in PAN-treated rats vs 0.023±0.007 in normal rats, P<0.05, n = 5, each sample was made from glomeruli isolated from two rats) (Figure 3). These findings indicate up-regulation of SMIT transcription before the onset of massive proteinuria.

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Fig. 3. Expression of SMIT mRNA in glomeruli from the normal kidneys and PAN nephrosis at day 3 detected by ribonuclease protection assay. Total RNA samples (10 µg each) were obtained from isolated glomeruli from the normal and PAN-treated kidneys. SMIT expression levels were quantified by densitometry, and shown as the ratio of the signal intensity for SMIT to that for GAPDH. Data represent means±SD (n = 5).
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SMIT expression in mAb 5-1-6 nephropathy
A single injection of mAb 5-1-6 induces massive proteinuria by antibodies binding to nephrin in the slit diaphragm [9,12]. In this nephropathy, no histological abnormalities are noted except for partial retraction of podocyte foot processes, suggesting that damage to podocytes is limited to a minimum [9]. We examined SMIT expression in the nephropathy characterized by minor podocyte abnormalities.
MAb 5-1-6 nephropathy was induced in Wistar rats because of strain variation in susceptibility for the development of mAb 5-1-6-induced proteinuria [13]. Overt proteinuria was produced 1 day after the injection in this model (Figure 4). Kidneys were removed and the glomeruli were isolated at day 3 of mAb 5-1-6 nephropathy, when the urinary protein levels (180.8±65.0 mg/day, n = 5) were similar to those of Wistar rats at day 10 of PAN nephrosis (181.7±79.6 mg/day, n = 6). SMIT expression in isolated glomeruli was compared among mAb 5-1-6-treated rats, PAN-treated ones and normal ones. The level of glomerular expression of SMIT in mAb 5-1-6 nephropathy was similar to that in normal kidneys, although a distinct increase in SMIT mRNA was detected in glomeruli in PAN nephrosis (0.026±0.005 in mAb 5-1-6 nephropathy, 0.022±0.007 in normal control, 0.139±0.074 in PAN nephrosis, n = 5, each sample was made from glomeruli isolated from one rat) (Figure 5).

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Fig. 4. Urinary protein in mAb 5-1-6 nephropathy and PAN nephrosis in Wistar rats. Data represent means±SD (n = 5 in mAb 5-1-6 nephropathy, n = 6 in PAN nephrosis).
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Fig. 5. Expression of SMIT mRNA in glomeruli from the normal control, mAb 5-1-6 nephropathy at day 3 and PAN nephrosis at day 10 shown by ribonuclease protection assay. A marked increase of SMIT transcripts is detected in total RNA samples (10 µg each) from PAN-treated glomeruli, but not from mAb 5-1-6 nephropathy. SMIT expression levels were quantified by densitometry, and shown as the ratio of the signal intensity for SMIT to that for GAPDH. Data represent means±SD (n = 5).
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Discussion
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Under the physiological condition and NaCl loading, SMIT expression in the kidney has been predominantly detected in the medullary and cortical thick ascending limb of Henle, inner medullary collecting duct and macula densa, but not in the glomerulus [5,14]. In the present study, we found a distinct increase of SMIT mRNA in isolated glomeruli in PAN nephrosis without a significant change in the total cortex and medulla. Consistent with the previous studies, in the normal control kidney, the levels of SMIT transcripts were the highest in the medulla, then in the total cortex, and the lowest in the glomerulus. Glomeruli were isolated from the cortex by the serial sieving method. The glomerular preparations do not yield a pure population of glomeruli because they are usually contaminated to low degrees with tubular fragments. Contamination of tubular fragments in the glomerular preparation could influence apparent levels of SMIT expression in isolated glomeruli. We, therefore, counted glomeruli in the preparations used in this study. They always consisted of
90% of glomeruli and 10% of tubular fragments. The percentages of glomeruli were at the same levels between normal control and nephrotic rats: 89.0% in control and 93.5% in PAN nephrosis at day10. The finding indicates that tubular contamination in glomerular preparations cannot explain the distinct increase of SMIT mRNAs in the glomeruli in PAN nephrosis, although there is no denying that low levels of SMIT mRNA detected in normal glomeruli are originated from tubular fragments. The in situ hybridization study corroborates SMIT expression in glomeruli in PAN nephrosis. Evident hybridization signals were detected in podocytes and parietal epithelial cells of Bowman's capsule in the nephrotic rats, whereas no significant signals were observed in normal glomeruli. Based on the finding that >80% of isolated glomeruli were decapsulated, it is proper to conclude that an 8.9-fold increase of SMIT expression in PAN nephrosis occurred mainly in podocytes.
Up-regulation of SMIT transcription in response to hypertonic stress has been demonstrated by using many types of cell in culture. In the case of MadinDarby canine kidney cells, when switched from isotonic to hypertonic medium, SMIT mRNA abundance rises 10-fold in 16 h [4]. The response of SMIT to hypertonicity has also been studied in vivo. In the rat kidney dehydrated by 3 days of water-deprivation, SMIT mRNA is 2.6-fold higher in the outer medulla and 2.5-fold higher in the inner medulla, compared with hydrated rats [14]. NaCl injection into rats rapidly increases SMIT mRNA, reaching a maximum 5 h after the injection (9.3-fold in the cortex and 4.6-fold in the medulla) [5]. Thus, extracellular hypertonicity is one of the possible causes for the up-regulation of SMIT transcription in podocytes in PAN nephrosis. In PAN nephrosis, plasma proteins are leaked from the glomerular capillary wall, resulting in an increase of the colloidal osmotic pressure in Bowman's space. It is plausible that the increased osmotic pressure contributes to the conspicuous SMIT up-regulation in podocytes at the nephrotic stage. However, the significant increase in SMIT transcripts in the glomerulus was detected at day 3 before the onset of massive proteinuria. In addition, no increase of SMIT mRNA was found in the massive proteinuria in mAb 5-1-6 nephropathy. These findings clearly show that SMIT expression in glomeruli is not directly related to urinary protein excretion. Taken together, hypertonicity in Bowman's space is unlikely to cause the increase of SMIT mRNA in podocytes in PAN nephrosis.
Morphologic changes of podocytes are strikingly different between PAN nephrosis and mAb 5-1-6 nephropathy. No histological abnormalities are observed except for partial retraction of foot processes in mAb 5-1-6 nephropathy [9]. In contrast, foot process retraction, vacuolation, ballooning and focal detachment of podocytes are prominent in PAN nephrosis [15]. A loss of the normal arrangement of foot processes began at day 2 of PAN injection. The morphologic changes indicate severe injury of podocytes induced by PAN injection and minimal injury in mAb 5-1-6 nephropathy. Recently, induction of the SMIT gene has been detected in brain injury models including cerebral ischaemia, haemorrhage, cryogenic injury and diffuse injury, suggesting that SMIT gene expression was also regulated by a mechanism other than extracellular tonicity [68]. In addition, Yamashita et al. have reported the interesting effect of veratridine, a voltage-gated sodium channel opener, on SMIT expression using neuroblastoma cells [16]. Veratridine evokes an accumulation of Na+ in the cells, which markedly increases the abundance of SMIT mRNA. When myo-inositol uptake is blocked, veratridine elicits cytotoxicity. Based on these findings, they have proposed the idea that SMIT gene is up-regulated by the influx of cations, and that increased myo-inositol uptake may play an important role in protection against cell toxicity induced by high intracellular electrolyte concentrations. The same situation could be applied to podocytes in PAN nephrosis. There is evidence that PAN-induced podocyte injury is mediated by reactive oxygen species. Extensive studies in vivo and in vitro have shown that antioxidants reduce proteinuria and inhibit foot process retraction [3]. Lipid peroxidation of cell membranes is postulated to be one of the major reasons for oxygen radical-induced tissue injury [17]. Actually, glomeruli and urine in PAN-treated rats contain markedly increased malondialdehyde, which is indicative of lipid peroxidation [18]. It has been reported that lipid peroxidation of cell membrane increases the permeability of the membrane bilayer to substances that do not normally cross it other than through specific channels and inactivates membrane-bound enzymes [19]. It has been demonstrated in cultured differentiated podocytes that PAN reduces function and expression of Na+Ca+ exchanger in the cell membrane, suggesting that inhibition of Ca+ extrusion via the Na+Ca+ exchanger may contribute to podocyte injury in PAN nephrosis [20]. It is fascinating to speculate that PAN-induced cell membrane damage increases influx of cations into podocytes resulting in up-regulation of SMIT gene expression, which may play a cytoprotective role against podocyte injury. Thus, this study presents the possibility that SMIT gene expression would be a molecular marker of podocyte injury. Further studies are needed to clarify the function of SMIT in injured podocyte.
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Acknowledgments
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Part of this work was presented at the 31st Annual Meeting of the American Society of Nephrology in Toronto, October 2000. The work was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Ministry for Education, Science, and Culture (14571017).
Conflict of interest statement. None declared.
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Received for publication: 7. 7.03
Accepted in revised form: 5.11.03