Role of integrin 11 in the regulation of renal medullary osmolyte concentration
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《美国生理学杂志》
1Renal Pathology Division, Department of Pathology and the 2Nephrology Division, Department of Medicine, Vanderbilt School of Medicine and 3Veterans Affairs Hospital, Nashville, Tennessee
ABSTRACT
The mechanism by which cells sense extracellular tonicity and trigger the accumulation of protective organic osmolytes is poorly understood. It has been proposed that changes in cell volume following alteration of extracellular toncity are important initiators of signaling events that lead to osmolyte accumulation. Because the extracellular matrix receptors integrins are linked to the cytoskeleton and can transduce signals that alter cell behavior, we investigated the role of these receptors in the modulation of osmolyte accumulation in the kidney medulla under different osmotic conditions. We show that integrin 1-null mice have impaired ability to accumulate organic osmolytes in the inner medulla due to altered signaling and decreased induction of osmolyte transporters or aldose reductase gene transcription. Utilizing inner medullary collecting duct cells, we demonstrate that the lack of integrin 11 results in an impaired ability to induce the tonicity enhancer-binding protein TonEBP under hypertonic conditions. Furthermore, under the same conditions, integrin 1-null cells show prolonged ERK1/2 phosphorylation and decreased inositol uptake compared with control cells. The reduction of inositol uptake is significantly reversed by treatment with the MEK inhibitor PD-98059. Finally, integrin 1-null mice develop morphological changes of early tubular necrosis and increased apoptosis of renal medullary cells following dehydration. Together, these results show that integrin 11 is an important mediator of the compatible osmolyte response in the medulla of the mammalian kidney.
volume regulation; signaling; tonEBP; MAPK
CELLS IN THE MAMMALIAN KIDNEY medulla are exposed to large alterations of extracellular tonicity that change with the hydration state of the animal (2). Cells respond to hypertonic stress by accumulating nonperturbing organic osmolytes such as the polyhydric alcohols myo-inositol and sorbitol, the methylamines betaine and glycerophosphorylcholine (GPC), and several amino acids (42). Intracellular concentrations of osmolytes are controlled by both rate of synthesis and intracellular accumulation. Myo-inositol and betaine are accumulated by sodium-coupled transporters, while sorbitol synthesis is catalyzed by the enzyme aldose reductase (AR). Both sodium-myo-inositol (SMIT) (41) and the betaine-GABA transporter 1 (BGT1) (38), as well as AR (35), are induced by hypertonicity in renal medullary cells both in vitro and in vivo, while GPC concentrations are regulated by reduction of the degradation enzyme GPC:choline p-hosphodiesterase (20). Increased abundance of mRNA for SMIT, BGT1, and AR in response to hypertonicity depends on the binding of a transcription factor TonE-binding protein (TonEBP/NFAT 5) (23) to tonicity enhancer element (TonE) (36).
The signaling mechanism whereby hyperosmolaltiy induces osmolyte transporter activity and/or enzyme transcription in the mammalian kidney medulla is still poorly understood. Activation of the MAP kinases might play a role, as JNK, ERK, and p38 MAP kinase pathways are induced when the renal medulla or collecting duct cells are exposed to hypertonicity (8, 13, 22, 27). Tonicity-dependent changes in cytoskeletal organization might activate the MAP kinase pathways in a c-src-dependent fashion (1, 17).
Interestingly, integrins, transmembrane receptors for extracellular matrix components, play a critical role in regulating the cytoskeletal organization (29) as well as src-dependent activation of MAP kinases (12).
Integrin 11, a major collagen-binding receptor, is highly expressed in both glomerular and tubular cells in the kidney (19, 39). Kidneys of integrin 1-null mice are abnormal with small and dysmorphic glomeruli that become severely sclerosed following renal injury (7). In addition, mesangial cells lacking integrin 1 have an altered cytoskeleton characterized by increased F-actin (Pozzi A, unpublished observations). Moreover, inner medullary collecting (IMCD) cells treated with blocking anti-1 antibodies or siRNA show decreased adhesion, migration, survival, and tubule formation on collagen I (6). Together, these data suggest that integrin 11 plays a critical role in normal renal cell morphology and physiological homeostasis.
Since integrin 11 appears to be important not only for IMCD survival in vitro (6) but also for mechanical-induced stress responses (3, 8), we determined the role of this collagen receptor in mediating protection of the host against hyperosmotic stress. We demonstrate that following dehydration integrin 1-null mice, but not their wild-type counterparts, show early focal medullary tubular and interstitial cell injury that correlates with an inability to upregulate the expression of osmolyte transporters and/or enzymes. Thus we provide evidence that integrin 11 is required for the induction of hyperosmotic stress-induced osmolyte response that might protect the kidney against renal tubular injury.
MATERIALS AND METHODS
Mice and experimental procedure. Wild-type and integrin 1-null 129Sv/ter female mice (8 wk old, 25 g body wt) were used for all the experiments performed according to institutional animal care guidelines. All animal experiments in this study were reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee protocol review board. Inner medullary osmolyte concentrations were quantified in kidney medullas of wild-type and integrin 1-null mice which were either untreated, dehydrated for 24 h, or treated with furosemide (LyphoMed, Rosemont, IL; 1 mg/kg body wt ip) twice daily for 3 days before death (25). Briefly, neutralized perchloric acid extracts from renal inner medullary tissues were prepared as described (25) and cellular organic osmolyte concentrations were quantified by an isocratic HPLC system (40). Intracellular osmolyte content was normalized to total protein concentrations in each of the samples. Protein concentrations were determined using the Bradford method (4). For collection of urine samples, mice were kept in metabolic cages and urine samples were collected over 24-h time periods. Urine osmolalities were measured using The Advanced Micro Osmometer (Advanced Instruments, Norwood, MA). Blood urea nitrogen (BUN) levels were determined using urea nitrogen kit (Sigma, St. Louis, MO). Three mice per genotype were used for single experiments. Four independent experiments were performed.
Histological evaluation of tubular injury. Paraffin sections of kidneys from wild-type and integrin 1-null mice untreated or dehydrated for 24 h were stained with hematoxylin and eosin or PAS and quantitatively assessed for tubular injury. Tubular injury criteria included dilatation of lumen, flattening of epithelium, and sloughing of proximal tubule brush border. Severity of injury was graded on a 1–4 scale (1 = less than 25%, 2 = 25–50%, 3 = 51–75%, and 4 = 76–100% of tubular profiles with injury). Three kidneys per genotype per treatment were analyzed.
Apoptosis within tubules was evaluated by staining paraffin kidney sections using ApopTag Plus Peroxidase in SITU Apoptosis Detection Kit (Serologicals). Degree of apoptosis was graded on a scale of 1–4 as indicated above. To evaluate epithelial injury in thick ascending loop of Henle (TALH), paraffin kidney sections were stained with anti-human Tamm-Horsfall protein (THP) antibody (MP biomedical #55140) as described (5).
Isolation of IMCD cells. Primary IMCD cells were isolated from wild-type and 1-integrin null mice as described (30). Briefly, kidneys were removed immediately at death and processed under aseptic conditions. Kidney medullas of two to four mice were dissected and transferred to hyperosmotic enzyme buffer (120 mM NaCl and 80 mM urea with a total osmolality of 630 mosmol/kgH2O) containing 12 ml DMEM-Ham's F-12 medium (GIBCO BRL), plus 24 mg collagenase B (Roche, Indianapolis, IN) and 8.5 hyaluronidase (Worthington Biochemical, Lakewood, NJ). All solutions in this process were hyperosmotic (640 mosmol/kgH2O). Inner medullas were minced and digested in enzyme solution for 90 min at 37°C under continuous agitation (300 rev/min) in a humidified incubator (5% CO2-95% O2). The resulting cell suspension was centrifuged at 160 g for 1 min, the cellular pellet washed in prewarmed, enzyme-free hyperosmotic DMEM-Ham's F-12 medium and resuspended in hyperosmotic medium that contained 50% low glucose DMEM (Irvine Scientific, Santa Ana, CA), 50% Coon's Improved Ham's F-12 medium (Cellgro, Mediatech, Herndon, VA), 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin G, 100 U/ml streptomycin sulfate, 50 nM hydrocortisone, 5 pM 3,3,5-triiodo-L-thyronine, 1 nM sodium selenate, 5 mg/l transferrin, and 10% fetal bovine serum (vol/vol).
Myo-[3H]inositol uptake study. 25 x 103 IMCD cells from wild-type and integrin 1-null mice were plated on a 96-well plate in DMEM/F12 containing 10% FCS and kept under isotonic conditions for 12 h (Costar 3598, Corning). Cells (100% confluent) were then washed with PBS and incubated in serum-free medium kept isotonic or in 100-mosmol/kgH2O increments to a final osmolarity of 500 mosmol/kgH2O. Cells were incubated for 60 min with different concentrations of unlabeled and 3H-labeled inositol (ICN, Boston, MA) (125 and 250 μmol/l unlabeled inositol mixed with 1 nmol of [3H]inositol, specific activity 10–20 Ci·mmol–1·l–1). In one experimental group, cells were pretreated with 20 μM PD-98059 (Calbiochem, La Jolla, CA) for 24 h before addition of inositol (43). Cells were then washed twice with cold iso- or hypertonic PBS, lysed in 1% SDS, and [3H]inositol uptake was measured using a -scintillation counter (Beckman, Fullerton, CA). Total inositol uptake was calculated, after subtraction of isotonic from hypertonic [3H]inositol uptake, using specific activity and respective labeled/unlabeled inositol ratio. Manual counts were performed in some wells to confirmed equal number of wild-type and integrin 1-null IMCD cells. Three independent experiments were performed in duplicate.
Generation of probes for Northern blot analysis. Total RNA from mouse inner medulla (1 μg) was reversed transcribed using Ready To Go T-Primed First-Strand Kit (Amersham, Piscataway, NJ). cDNAs were amplified using the following selective primers: AR (545 bp): sense, 5'-GCATGGTGAAAGGGGCCTGCC-3', antisense, 5'GCTGGTGTCACAGACTTGG-3'. The cDNA probe for SMIT was a generous gift from Dr. M. Kwon (University of Maryland, MD). All probes were 32P-labeled using Prime-it RII Random Primer Labeling Kit (Stratagene, La Jolla, CA).
Northern blot. IMCD cells were grown to confluency in DMEM/F12 containing 10% FCS and then adapted to a final hyperosmolarity of 500 osM over a 12-h period (100-osM increment in DMEM/F12 containing 2% FCS). IMCD cells were then kept at 500 osM for further 24 h and subsequently harvested for RNA isolation.
Total RNA from mouse renal inner medulla (3 animals/genotype per experiment) or IMCD cells was isolated using guanidinium thiocyanate as described (9). Twenty micrograms of total RNA were separated in formaldehyde-containing 1% agarose gels, transferred to nylon membranes (Nytran Supercharge, Schleicher & Schuell, Keene, NH), and hybridized with the 32P-labeled cDNAs described above. Mouse GAPDH cDNA was used for normalization. Transporter or enzyme and GAPDH bands were quantified by densitometry analysis using an Alpha Imager 2000 (Alpha Innotech, San Leandro, CA) and signals were expressed as transporter or enzyme/GAPDH.
Western blot analysis. Confluent IMCD cells were serum starved for 24 h and then incubated for 0, 10, 30, and 60 min in hypertonic serum-free medium. The cells were then washed with PBS, scraped, suspended in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, and centrifuged for 10 min at 14,000 rpm. Cell or tissue lysates were resolved by 4–20% gradient SDS/PAGE (50 μg total protein/lane) and transferred to Immobilon-P membranes (Millipore, Billerica, MA). Membranes were incubated with either rabbit TonEBP antiserum (1:2,000; generous gift from Dr. S. K. Woo, University of Maryland), rabbit anti-phospho ERK (1:1,000), anti-phospho-p38 (1:1,000), and anti-phospho-AKT antibodies (all from Cell Signaling Technology, Beverly, MA). Immunoreactive proteins were visualized using a peroxidase-conjugated goat anti-rabbit and an ECL kit (Pierce, Rockford, IL). Total ERK, p38, and AKT content was verified by stripping the membranes in 50 mM Tris·HCl (pH 6.5) containing 2% SDS and 0.4% -mercaptoethanol for 1 h at 55°C and reprobing them with rabbit anti-ERK, -p38, and -AKT antibodies (Cell Signaling Technology).
For the PKA-specific kinase assay, IMCD cells were scraped in 25 mM Tris·HCl, pH 7.4 with 0.5 mM EDTA, 0.5 mM EGTA and protease cocktail buffer (Worthington Biochemical). Cell lysates were subsequently sonicated and 5 μl lysate, corresponding to 10 μg total protein, were assayed for PKA-specific kinase activity using a specific PKA kit (Promega, Madison, WI) according to the manufacturer's instruction. Phosphorylated and unphosphorylated substrates were separated by agarose gel electrophoresis (0.8% agarose in 50 mM Tris·HCl, pH 8.0 buffer). The gels were photographed after placing on a ultraviolet transilluminator.
Statistical analysis. We used the t-test for comparisons between two groups and ANOVA using Sigma-Stat software for statistical differences between multiple groups. P 0.05 was considered statistically significant.
RESULTS
Tubular injury following dehydration. Kidney tissue sections from wild-type and 1-null mice, either allowed free access to fluids or dehydrated for 24 h, were examined by light microscopy for morphological evidence of tubular epithelial injury. While no significant tubular injury was observed in dehydrated wild-type mice (Fig. 1, A, C, E), integrin 1-null mice had mild focal morphological changes of early tubular necrosis in the S3 segment of outer medulla. This was characterized by tubular lumen dilatation, disintegration of individual epithelial cells, sloughing of apical brush border, and nuclear dropout involving 25–50% (grade 2) of tubular profiles (Fig. 1B). Epithelial cell sloughing and decreased staining intensity for THP was also observed in some outer medulla TALH of dehydrated 1-null mice (Fig. 1D). Both tubular epithelial and interstitial cells in the inner medulla of integrin 1-null mice showed moderate apoptosis involving 50–75% of papillary cells (Fig. 1F). No significant TUNEL positivity was seen in medullary cells of dehydrated wild-type animals (Fig. 1E). Despite these focal morphological differences, BUN levels were similar in the two genotypes.
Deficient osmolyte accumulation in integrin 1-null mice kidney. To determine whether wild-type and integrin 1-null mice accumulate the organic osmolytes sorbitol, betaine and inositol, we quantitated these osmolytes in kidney inner medulla tissue sections from untreated, dehydrated and diuretic animals. Urine osmolality was significantly decreased in 1-null mice compared with their wild-type counterparts under control conditions (water ad libitum, Table 1). Also, significant differences in sorbitol and betaine, but not inositol, concentrations were observed between wild-type and 1-null mice under control conditions (Fig. 2). As expected, significant increases in inner medullary betaine and inositol concentrations were deteceted in dehydrated wild-type mice compared with their untreated counterparts (Table 2 and Fig. 2). In contrast, medullary osmolyte accumulation, with exception to inositol, remained constant in dehydrated 1-null mice compared with their untreated counterparts (Table 2 and Fig. 2). Surprisingly, urine osmolarities and urine volumes were not significantly different in dehydrated 1-null mice compared with dehydrated wild-type mice (Table 1).
Because osmolyte concentrations closely follow NaCl concentrations in mammalian kidney medulla, treatment with the diuretic furosemide significantly reduces medullary osmolyte content in vivo (24). As expected, furosemide treatment in this study reduced medullary concentrations of all three organic osmolytes in wild-type mice (Fig. 3 and Table 2) compared with their untreated counterparts. The largest reduction was seen for inositol (62%), whereas sorbitol and betaine concentrations were reduced to a lesser extent. Interestingly, in diuretic 1-null mice, only inositol concentrations were significantly reduced compared with control animals (Fig. 3). Urine osmolality was significantly lower in furosemide-treated 1- null mice compared with their wild-type counterparts (575 ± 31 vs. 818 ± 17 mosmol/kgH2O, respectively; Table 1).
Integrin 1-null mice are deficient in osmolyte transporter and AR enzyme expression following hypertonic conditions. We previously reported that hyperosmotic conditions lead to increased transcriptional activity of osmolyte transporters and enzymes both in vivo and in vitro (24, 26). To determine whether failure of integrin 1-null mice to accumulate osmolytes following dehydration was due to impaired transcriptional regulation of osmolyte transporter and/or enzyme genes, we analyzed mRNA levels of SMIT and AR in renal medullary sections of wild-type and integrin 1-null mice. Furthermore, in the same tissue sections, we determined protein levels of TonEBP, the predominant transcription factor that regulates osmolyte transporters and enzymes in the kidney (23).
Dehydration significantly increased SMIT mRNA levels in wild-type but only marginally in 1-null mice compared with their respective controls (Fig. 4, A and B). Diuresis, on the other hand, did not change SMIT mRNA levels in either wild-type or 1-null mice compared with their untreated controls. Wild-type and integrin 1-null mice showed similar AR mRNA levels under control conditions, while dehydration led to significantly increased AR mRNA levels in wild-type mice (Fig. 4, A and B) compared with controls. In contrast, increase in AR mRNA levels was significantly less in dehydrated integrin 1-null mice compared with their untreated counterparts. On the other hand, diuresis significantly reduced the AR mRNA levels in wild-type, but not in 1-null animals (Fig. 4, A and B). Increase in TonEBP levels was reduced by 50% in dehydrated 1-null mice compared with dehydrated wild-type animals. Furosemide diuresis significantly decreased TonEBP in wild-type animals only (see Fig. 5, A and B).
Integrin 1-null IMCD cells are unable to modulate TonEBP levels and inositol uptake following hypertonic exposure. To better understand the underlying cellular mechanisms that lead to deficient osmolyte uptake in the renal medulla of integrin 1-null mice, we utilized primary IMCD cell cultures from both wild-type and 1-null mice. As TonEBP is the predominant transcription factor that regulates osmolyte transporters and enzymes in the kidney (23), we assessed the levels of TonEBP in IMCD cells following exposure to isotonic or hypertonic conditions. No significant differences in TonEBP levels were observed between wild-type and integrin 1-null IMCD cells cultured under isotonic conditions (Fig. 6, A and B). However, under hypertonic conditions, TonEBP levels increased 62% in wild-type, but only 31% in 1-null IMCD cells compared with their isotonic control cells (Fig. 6, A and B).
As MAP kinase and ERK pathways are induced when collecting duct cells are exposed to hypertonicity (13, 16, 22, 27) and integrin 11 can stimulate activation of the ERK pathway in certain cell types (28), we investigated ERK signaling in wild-type and integrin 1-null IMCD cells exposed to hyperosmotic conditions. Furthermore, we investigated AKT and PKA activity, as these kinases have recently been shown to either play a role in TonEBP activation (PKA) or induced under hyperosmotic conditions (11, 37). As shown in Fig. 7, increased activation of ERK, as evaluated by analyzing the levels of phophorylated protein, was observed in wild-type IMCD cells within 20 min of hypertonic stress, but rapidly returned to baseline within 40 min of onset of hyperosmotic conditions. In contrast, 1-null IMCD cell activation of ERK peaked at 40 min posthyperosmotic stress induction and remained elevated over the entire study period. In contrast, while no or minimal activation of p38 was observed in 1-null IMCD exposed to hypertonic stress, increased levels of p38 phosphorylation were observed at 40 and 60 min posthyperosmotic stress in wild-type cells. There was no significant difference in AKT phosphorylation between the two cell types treated with hypertonic medium (Fig. 7). Similary, no PKA activity was evident in either cell type cultured under normal and hyperosmotic conditions (Fig. 8).
TonEBP has previously been shown to regulate SMIT mRNA levels and inositol uptake in kidney cells (14). We therefore measured [3H]inositol uptake in wild-type and integrin 1-null IMCD cells cultured under hyperosmotic conditions. Although [3H]inositol uptake increased proportional to rising medium inositol concentrations (125 and 250 μmol/l) in both wild-type and 1-null cells, inositol uptake in the 1-null IMCD cells was markedly decreased compared with wild-type cells (Fig. 9).
To determine whether the prolonged increased ERK activation might be a cause for impaired inositol uptake, we performed inositol uptake experiment in the presence of the MEK inhibitor PD-98059. Incubation with PD-98059 restored inositol uptake in 1-null IMCD cells to levels close to those observed in wild-type cells (Fig. 9). No differences in inositol uptake were observed between wild-type cells untreated or treated with the MEK inhibitor. Thus prolonged ERK activation in integrin 1-null IMCD cells affects inositol uptake from the surrounding culture medium.
DISCUSSION
Our study was aimed at investigating the hypothesis that integrin 11 is an important mediator in renal medullary osmolyte regulation. We show that integrin 11 plays a regulatory role in hypertonicity-mediated osmolyte accumulation in the kidney inner medulla both in vivo and in vitro. Mice lacking integrin 11 not only have less osmolyte accumulated under normal conditions but also show impaired regulation of osmolytes in states of dehydration and diuresis. These deficiencies in 1-null mice appear to be based on inability of these animals to induce adequate p38 activation under dehydration conditions, while ERK activity at the same time is exaggerated. These changes in hyperosmotically-induced signaling pathways lead to impaired expression of TonEBP, the transcription factor that regulates the osmolyte transporter SMIT and the sorbitol-catalyzing enzyme aldose reductase in the kidney.
This impairment of the compatible osmolyte response results in mild, focal tubular cell injury in the 1-null mice following 24-h dehydration. Our findings are consistent with a previously published study showing that impaired inositol uptake induces cell death in TALH cells in dehydrated animals in vivo (18). Surprisingly, BUN levels did not increase significantly in acutely dehydrated 1-null mice. Moreover, although dehydrated 1-null animals showed focal injury of TALH segments, no impairment of urinary concentration capability was noticed. The reason for this lack of physiological significant injury is that only 25–50% of the assessed renal parenchyma showed signs of mild, early acute tubular necrosis in S3 and TALH segments. Longer periods of dehydration or administration of additional injurious agents would produce more extensive injury with impairment of physiological renal function.
Furthermore, findings of a recent study by Lam et al. (21) showed that urinary concentration defect in transgenic mice that overexpressed OREBP/TonEBPdn (a dominant negative form of TonEBP) was most likely due to reduced expression of aquaporin AQP2 and the urea tranporter UT-A1 and UT-A2 mRNAs, rather than reduction in intracellular osmolyte concentrations.
We demonstrate that integrin 1 null mice, unlike wild-type animals, are less able to upregulate expression of TonEBP in vivo following dehydration. Similarly, increases in TonEBP expression in 1-null IMCD cells exposed to hypertonic medium are not of the same magnitude as those seen in wild-type IMCD cells. In both experiments, the impairment of TonEBP expression was 50%. A previous study by Sheikh-Hamad et al. (34) showed the importance of 1-integrin in cellular hyperosmotic stress response.
The authors argue that signaling pathways in osmotically stressed cells may be initiated from the HB-EGF/CD9/1-integrin protein complex. Our results support their hypothesis and further indicate that inefficient signaling in integrin 1-null IMCD cells leads to deficient TonEBP expression and subsequently decreased osmolyte accumulation. Together, these findings suggest that integrin 1, which is one of the 12 1- integrins (15), may be required for mediating normal physiological signaling responses in renal medullary collecting duct cells following hypertonic stress.
We demonstrated that the lack of ability of 1-null IMCD cells to accumulate osmolytes correlates with a persistence of ERK signaling. In addition, treatment with the MEK inhibitor PD-98059 rescues the ability of 1-null cells to accumulate inositol. These results suggest that the persistence of ERK signaling is at least one of the abnormal signaling events that prevent osmolyte accumulation under hyperosmotic conditions in 1-null IMCD cells. A previous study suggested that both p38 and ERK might play a role in osmolyte accumulation and that inhibition of p38 correlates with upregulation of other MAPKs (32). Our findings support this hypothesis as 1-null IMCD cells showed very little p38 activation but strong and persistent ERK phosphorylation under hyperosmotic conditions. This activation pattern was different from wild-type cells, which showed both ERK and p38 activated following hyperosmotic stress. These findings indicate that integrin 11 might be an important regulator of signal responses to hyperosmotic stress.
A recent study indicated a role of PKA signaling in TonEBP regulation and osmolyte accumulation (11). Furthermore, AKT is activated following hypertonic conditions (37). Surprisingly, we did not observe significant difference in AKT activation between wild-type and 1-null IMCD cells. This indicates that AKT is not a major pathway by which integrin 11 regulates osmolyte accumulation under hyperosmotic conditions. Also, we could not detect PKA activity in our wild-type or null IMCD cells following hyperosmotic stimulation, suggesting that PKA is not involved in 11-dependent regulation of osmolyte accumulation.
In conclusion our study suggests that integrin 11 is an important regulator of signaling events that lead to osmolyte accumulation in mammalian kidney inner medulla cells, both in vivo and in vitro. Integrin 11-mediated signaling might be important for stimulating the transcription factor TonEBP expression and subsequently for inducing osmolyte transporter and enzyme gene expression in kidney medulla. Furthermore, 1-mediated ERK1/2 phosphorylation appears to be a critical regulatory step for the maintenance of intracellular osmolyte content in IMCD cells. Lack of integrin 1 predisposes mice kidney medullary cells to apoptosis, likely due to impaired osmolyte accumulation and impaired protection against hyperosmotic stress.
GRANTS
This work was supported by National Institutes of Health (NIH) Grant KO8-DK-59975 (to G. W. Moeckel); by the National Kidney Foundation Career Development Award, Veterans Administration Advanced Career Development and Merit Awards, RO1-DK-069921–01 and the American Heart Association Grant in Aid Award (to R. Zent); by NIH Grant NCI/RO1 CA-94849–01 (to A. Pozzi), and by the Vanderbilt George M. O'Brien Center for Kidney and Urologic Research Grant DK-39261 (to G. W. Moeckel), and NIH/National Institute of Diabetes and Digestive and Kidney Diseases O'Brien Center Grant P50-DK-39261–16 (to R. Zent and A. Pozzi).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Banes AJ, Tsuzaki M, Yamamoto J, Fischer T, Brigman B, Brown T, and Miller L. Mechanoreception at the cellular level: the detection, interpretation and diversity of responses to mechanical signals. Biochem Cell Biol 73: 349–365, 1995.
Beck FX, Schmolke M, and Guder WG. Osmolytes. Curr Opin Nephrol Hypertens 1: 43–52, 1992.
Blaschke F, Stawowy P, Goetze S, Hintz O, Grfe M, Kintscher U, Fleck E, and Graf K. Hypoxia activates 1-integrin via ERK 1/2 and p38 MAP kinase in human vascular smooth muscle cells. Biochem Biophys Res Comm 296: 890–896, 2002.
Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.
Burger-Kentischer A, Müller E, Mrz J, Fraek ML, Thurau K, and Beck FX. Hypertonicity-induced accumulation of organic osmolytes in papillary interstitial cells. Kidney Int 55: 1417–1425, 1999.
Chen D, Roberts R, Pohl M, Nigam S, Kreidberg J, Wang Z, Heino J, Ivaska J, Coffa S, Harris RC, Pozzi A, and Zent R. Differential expression of collagen- and laminin-binding integrins mediates ureteric bud and inner medullary collecting duct cell tubulogenesis. Am J Physiol Renal Physiol 287: F602–F611, 2004.
Chen X, Moeckel G, Morrow JD, Cosgrove D, Harris RC, Fogo AB, Zent R, and Pozzi A. Lack of integrin 11 leads to severe glomerulosclerosis after glomerular injury. Am J Pathol 165: 617–630, 2004.
Chiquet M, Renedo AS, Huber F, and Flück M. How do fibroblasts translate mechanical signals into changes in extracellular matrix production Matrix Biol 22: 73–80, 2003.
Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.
Damsky CH, Knudsen KA, Bradley D, Buck CA, and Horwitz AF. Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblastic cells in culture. J Cell Biol 100: 1528–1539, 1985.
Ferraris JD, Persaud P, Williams CK, Chen Y, and Burg MB. cAMP-independent role of PKA in tonicity-induced transactivation of tonicity-responsive enhancer/osmotic response element-binding protein. PNAS 99: 16800–16805, 2002.
Guan JL. Focal adhesion kinase in integrin signaling. Matrix Biol 16: 195–200, 1997.
Han J, Lee JD, Bibbs L, and Ulevitch RJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265: 808–811, 1994.
Handler JS and Kwon HM. Cell and molecular biology of organic osmolyte accumulation in hypertonic renal cells. Nephron 87: 106–110, 2001.
Hynes R. Integrins: bidirectional, allosteric signaling machines. Cell 110: 673–687, 2002.
Itoh T, Yamauchi A, Miyai A, Yokoyama K, Kamada T, Ueda N, and Fujiwara Y. Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells. J Clin Invest 93: 2387–2392, 1994.
Jalali S, Li YS, Sotoudeh M, Yuan S, Li S, Chen S, and Shyy JY. Shear stress activates p60src-Ras-MAPK signaling pathways in vascular endothelial cells. Arterioscler Thromb Vasc Biol 18: 227–234, 1998.
Kitamura H, Yamauchi A, Sugiura T, Matsuoka Y, Horio M, Tohyama M, Shimada S, Imai E, and Hori M. Inhibition of myo-inositol transport causes acute renal failure with selective medullary injury in rat. Kidney Int 53: 146–153, 1998.
Korhonen M, Ylanne J, Laitinen L, and Virtanen I. The 1-6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron. J Cell Biol 111: 1245–1254, 1990.
Kwon ED, Zablocki K, Jung KY, Peters EM, Garcia-Perez A, and Burg MB. Osmoregulation of GPC:choline phosphaodiesterase in MDCK cells: different effects of urea and NaCl. Am J Physiol Cell Physiol 269: C35–C41, 1995.
Lam AK, Ko BC, Tam S, Morris R, Yang JY, Chung SK, and Chung SS. Osmotic response element-binding protein (OREBP) is an essential regulator of the urine concentrating mechanism. J Biol Chem 279: 48048–48054, 2004.
Matsuda S, Kawasaki H, Moriguchi T, Gotoh Y, and Nishida E. Activation of protein kinase cascades by osmotic shock. J Biol Chem 270: 12781–12786, 1995.
Miyakawa H, Woo SK, Dahl SC, Handler JS, and Kwon HM. Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity. Proc Natl Acad Sci 96: 2538–2542, 1999.
Moeckel GW, Lai LW, Guder WG, Kwon HM, and Lien YHH. Kinetics and osmoregulation of Na+ and Cl–-dependent betaine transporter in rat renal medulla. Am J Physiol Renal Physiol 272: F100–F106, 1997.
Moeckel GW and Lien YHH. Distribution of de novo synthesized betaine in rat kidney: role of renal synthesis on medullary betaine accumulation. Am J Physiol Renal Physiol 272: F94–F99, 1997.
Moeckel GW, Zhang L, Fogo A, Hao C, Pozzi A, and Breyer MD. COX2 activity promotes organic osmolyte accumulation and adaptation of renal medullary interstitial cells to hypertonic stress. J Biol Chem 278: 19352–19357, 2003.
Moriguchi T, Kawasaki H, Matsuda S, Gotoh Y, and Nishida E. Evidence for multiple activators for stress-activated protein kinase/c-Jun amino-terminal kinases. Existence of novel activators. J Biol Chem 270: 12969–12972, 1995.
Pozzi A, Wary KK, Giancotti FG, and Gardner HA. Integrin 11 mediates a unique collagen-dependent proliferation pathway in vivo. J Cell Biol 142: 587–594, 1998.
Pozzi A and Zent R. Integrins: sensors of extracellular matrix and modulators of cell function. Nephron Exp Nephrol 94: 77–84, 2003.
Rauchman MI, Nigam SK, Delpire E, and Gullans SR. An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse. Am J Physiol Renal Fluid Electrolyte Physiol 265: F416–F424, 1993.
Schliess F, Schreiber R, and Haussinger D. Activation of extracellular signal-regulated kinases Erk-1 and Erk-2 by cell swelling in H4IIE hepatoma cells. Biochem J 309: 13–17, 1996.
Sheikh-Hamad D, Di Mari J, Suki WN, Safirstein R, Watts BA, and Rouse D. p38 Kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells. J Biol Chem 273: 1832–1837, 1998.
Sheikh-Hamad D, Nadkarni V, Choi Y, Truong LD, Wideman C, Hodjati R, and Gabbay KH. Cyclosporine A inhibits the adaptive responses to hypertonicity: a potential mechanism of nephrotoxicity. J Am Soc Nephrol 12: 2732–2741, 2001.
Sheikh-Hamad D, Suki WN, and Zhao W. Hypertonic induction of the cell adhesion molecule beta1-integrin in MDCK cells. Am J Physiol Cell Physiol 273: C902–C908, 1997.
Smardo FL, Burg MB, and Garcia-Perez A. Kidney aldose reductase gene transcription is osmotically regulated. Am J Physiol Cell Physiol 262: C776–C782, 1992.
Takenaka M, Preston AS, Kwon HM, and Handler JS. The tonicity-sensitive element that mediates increased transcription of the betaine transporter gene in response to hypertonic stress. J Biol Chem 269: 29379–29381, 1994.
Terada Y, Inoshita S, Hanada S, Shimamura H, Kuwahara M, Ogawa W, Kasuga M, Sasaki S, and Marumo F. Hyperosmolality activates Akt and regulates apoptosis in renal tubule cells. Kidney Int 60: 553–567, 2001.
Uchida SA, Yamauchi AS, Preston AS, Kwon MH, and Handler JS. Medium tonicity regulates expression of the Na+ and Cl–-dependent betaine transporter in Madin-Darby canine kidney cells by increasing transcription of the transporter gene. J Clin Invest 91: 1604–1607, 1993.
Voigt S, Gossrau R, Baum O, Loster K, Hofmann W, and Reutter W. Distribution and quantification of 1-integrin subunit in rat organs. Histochem J 27: 123–132, 1995.
Wolff SD, Yancey PH, Stanton TS, and Balaban RS. A simple HPLC method for quantitating major organic solutes of renal medulla. Am J Physiol Renal Fluid Electrolyte Physiol 256: F954–F956, 1989.
Yamauchi AS, Uchida AS, Preston AS, Kwon HM, and Handler JS. Hypertonicity stimulates transcription of gene for Na+-myo-inositol cotransporter in MDCK cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F20–F23, 1993.
Yancey PH, Clark ME, Hand SC, Bowlus RD, and Somero GN. Living with water stress: evolution of osmolyte systems. Science 217: 1214–1222, 1982.
Yang X, Zhang Z, and Cohen DM. ERK activation by urea in the renal inner medullary mIMCD3 cell line. Am J Physiol Renal Physiol 277: F176–F185, 1999.(Gilbert W. Moeckel, Li Zhang, Xiwu Chen,)
ABSTRACT
The mechanism by which cells sense extracellular tonicity and trigger the accumulation of protective organic osmolytes is poorly understood. It has been proposed that changes in cell volume following alteration of extracellular toncity are important initiators of signaling events that lead to osmolyte accumulation. Because the extracellular matrix receptors integrins are linked to the cytoskeleton and can transduce signals that alter cell behavior, we investigated the role of these receptors in the modulation of osmolyte accumulation in the kidney medulla under different osmotic conditions. We show that integrin 1-null mice have impaired ability to accumulate organic osmolytes in the inner medulla due to altered signaling and decreased induction of osmolyte transporters or aldose reductase gene transcription. Utilizing inner medullary collecting duct cells, we demonstrate that the lack of integrin 11 results in an impaired ability to induce the tonicity enhancer-binding protein TonEBP under hypertonic conditions. Furthermore, under the same conditions, integrin 1-null cells show prolonged ERK1/2 phosphorylation and decreased inositol uptake compared with control cells. The reduction of inositol uptake is significantly reversed by treatment with the MEK inhibitor PD-98059. Finally, integrin 1-null mice develop morphological changes of early tubular necrosis and increased apoptosis of renal medullary cells following dehydration. Together, these results show that integrin 11 is an important mediator of the compatible osmolyte response in the medulla of the mammalian kidney.
volume regulation; signaling; tonEBP; MAPK
CELLS IN THE MAMMALIAN KIDNEY medulla are exposed to large alterations of extracellular tonicity that change with the hydration state of the animal (2). Cells respond to hypertonic stress by accumulating nonperturbing organic osmolytes such as the polyhydric alcohols myo-inositol and sorbitol, the methylamines betaine and glycerophosphorylcholine (GPC), and several amino acids (42). Intracellular concentrations of osmolytes are controlled by both rate of synthesis and intracellular accumulation. Myo-inositol and betaine are accumulated by sodium-coupled transporters, while sorbitol synthesis is catalyzed by the enzyme aldose reductase (AR). Both sodium-myo-inositol (SMIT) (41) and the betaine-GABA transporter 1 (BGT1) (38), as well as AR (35), are induced by hypertonicity in renal medullary cells both in vitro and in vivo, while GPC concentrations are regulated by reduction of the degradation enzyme GPC:choline p-hosphodiesterase (20). Increased abundance of mRNA for SMIT, BGT1, and AR in response to hypertonicity depends on the binding of a transcription factor TonE-binding protein (TonEBP/NFAT 5) (23) to tonicity enhancer element (TonE) (36).
The signaling mechanism whereby hyperosmolaltiy induces osmolyte transporter activity and/or enzyme transcription in the mammalian kidney medulla is still poorly understood. Activation of the MAP kinases might play a role, as JNK, ERK, and p38 MAP kinase pathways are induced when the renal medulla or collecting duct cells are exposed to hypertonicity (8, 13, 22, 27). Tonicity-dependent changes in cytoskeletal organization might activate the MAP kinase pathways in a c-src-dependent fashion (1, 17).
Interestingly, integrins, transmembrane receptors for extracellular matrix components, play a critical role in regulating the cytoskeletal organization (29) as well as src-dependent activation of MAP kinases (12).
Integrin 11, a major collagen-binding receptor, is highly expressed in both glomerular and tubular cells in the kidney (19, 39). Kidneys of integrin 1-null mice are abnormal with small and dysmorphic glomeruli that become severely sclerosed following renal injury (7). In addition, mesangial cells lacking integrin 1 have an altered cytoskeleton characterized by increased F-actin (Pozzi A, unpublished observations). Moreover, inner medullary collecting (IMCD) cells treated with blocking anti-1 antibodies or siRNA show decreased adhesion, migration, survival, and tubule formation on collagen I (6). Together, these data suggest that integrin 11 plays a critical role in normal renal cell morphology and physiological homeostasis.
Since integrin 11 appears to be important not only for IMCD survival in vitro (6) but also for mechanical-induced stress responses (3, 8), we determined the role of this collagen receptor in mediating protection of the host against hyperosmotic stress. We demonstrate that following dehydration integrin 1-null mice, but not their wild-type counterparts, show early focal medullary tubular and interstitial cell injury that correlates with an inability to upregulate the expression of osmolyte transporters and/or enzymes. Thus we provide evidence that integrin 11 is required for the induction of hyperosmotic stress-induced osmolyte response that might protect the kidney against renal tubular injury.
MATERIALS AND METHODS
Mice and experimental procedure. Wild-type and integrin 1-null 129Sv/ter female mice (8 wk old, 25 g body wt) were used for all the experiments performed according to institutional animal care guidelines. All animal experiments in this study were reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee protocol review board. Inner medullary osmolyte concentrations were quantified in kidney medullas of wild-type and integrin 1-null mice which were either untreated, dehydrated for 24 h, or treated with furosemide (LyphoMed, Rosemont, IL; 1 mg/kg body wt ip) twice daily for 3 days before death (25). Briefly, neutralized perchloric acid extracts from renal inner medullary tissues were prepared as described (25) and cellular organic osmolyte concentrations were quantified by an isocratic HPLC system (40). Intracellular osmolyte content was normalized to total protein concentrations in each of the samples. Protein concentrations were determined using the Bradford method (4). For collection of urine samples, mice were kept in metabolic cages and urine samples were collected over 24-h time periods. Urine osmolalities were measured using The Advanced Micro Osmometer (Advanced Instruments, Norwood, MA). Blood urea nitrogen (BUN) levels were determined using urea nitrogen kit (Sigma, St. Louis, MO). Three mice per genotype were used for single experiments. Four independent experiments were performed.
Histological evaluation of tubular injury. Paraffin sections of kidneys from wild-type and integrin 1-null mice untreated or dehydrated for 24 h were stained with hematoxylin and eosin or PAS and quantitatively assessed for tubular injury. Tubular injury criteria included dilatation of lumen, flattening of epithelium, and sloughing of proximal tubule brush border. Severity of injury was graded on a 1–4 scale (1 = less than 25%, 2 = 25–50%, 3 = 51–75%, and 4 = 76–100% of tubular profiles with injury). Three kidneys per genotype per treatment were analyzed.
Apoptosis within tubules was evaluated by staining paraffin kidney sections using ApopTag Plus Peroxidase in SITU Apoptosis Detection Kit (Serologicals). Degree of apoptosis was graded on a scale of 1–4 as indicated above. To evaluate epithelial injury in thick ascending loop of Henle (TALH), paraffin kidney sections were stained with anti-human Tamm-Horsfall protein (THP) antibody (MP biomedical #55140) as described (5).
Isolation of IMCD cells. Primary IMCD cells were isolated from wild-type and 1-integrin null mice as described (30). Briefly, kidneys were removed immediately at death and processed under aseptic conditions. Kidney medullas of two to four mice were dissected and transferred to hyperosmotic enzyme buffer (120 mM NaCl and 80 mM urea with a total osmolality of 630 mosmol/kgH2O) containing 12 ml DMEM-Ham's F-12 medium (GIBCO BRL), plus 24 mg collagenase B (Roche, Indianapolis, IN) and 8.5 hyaluronidase (Worthington Biochemical, Lakewood, NJ). All solutions in this process were hyperosmotic (640 mosmol/kgH2O). Inner medullas were minced and digested in enzyme solution for 90 min at 37°C under continuous agitation (300 rev/min) in a humidified incubator (5% CO2-95% O2). The resulting cell suspension was centrifuged at 160 g for 1 min, the cellular pellet washed in prewarmed, enzyme-free hyperosmotic DMEM-Ham's F-12 medium and resuspended in hyperosmotic medium that contained 50% low glucose DMEM (Irvine Scientific, Santa Ana, CA), 50% Coon's Improved Ham's F-12 medium (Cellgro, Mediatech, Herndon, VA), 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin G, 100 U/ml streptomycin sulfate, 50 nM hydrocortisone, 5 pM 3,3,5-triiodo-L-thyronine, 1 nM sodium selenate, 5 mg/l transferrin, and 10% fetal bovine serum (vol/vol).
Myo-[3H]inositol uptake study. 25 x 103 IMCD cells from wild-type and integrin 1-null mice were plated on a 96-well plate in DMEM/F12 containing 10% FCS and kept under isotonic conditions for 12 h (Costar 3598, Corning). Cells (100% confluent) were then washed with PBS and incubated in serum-free medium kept isotonic or in 100-mosmol/kgH2O increments to a final osmolarity of 500 mosmol/kgH2O. Cells were incubated for 60 min with different concentrations of unlabeled and 3H-labeled inositol (ICN, Boston, MA) (125 and 250 μmol/l unlabeled inositol mixed with 1 nmol of [3H]inositol, specific activity 10–20 Ci·mmol–1·l–1). In one experimental group, cells were pretreated with 20 μM PD-98059 (Calbiochem, La Jolla, CA) for 24 h before addition of inositol (43). Cells were then washed twice with cold iso- or hypertonic PBS, lysed in 1% SDS, and [3H]inositol uptake was measured using a -scintillation counter (Beckman, Fullerton, CA). Total inositol uptake was calculated, after subtraction of isotonic from hypertonic [3H]inositol uptake, using specific activity and respective labeled/unlabeled inositol ratio. Manual counts were performed in some wells to confirmed equal number of wild-type and integrin 1-null IMCD cells. Three independent experiments were performed in duplicate.
Generation of probes for Northern blot analysis. Total RNA from mouse inner medulla (1 μg) was reversed transcribed using Ready To Go T-Primed First-Strand Kit (Amersham, Piscataway, NJ). cDNAs were amplified using the following selective primers: AR (545 bp): sense, 5'-GCATGGTGAAAGGGGCCTGCC-3', antisense, 5'GCTGGTGTCACAGACTTGG-3'. The cDNA probe for SMIT was a generous gift from Dr. M. Kwon (University of Maryland, MD). All probes were 32P-labeled using Prime-it RII Random Primer Labeling Kit (Stratagene, La Jolla, CA).
Northern blot. IMCD cells were grown to confluency in DMEM/F12 containing 10% FCS and then adapted to a final hyperosmolarity of 500 osM over a 12-h period (100-osM increment in DMEM/F12 containing 2% FCS). IMCD cells were then kept at 500 osM for further 24 h and subsequently harvested for RNA isolation.
Total RNA from mouse renal inner medulla (3 animals/genotype per experiment) or IMCD cells was isolated using guanidinium thiocyanate as described (9). Twenty micrograms of total RNA were separated in formaldehyde-containing 1% agarose gels, transferred to nylon membranes (Nytran Supercharge, Schleicher & Schuell, Keene, NH), and hybridized with the 32P-labeled cDNAs described above. Mouse GAPDH cDNA was used for normalization. Transporter or enzyme and GAPDH bands were quantified by densitometry analysis using an Alpha Imager 2000 (Alpha Innotech, San Leandro, CA) and signals were expressed as transporter or enzyme/GAPDH.
Western blot analysis. Confluent IMCD cells were serum starved for 24 h and then incubated for 0, 10, 30, and 60 min in hypertonic serum-free medium. The cells were then washed with PBS, scraped, suspended in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, and centrifuged for 10 min at 14,000 rpm. Cell or tissue lysates were resolved by 4–20% gradient SDS/PAGE (50 μg total protein/lane) and transferred to Immobilon-P membranes (Millipore, Billerica, MA). Membranes were incubated with either rabbit TonEBP antiserum (1:2,000; generous gift from Dr. S. K. Woo, University of Maryland), rabbit anti-phospho ERK (1:1,000), anti-phospho-p38 (1:1,000), and anti-phospho-AKT antibodies (all from Cell Signaling Technology, Beverly, MA). Immunoreactive proteins were visualized using a peroxidase-conjugated goat anti-rabbit and an ECL kit (Pierce, Rockford, IL). Total ERK, p38, and AKT content was verified by stripping the membranes in 50 mM Tris·HCl (pH 6.5) containing 2% SDS and 0.4% -mercaptoethanol for 1 h at 55°C and reprobing them with rabbit anti-ERK, -p38, and -AKT antibodies (Cell Signaling Technology).
For the PKA-specific kinase assay, IMCD cells were scraped in 25 mM Tris·HCl, pH 7.4 with 0.5 mM EDTA, 0.5 mM EGTA and protease cocktail buffer (Worthington Biochemical). Cell lysates were subsequently sonicated and 5 μl lysate, corresponding to 10 μg total protein, were assayed for PKA-specific kinase activity using a specific PKA kit (Promega, Madison, WI) according to the manufacturer's instruction. Phosphorylated and unphosphorylated substrates were separated by agarose gel electrophoresis (0.8% agarose in 50 mM Tris·HCl, pH 8.0 buffer). The gels were photographed after placing on a ultraviolet transilluminator.
Statistical analysis. We used the t-test for comparisons between two groups and ANOVA using Sigma-Stat software for statistical differences between multiple groups. P 0.05 was considered statistically significant.
RESULTS
Tubular injury following dehydration. Kidney tissue sections from wild-type and 1-null mice, either allowed free access to fluids or dehydrated for 24 h, were examined by light microscopy for morphological evidence of tubular epithelial injury. While no significant tubular injury was observed in dehydrated wild-type mice (Fig. 1, A, C, E), integrin 1-null mice had mild focal morphological changes of early tubular necrosis in the S3 segment of outer medulla. This was characterized by tubular lumen dilatation, disintegration of individual epithelial cells, sloughing of apical brush border, and nuclear dropout involving 25–50% (grade 2) of tubular profiles (Fig. 1B). Epithelial cell sloughing and decreased staining intensity for THP was also observed in some outer medulla TALH of dehydrated 1-null mice (Fig. 1D). Both tubular epithelial and interstitial cells in the inner medulla of integrin 1-null mice showed moderate apoptosis involving 50–75% of papillary cells (Fig. 1F). No significant TUNEL positivity was seen in medullary cells of dehydrated wild-type animals (Fig. 1E). Despite these focal morphological differences, BUN levels were similar in the two genotypes.
Deficient osmolyte accumulation in integrin 1-null mice kidney. To determine whether wild-type and integrin 1-null mice accumulate the organic osmolytes sorbitol, betaine and inositol, we quantitated these osmolytes in kidney inner medulla tissue sections from untreated, dehydrated and diuretic animals. Urine osmolality was significantly decreased in 1-null mice compared with their wild-type counterparts under control conditions (water ad libitum, Table 1). Also, significant differences in sorbitol and betaine, but not inositol, concentrations were observed between wild-type and 1-null mice under control conditions (Fig. 2). As expected, significant increases in inner medullary betaine and inositol concentrations were deteceted in dehydrated wild-type mice compared with their untreated counterparts (Table 2 and Fig. 2). In contrast, medullary osmolyte accumulation, with exception to inositol, remained constant in dehydrated 1-null mice compared with their untreated counterparts (Table 2 and Fig. 2). Surprisingly, urine osmolarities and urine volumes were not significantly different in dehydrated 1-null mice compared with dehydrated wild-type mice (Table 1).
Because osmolyte concentrations closely follow NaCl concentrations in mammalian kidney medulla, treatment with the diuretic furosemide significantly reduces medullary osmolyte content in vivo (24). As expected, furosemide treatment in this study reduced medullary concentrations of all three organic osmolytes in wild-type mice (Fig. 3 and Table 2) compared with their untreated counterparts. The largest reduction was seen for inositol (62%), whereas sorbitol and betaine concentrations were reduced to a lesser extent. Interestingly, in diuretic 1-null mice, only inositol concentrations were significantly reduced compared with control animals (Fig. 3). Urine osmolality was significantly lower in furosemide-treated 1- null mice compared with their wild-type counterparts (575 ± 31 vs. 818 ± 17 mosmol/kgH2O, respectively; Table 1).
Integrin 1-null mice are deficient in osmolyte transporter and AR enzyme expression following hypertonic conditions. We previously reported that hyperosmotic conditions lead to increased transcriptional activity of osmolyte transporters and enzymes both in vivo and in vitro (24, 26). To determine whether failure of integrin 1-null mice to accumulate osmolytes following dehydration was due to impaired transcriptional regulation of osmolyte transporter and/or enzyme genes, we analyzed mRNA levels of SMIT and AR in renal medullary sections of wild-type and integrin 1-null mice. Furthermore, in the same tissue sections, we determined protein levels of TonEBP, the predominant transcription factor that regulates osmolyte transporters and enzymes in the kidney (23).
Dehydration significantly increased SMIT mRNA levels in wild-type but only marginally in 1-null mice compared with their respective controls (Fig. 4, A and B). Diuresis, on the other hand, did not change SMIT mRNA levels in either wild-type or 1-null mice compared with their untreated controls. Wild-type and integrin 1-null mice showed similar AR mRNA levels under control conditions, while dehydration led to significantly increased AR mRNA levels in wild-type mice (Fig. 4, A and B) compared with controls. In contrast, increase in AR mRNA levels was significantly less in dehydrated integrin 1-null mice compared with their untreated counterparts. On the other hand, diuresis significantly reduced the AR mRNA levels in wild-type, but not in 1-null animals (Fig. 4, A and B). Increase in TonEBP levels was reduced by 50% in dehydrated 1-null mice compared with dehydrated wild-type animals. Furosemide diuresis significantly decreased TonEBP in wild-type animals only (see Fig. 5, A and B).
Integrin 1-null IMCD cells are unable to modulate TonEBP levels and inositol uptake following hypertonic exposure. To better understand the underlying cellular mechanisms that lead to deficient osmolyte uptake in the renal medulla of integrin 1-null mice, we utilized primary IMCD cell cultures from both wild-type and 1-null mice. As TonEBP is the predominant transcription factor that regulates osmolyte transporters and enzymes in the kidney (23), we assessed the levels of TonEBP in IMCD cells following exposure to isotonic or hypertonic conditions. No significant differences in TonEBP levels were observed between wild-type and integrin 1-null IMCD cells cultured under isotonic conditions (Fig. 6, A and B). However, under hypertonic conditions, TonEBP levels increased 62% in wild-type, but only 31% in 1-null IMCD cells compared with their isotonic control cells (Fig. 6, A and B).
As MAP kinase and ERK pathways are induced when collecting duct cells are exposed to hypertonicity (13, 16, 22, 27) and integrin 11 can stimulate activation of the ERK pathway in certain cell types (28), we investigated ERK signaling in wild-type and integrin 1-null IMCD cells exposed to hyperosmotic conditions. Furthermore, we investigated AKT and PKA activity, as these kinases have recently been shown to either play a role in TonEBP activation (PKA) or induced under hyperosmotic conditions (11, 37). As shown in Fig. 7, increased activation of ERK, as evaluated by analyzing the levels of phophorylated protein, was observed in wild-type IMCD cells within 20 min of hypertonic stress, but rapidly returned to baseline within 40 min of onset of hyperosmotic conditions. In contrast, 1-null IMCD cell activation of ERK peaked at 40 min posthyperosmotic stress induction and remained elevated over the entire study period. In contrast, while no or minimal activation of p38 was observed in 1-null IMCD exposed to hypertonic stress, increased levels of p38 phosphorylation were observed at 40 and 60 min posthyperosmotic stress in wild-type cells. There was no significant difference in AKT phosphorylation between the two cell types treated with hypertonic medium (Fig. 7). Similary, no PKA activity was evident in either cell type cultured under normal and hyperosmotic conditions (Fig. 8).
TonEBP has previously been shown to regulate SMIT mRNA levels and inositol uptake in kidney cells (14). We therefore measured [3H]inositol uptake in wild-type and integrin 1-null IMCD cells cultured under hyperosmotic conditions. Although [3H]inositol uptake increased proportional to rising medium inositol concentrations (125 and 250 μmol/l) in both wild-type and 1-null cells, inositol uptake in the 1-null IMCD cells was markedly decreased compared with wild-type cells (Fig. 9).
To determine whether the prolonged increased ERK activation might be a cause for impaired inositol uptake, we performed inositol uptake experiment in the presence of the MEK inhibitor PD-98059. Incubation with PD-98059 restored inositol uptake in 1-null IMCD cells to levels close to those observed in wild-type cells (Fig. 9). No differences in inositol uptake were observed between wild-type cells untreated or treated with the MEK inhibitor. Thus prolonged ERK activation in integrin 1-null IMCD cells affects inositol uptake from the surrounding culture medium.
DISCUSSION
Our study was aimed at investigating the hypothesis that integrin 11 is an important mediator in renal medullary osmolyte regulation. We show that integrin 11 plays a regulatory role in hypertonicity-mediated osmolyte accumulation in the kidney inner medulla both in vivo and in vitro. Mice lacking integrin 11 not only have less osmolyte accumulated under normal conditions but also show impaired regulation of osmolytes in states of dehydration and diuresis. These deficiencies in 1-null mice appear to be based on inability of these animals to induce adequate p38 activation under dehydration conditions, while ERK activity at the same time is exaggerated. These changes in hyperosmotically-induced signaling pathways lead to impaired expression of TonEBP, the transcription factor that regulates the osmolyte transporter SMIT and the sorbitol-catalyzing enzyme aldose reductase in the kidney.
This impairment of the compatible osmolyte response results in mild, focal tubular cell injury in the 1-null mice following 24-h dehydration. Our findings are consistent with a previously published study showing that impaired inositol uptake induces cell death in TALH cells in dehydrated animals in vivo (18). Surprisingly, BUN levels did not increase significantly in acutely dehydrated 1-null mice. Moreover, although dehydrated 1-null animals showed focal injury of TALH segments, no impairment of urinary concentration capability was noticed. The reason for this lack of physiological significant injury is that only 25–50% of the assessed renal parenchyma showed signs of mild, early acute tubular necrosis in S3 and TALH segments. Longer periods of dehydration or administration of additional injurious agents would produce more extensive injury with impairment of physiological renal function.
Furthermore, findings of a recent study by Lam et al. (21) showed that urinary concentration defect in transgenic mice that overexpressed OREBP/TonEBPdn (a dominant negative form of TonEBP) was most likely due to reduced expression of aquaporin AQP2 and the urea tranporter UT-A1 and UT-A2 mRNAs, rather than reduction in intracellular osmolyte concentrations.
We demonstrate that integrin 1 null mice, unlike wild-type animals, are less able to upregulate expression of TonEBP in vivo following dehydration. Similarly, increases in TonEBP expression in 1-null IMCD cells exposed to hypertonic medium are not of the same magnitude as those seen in wild-type IMCD cells. In both experiments, the impairment of TonEBP expression was 50%. A previous study by Sheikh-Hamad et al. (34) showed the importance of 1-integrin in cellular hyperosmotic stress response.
The authors argue that signaling pathways in osmotically stressed cells may be initiated from the HB-EGF/CD9/1-integrin protein complex. Our results support their hypothesis and further indicate that inefficient signaling in integrin 1-null IMCD cells leads to deficient TonEBP expression and subsequently decreased osmolyte accumulation. Together, these findings suggest that integrin 1, which is one of the 12 1- integrins (15), may be required for mediating normal physiological signaling responses in renal medullary collecting duct cells following hypertonic stress.
We demonstrated that the lack of ability of 1-null IMCD cells to accumulate osmolytes correlates with a persistence of ERK signaling. In addition, treatment with the MEK inhibitor PD-98059 rescues the ability of 1-null cells to accumulate inositol. These results suggest that the persistence of ERK signaling is at least one of the abnormal signaling events that prevent osmolyte accumulation under hyperosmotic conditions in 1-null IMCD cells. A previous study suggested that both p38 and ERK might play a role in osmolyte accumulation and that inhibition of p38 correlates with upregulation of other MAPKs (32). Our findings support this hypothesis as 1-null IMCD cells showed very little p38 activation but strong and persistent ERK phosphorylation under hyperosmotic conditions. This activation pattern was different from wild-type cells, which showed both ERK and p38 activated following hyperosmotic stress. These findings indicate that integrin 11 might be an important regulator of signal responses to hyperosmotic stress.
A recent study indicated a role of PKA signaling in TonEBP regulation and osmolyte accumulation (11). Furthermore, AKT is activated following hypertonic conditions (37). Surprisingly, we did not observe significant difference in AKT activation between wild-type and 1-null IMCD cells. This indicates that AKT is not a major pathway by which integrin 11 regulates osmolyte accumulation under hyperosmotic conditions. Also, we could not detect PKA activity in our wild-type or null IMCD cells following hyperosmotic stimulation, suggesting that PKA is not involved in 11-dependent regulation of osmolyte accumulation.
In conclusion our study suggests that integrin 11 is an important regulator of signaling events that lead to osmolyte accumulation in mammalian kidney inner medulla cells, both in vivo and in vitro. Integrin 11-mediated signaling might be important for stimulating the transcription factor TonEBP expression and subsequently for inducing osmolyte transporter and enzyme gene expression in kidney medulla. Furthermore, 1-mediated ERK1/2 phosphorylation appears to be a critical regulatory step for the maintenance of intracellular osmolyte content in IMCD cells. Lack of integrin 1 predisposes mice kidney medullary cells to apoptosis, likely due to impaired osmolyte accumulation and impaired protection against hyperosmotic stress.
GRANTS
This work was supported by National Institutes of Health (NIH) Grant KO8-DK-59975 (to G. W. Moeckel); by the National Kidney Foundation Career Development Award, Veterans Administration Advanced Career Development and Merit Awards, RO1-DK-069921–01 and the American Heart Association Grant in Aid Award (to R. Zent); by NIH Grant NCI/RO1 CA-94849–01 (to A. Pozzi), and by the Vanderbilt George M. O'Brien Center for Kidney and Urologic Research Grant DK-39261 (to G. W. Moeckel), and NIH/National Institute of Diabetes and Digestive and Kidney Diseases O'Brien Center Grant P50-DK-39261–16 (to R. Zent and A. Pozzi).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Banes AJ, Tsuzaki M, Yamamoto J, Fischer T, Brigman B, Brown T, and Miller L. Mechanoreception at the cellular level: the detection, interpretation and diversity of responses to mechanical signals. Biochem Cell Biol 73: 349–365, 1995.
Beck FX, Schmolke M, and Guder WG. Osmolytes. Curr Opin Nephrol Hypertens 1: 43–52, 1992.
Blaschke F, Stawowy P, Goetze S, Hintz O, Grfe M, Kintscher U, Fleck E, and Graf K. Hypoxia activates 1-integrin via ERK 1/2 and p38 MAP kinase in human vascular smooth muscle cells. Biochem Biophys Res Comm 296: 890–896, 2002.
Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.
Burger-Kentischer A, Müller E, Mrz J, Fraek ML, Thurau K, and Beck FX. Hypertonicity-induced accumulation of organic osmolytes in papillary interstitial cells. Kidney Int 55: 1417–1425, 1999.
Chen D, Roberts R, Pohl M, Nigam S, Kreidberg J, Wang Z, Heino J, Ivaska J, Coffa S, Harris RC, Pozzi A, and Zent R. Differential expression of collagen- and laminin-binding integrins mediates ureteric bud and inner medullary collecting duct cell tubulogenesis. Am J Physiol Renal Physiol 287: F602–F611, 2004.
Chen X, Moeckel G, Morrow JD, Cosgrove D, Harris RC, Fogo AB, Zent R, and Pozzi A. Lack of integrin 11 leads to severe glomerulosclerosis after glomerular injury. Am J Pathol 165: 617–630, 2004.
Chiquet M, Renedo AS, Huber F, and Flück M. How do fibroblasts translate mechanical signals into changes in extracellular matrix production Matrix Biol 22: 73–80, 2003.
Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.
Damsky CH, Knudsen KA, Bradley D, Buck CA, and Horwitz AF. Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblastic cells in culture. J Cell Biol 100: 1528–1539, 1985.
Ferraris JD, Persaud P, Williams CK, Chen Y, and Burg MB. cAMP-independent role of PKA in tonicity-induced transactivation of tonicity-responsive enhancer/osmotic response element-binding protein. PNAS 99: 16800–16805, 2002.
Guan JL. Focal adhesion kinase in integrin signaling. Matrix Biol 16: 195–200, 1997.
Han J, Lee JD, Bibbs L, and Ulevitch RJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265: 808–811, 1994.
Handler JS and Kwon HM. Cell and molecular biology of organic osmolyte accumulation in hypertonic renal cells. Nephron 87: 106–110, 2001.
Hynes R. Integrins: bidirectional, allosteric signaling machines. Cell 110: 673–687, 2002.
Itoh T, Yamauchi A, Miyai A, Yokoyama K, Kamada T, Ueda N, and Fujiwara Y. Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells. J Clin Invest 93: 2387–2392, 1994.
Jalali S, Li YS, Sotoudeh M, Yuan S, Li S, Chen S, and Shyy JY. Shear stress activates p60src-Ras-MAPK signaling pathways in vascular endothelial cells. Arterioscler Thromb Vasc Biol 18: 227–234, 1998.
Kitamura H, Yamauchi A, Sugiura T, Matsuoka Y, Horio M, Tohyama M, Shimada S, Imai E, and Hori M. Inhibition of myo-inositol transport causes acute renal failure with selective medullary injury in rat. Kidney Int 53: 146–153, 1998.
Korhonen M, Ylanne J, Laitinen L, and Virtanen I. The 1-6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron. J Cell Biol 111: 1245–1254, 1990.
Kwon ED, Zablocki K, Jung KY, Peters EM, Garcia-Perez A, and Burg MB. Osmoregulation of GPC:choline phosphaodiesterase in MDCK cells: different effects of urea and NaCl. Am J Physiol Cell Physiol 269: C35–C41, 1995.
Lam AK, Ko BC, Tam S, Morris R, Yang JY, Chung SK, and Chung SS. Osmotic response element-binding protein (OREBP) is an essential regulator of the urine concentrating mechanism. J Biol Chem 279: 48048–48054, 2004.
Matsuda S, Kawasaki H, Moriguchi T, Gotoh Y, and Nishida E. Activation of protein kinase cascades by osmotic shock. J Biol Chem 270: 12781–12786, 1995.
Miyakawa H, Woo SK, Dahl SC, Handler JS, and Kwon HM. Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity. Proc Natl Acad Sci 96: 2538–2542, 1999.
Moeckel GW, Lai LW, Guder WG, Kwon HM, and Lien YHH. Kinetics and osmoregulation of Na+ and Cl–-dependent betaine transporter in rat renal medulla. Am J Physiol Renal Physiol 272: F100–F106, 1997.
Moeckel GW and Lien YHH. Distribution of de novo synthesized betaine in rat kidney: role of renal synthesis on medullary betaine accumulation. Am J Physiol Renal Physiol 272: F94–F99, 1997.
Moeckel GW, Zhang L, Fogo A, Hao C, Pozzi A, and Breyer MD. COX2 activity promotes organic osmolyte accumulation and adaptation of renal medullary interstitial cells to hypertonic stress. J Biol Chem 278: 19352–19357, 2003.
Moriguchi T, Kawasaki H, Matsuda S, Gotoh Y, and Nishida E. Evidence for multiple activators for stress-activated protein kinase/c-Jun amino-terminal kinases. Existence of novel activators. J Biol Chem 270: 12969–12972, 1995.
Pozzi A, Wary KK, Giancotti FG, and Gardner HA. Integrin 11 mediates a unique collagen-dependent proliferation pathway in vivo. J Cell Biol 142: 587–594, 1998.
Pozzi A and Zent R. Integrins: sensors of extracellular matrix and modulators of cell function. Nephron Exp Nephrol 94: 77–84, 2003.
Rauchman MI, Nigam SK, Delpire E, and Gullans SR. An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse. Am J Physiol Renal Fluid Electrolyte Physiol 265: F416–F424, 1993.
Schliess F, Schreiber R, and Haussinger D. Activation of extracellular signal-regulated kinases Erk-1 and Erk-2 by cell swelling in H4IIE hepatoma cells. Biochem J 309: 13–17, 1996.
Sheikh-Hamad D, Di Mari J, Suki WN, Safirstein R, Watts BA, and Rouse D. p38 Kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells. J Biol Chem 273: 1832–1837, 1998.
Sheikh-Hamad D, Nadkarni V, Choi Y, Truong LD, Wideman C, Hodjati R, and Gabbay KH. Cyclosporine A inhibits the adaptive responses to hypertonicity: a potential mechanism of nephrotoxicity. J Am Soc Nephrol 12: 2732–2741, 2001.
Sheikh-Hamad D, Suki WN, and Zhao W. Hypertonic induction of the cell adhesion molecule beta1-integrin in MDCK cells. Am J Physiol Cell Physiol 273: C902–C908, 1997.
Smardo FL, Burg MB, and Garcia-Perez A. Kidney aldose reductase gene transcription is osmotically regulated. Am J Physiol Cell Physiol 262: C776–C782, 1992.
Takenaka M, Preston AS, Kwon HM, and Handler JS. The tonicity-sensitive element that mediates increased transcription of the betaine transporter gene in response to hypertonic stress. J Biol Chem 269: 29379–29381, 1994.
Terada Y, Inoshita S, Hanada S, Shimamura H, Kuwahara M, Ogawa W, Kasuga M, Sasaki S, and Marumo F. Hyperosmolality activates Akt and regulates apoptosis in renal tubule cells. Kidney Int 60: 553–567, 2001.
Uchida SA, Yamauchi AS, Preston AS, Kwon MH, and Handler JS. Medium tonicity regulates expression of the Na+ and Cl–-dependent betaine transporter in Madin-Darby canine kidney cells by increasing transcription of the transporter gene. J Clin Invest 91: 1604–1607, 1993.
Voigt S, Gossrau R, Baum O, Loster K, Hofmann W, and Reutter W. Distribution and quantification of 1-integrin subunit in rat organs. Histochem J 27: 123–132, 1995.
Wolff SD, Yancey PH, Stanton TS, and Balaban RS. A simple HPLC method for quantitating major organic solutes of renal medulla. Am J Physiol Renal Fluid Electrolyte Physiol 256: F954–F956, 1989.
Yamauchi AS, Uchida AS, Preston AS, Kwon HM, and Handler JS. Hypertonicity stimulates transcription of gene for Na+-myo-inositol cotransporter in MDCK cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F20–F23, 1993.
Yancey PH, Clark ME, Hand SC, Bowlus RD, and Somero GN. Living with water stress: evolution of osmolyte systems. Science 217: 1214–1222, 1982.
Yang X, Zhang Z, and Cohen DM. ERK activation by urea in the renal inner medullary mIMCD3 cell line. Am J Physiol Renal Physiol 277: F176–F185, 1999.(Gilbert W. Moeckel, Li Zhang, Xiwu Chen,)