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Renal Division, Federal University of São Paulo, 04023-900 São Paulo, Brazil
Submitted 22 October 2003 ; accepted in final form 5 January 2004
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ABSTRACT |
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hyperglycemia; diabetic nephropathy; glomerular sclerosis; renin-angiotensin system
Renal tubular epithelial cells constitute a potential site of intrarenal ANG II synthesis (25, 35). In addition, it has been demonstrated that mesangial cells express mRNA for renin, angiotensinogen, and ACE (4, 5). Renin is a rate-limiting enzyme in the synthesis of ANG II (34), and we have recently demonstrated that mesangial cells in culture are able to synthesize, store, and secrete both forms of the enzyme, i.e., active renin and inactive prorenin (4).
Hyperglycemia has been recognized to be the pathogenic factor of the long-term complications of diabetes mellitus. Thus in the present study we examined whether a high glucose concentration in the culture medium is able to modify renin synthesis, the secretion of the renin/prorenin ratio, and the intracellular renin activity in mesangial cells in culture. Also, we examined whether this "hyperglycemic" condition alters the levels of mRNA expression of RAS components, including angiotensinogen, prorenin, ACE, types I and II (AT1 and AT2) angiotensin receptors, and cathepsin B, a potential enzyme involved in renin activation. Finally, we also determined whether glucose-inducing modifications in renin activity and in the expression of RAS components result in an increase in local ANG II generation.
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MATERIALS AND METHODS |
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300 glomeruli/cm onto RPMI 1640 containing 10 mM D-glucose and supplemented with 20% fetal calf serum, 50 U/ml penicillin, 2.6 g acid HEPES, and 2 mM glutamine. Culture flasks were kept in a 95% air-5% CO2 humidified environment at 37°C. The culture medium was replaced every 36 h. After 3 wk, cells were harvested with trypsin and the subcultures were grown in the same culture medium. Cells were used between the third and fifth subculture and characterized by classic methods using the following criteria: morphological appearance of stellate cells, immunofluorescence staining of the extracellular matrix for type IV collagen and fibronectin, negative immunofluorescence staining for human factor VIII antigens (glomerular endothelial cells) and cytokeratin (parietal epithelial cells), and positive immunofluorescence staining for actin and myosin. In particular, the presence of contaminating juxtaglomerular cells was excluded on the basis of their characteristic phenotype including their spherical shape and the presence of a high density of granules. Experimental protocol. At subconfluence, mesangial cells were divided into three groups as follows: control, where cells were kept in RPMI 1640 medium supplemented with FBS, containing normal glucose concentration of 10 mM D-glucose (NG group); high glucose, where cells were cultured in RPMI 1640 supplemented with FBS containing 30 mM D-glucose (HG group); and mannitol, where cells were submitted to the same procedure as used for the cell culture in the presence of glucose, but 30 mM mannitol was added instead of glucose and served as osmolarity control (M group). Cells were exposed to 30 mM D-glucose or mannitol for a total of 72 h (16). After 48 h, cells were rinsed twice with PBS and the culture medium was replaced with a medium containing the respective normal- or high-glucose and mannitol concentrations, but no FBS, to keep the cells in the G0 phase of the cell cycle and without any interference from serum proteins. The culture medium was then collected over the last 24 h and stored at –70°C until use. Cells were rinsed with PBS, lysed with 1 mM Tris·HCl buffer, pH 7.5, and stored at –70°C until use. An additional group of cells were exposed to high glucose concentration for a shorter period of 24 h.
Determination of active and inactive renin (prorenin) content and renin activity. The renin content of the active and inactive forms of renin was estimated by measuring the amount of ANG I present in the cell lysate and culture medium by HPLC, as described below. Total renin content included active renin and the inactive prorenin forms. Prorenin was activated by adding 10 µl of trypsin (50 µg/ml) for 16–18 h at 37°C to both the cell lysate and culture medium. Active renin was evaluated in the absence of trypsin. To prevent further cleavage of prorenin, angiotensinogen, and ANG I, a pool of enzymatic inhibitors was added to the cell homogenate and culture medium: 50 mM EDTA, 1 mM OPhe, 32 mM PMFS, and 200 mM DTT. These inhibitors act efficiently on a broad range of metallo-, serine, and cysteine proteases, respectively. Renin activity was estimated by ANG I generation when the cell lysate and culture medium (in the presence and absence of trypsin) were incubated with 10 µl of 1 mg/ml synthetic tetradecapeptide substrate (Sigma, St. Louis, MO) for 4 h at 37°C, as previously described (24). The reaction was stopped by adding 10 µl of 50% H3PO4. One hundred microliters of each sample were filtered and injected into the HPLC system. The released ANG I peptide was quantified by reverse-phase HPLC using an aquapore ODS 300 column equilibrated with 0.1% phosphoric acid containing 5% acetonitrile (vol/vol). ANG I was separated by isocratic elution for 5 min, followed by a 20-min linear gradient of 5–35% acetonitrile in 0.1% phosphoric acid (vol/vol) at 1.5 ml/min. The chromatographic profile of each sample was compared with that obtained for standard samples containing angiotensinogen and ANG I at an absorbance of 214 nm. Peptide fragments were identified by elution position and quantified by integration area using repeated injections of standard peptide solution to correct for small differences in retention time (<6%) and peak height (<5%). The results were corrected by the amount of intracellular protein determined by the method of Lowry. All reagents used for renin analysis were purchased from Sigma.
Identification of RAS component mRNA expression by RT-PCR.
The expression of mRNAs of the RAS components in mesangial cells was determined by RT-PCR. Total RNA was purified from cells by the phenol and guanidine isothiocyanate-cesium chloride method using an appropriate kit (TRIzol, Life Tecnologies). The RNA pellet was resuspended in RNase-free water. Total RNA concentration was estimated with a spectrophotometer (Gene Quant RNA/DNA calculator, Amersham Pharmacia Biotech, Uppsala, Sweden). Two micrograms of total RNA were reverse transcribed into cDNA by the addition of a mix containing 0.5 mg/ml oligo dT, 10 mM DTT, 0.5 mM dNTPs (Pharmacia Biotech), and 200 U of RT enzyme (SuperScript RT, GIBCO BRL). The mixture was incubated at 37°C for 1 h and then at 95°C for 5 min. PCR was performed in a thermal cycler (model PTC-100, MJ Research, Watertown, MA) using 2 µl of reverse-transcribed cDNA in a total volume of 20 µl containing 1.0–2.5 mM MgCl (optimized for each primer pair), 0.5 mM of each primer, 0.5 mM dNTP mix, and 0.5 U Taq DNA polymerase (Pharmacia Biotech). Primer sequences for amplification of angiotensinogen, prorenin, ACE, and AT1 and AT2 receptors were designed using appropriate software (PCR-Designer, Research Genetics) based on each cDNA sequence obtained from GenBank. Primer sequences and the amplification conditions for each primer are shown in Table 1. Negative controls were included in each PCR set where cDNA was replaced with water as a control for contamination from exogenous sources. In addition, the RT enzyme was omitted in some samples as a negative control for amplification of genomic DNA. Positive controls included kidney cortex cDNA and the efficiency of the RT reaction was monitored by the amplification of the constitutively expressed gene for 
-actin. PCR products were electrophoresed on 1% agarose gels and visualized by ethidium bromide staining under UV light.
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ANG II measurement by ELISA. ANG II levels in the cell lysate were determined by ELISA using the avidin-streptavidin method as previously described (38). Five micrograms of protein samples were added to the wells of a microtiter plate in duplicate. Protein was allowed to adsorb for 1 h at 37°C and then blocked with 200 µl of PBS-Tween 20 containing 0.5% casein for 2 h at 37°C, and the anti-ANG II antibody (1:100 dilution) was added to the respective well and incubated for 1.5 h at 37°C. Plates were washed three times with PBS-Tween 20, and 150 µl of biotinylated anti-mouse IgG (1:500 dilution, Life Science) were added to each well and incubated for 1 h at 37°C. Samples were washed and then incubated with 100 µl of streptavidin-peroxidase (1:2,000 dilution, Amersham Life Science) for 45 min at 37°C. The final wash was followed by development using o-phenylenedimine dihydrochloride (Abbott Laboratories) and hydrogen peroxidase reagents for 20 min at room temperature. Absorbance was recorded at 495 nm, and ANG II concentrations were calculated from the standard curve.
Statistical analysis. Results are expressed as means ± SE. Data were analyzed by one-way ANOVA followed by the Tukey or Newman-Keuls test. Nonparametric data were analyzed by the Dunn or Kruskal-Wallis test. P < 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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We have shown that mesangial cells are able to synthesize, store, and spontaneously secrete both the active and inactive forms of renin, as previously demonstrated by us in rat and mouse mesangial cells (4) and by Chansel et al. (10) in human mesangial cells. Moreover, it was shown that the secretion rate of active and inactive renin changes in response to the external stimulus induced by high glucose concentration in the culture medium. In control and mannitol-stimulated cells, prorenin was predominant in the culture medium, indicating that under basal conditions most of the renin secreted is in the inactive form, as also described for juxtaglomerular cells and other renin-secreting cells (9, 15, 18). The reason mesangial cells secrete large quantities of inactive renin is not known, but the presence of renin and prorenin receptors in the kidney (29) and mesangial cells (28) suggests an exciting possibility of autocrine and paracrine functions for prorenin and renin secreted by mesangial cells.
High glucose induced a reduction in renin content in the culture medium, mainly in prorenin secretion, roughly three times that observed in control cells, resulting in the accumulation of prorenin in the intracellular compartment. It was recently demonstrated that the mesangial renin receptor binds renin and prorenin, and the binding of renin to this receptor, in human mesangial cells, induced a fourfold increase in the efficiency of angiotensinogen conversion to ANG I (29) compared with renin in the soluble phase. Moreover, the renin/prorenin binding to the receptor would not be detected in the culture medium but in the cell lysate fraction. Whether the increased prorenin content in the intracellular compartment observed in the present study was a result of reduced secretion and/or a binding to the receptor deserves further investigation. In addition, intracellular prorenin accumulation was also a result of increased prorenin gene transcription, observed after a short period of high-glucose stimulation. Actually, one of the most important events observed in the present study was an impressive rise in intracellular renin content and activity induced by high-glucose exposure, suggesting that the accumulated prorenin was converted to active renin inside the cell, and thus high glucose also induced an increase in the rate of prorenin-to-renin conversion. This result is in contrast to that recently described by Singh and co-workers (38). They found that renin activity, measured as ANG I generation in the presence of an excess of angiotensinogen, was not altered by high glucose. We have no explanation for this discrepancy, but maybe some differences between the protocols used to measure renin activity would be considered. For instance, the aforementioned study does not mention whether the protease inhibitors were added to the renin assay, as in the present study, and this is particularly important because renin appears to be rapidly inactivated in vitro and probably by proteases (13).
The conversion of prorenin to active renin results from the proteolytic cleavage of 43 amino acids from the pro-segment of prorenin. Although many enzymes have been implicated in this mechanism, cathepsin B has been suggested to be the primary one involved in the activation of prorenin in vivo (27). The presence of cathepsin B-like activity was recently demonstrated in rat mesangial cells, in parallel to its ability to hydrolyze prorenin (1), making this enzyme a potential candidate involved in the conversion of prorenin to active renin in mesangial cells. We found a significant increase in cathepsin B mRNA expression levels after 24 h of high-glucose exposure, possibly indicating that the increase in the conversion of prorenin to active renin induced by high glucose was, at least initially, mediated by cathepsin B. On the other hand, cathepsin B mRNA returned to control levels after the more prolonged period of 72 h of glucose stimulation. Actually, previous reports have shown that the diabetic milieu is mostly associated with a decrease in the degradative enzymes, including cathepsin B, in glomeruli and mesangial cells (22, 23, 39). Taken together, these data raise the possibility that glucose may interfere with cathepsin B expression through a time-dependent pathway, initially stimulating and then suppressing cathepsin B gene transcription.
Similar to cathepsin B mRNA expression, high glucose induced an initial increase in prorenin mRNA, detected after 24 h, with a return to control levels after 72 h of glucose exposure, suggesting that renin synthesis by mesangial cells is probably subjected to the usual negative feedback control induced by ANG II as classically observed in juxtaglomerular cells (26).
In contrast to time-dependent upregulation of prorenin and cathepsin B gene transcription, the expression levels of ACE mRNA were significantly elevated after both 24 and 72 h of high-glucose stimulation, suggesting that ACE is probably involved in the increased mesangial ANG II generation by glucose. However, whether ACE gene transcription is influenced by high glucose and/or by ANG I concentration deserves additional investigation.
High glucose induced an upregulation of angiotensinogen gene expression detected after 72 h. Singh et al. (38) recently demonstrated that mesangial cells incubated in high-glucose media increased both angiotensinogen mRNA and protein levels. Taken together, these data suggest that, in parallel to increased renin activity, high glucose also increased the availability of the renin substrate.
The results showed that mesangial cells in culture are able to constitutively express mRNA for both AT1 and AT2 receptors, as also previously demonstrated (6, 11, 20, 35). In contrast to intracellular components, high glucose did not change mRNA expression levels for AT1 or AT2 receptors. Conflicting results have been reported concerning the effect of glucose or diabetes on the expression of ANG II receptors in kidney cells. Downregulation of the AT1 receptor has been demonstrated in glomeruli and proximal tubules of diabetic rats (7, 30, 40), as well as in mesangial cells chronically incubated in culture medium containing high glucose levels (2). In contrast, Chouinard and co-workers (11) recently demonstrated that high glucose did not modify AT1 mRNA expression in mesangial cells but increased the expression of AT2 receptors. On the other hand, the absence of changes in ANG II receptor mRNA observed in the present study does not rule out the possibility of an upregulation of gene transcription during shorter periods of glucose stimulation. Finally, it is important to reflect on the possibility that ANG II may not leave the cells to induce its effects. Previous data from our laboratory (unpublished observations) showed ANG II immunostaining in the nucleus of mesangial cells, which was strongly increased after exposure to high glucose levels. ANG II accumulation inside the nucleus has been found in other tissues, including myocardium, brain, smooth muscle, and adrenal glands (8, 14, 32). The presence of ANG II inside the nucleus may represent an exciting mechanism involved in the well-known effects of ANG II-inducing gene expression, particularly those involved in cell growth, metabolism, and synthesis of extracellular matrix components, manifestations that are typical of diabetic nephropathy.
In summary, the present study showed a significant increase in ANG II generation in mesangial cells exposed to a high glucose concentration. The results suggest that the mechanisms involved in the upregulation of mesangial ANG II primarily include an increase in intracellular renin activity, which resulted from at least three mechanisms, i.e., a time-dependent stimulation of prorenin gene transcription, a reduction in prorenin secretion, and an increase in the rate of prorenin conversion to active renin, probably mediated by cathepsin B. The increased renin activity was paralleled by a rise in angiotensinogen gene expression, indicating availability of the substrate to renin. In contrast to time-dependent upregulation of prorenin and cathepsin B gene transcription, the expression levels of ACE mRNA were significantly elevated after both 24 and 72 h of high-glucose stimulation, indicating that ACE upregulation is probably necessary to increase ANG II synthesis. The mRNA levels for ANG II receptors were unchanged, at least after 72 h of exposure to glucose, suggesting that ANG II production by mesangial cells under glucose stimulation may serve as an intracrine action, thus directly mediating the proliferative and inflammatory effects of ANG II, with consequent cell proliferation, matrix expansion, and inflammation contributing to the glomerular sclerosis observed in diabetic nephropathy.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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expression in rat glomerular mesangial cells. J Clin Invest 93: 2431–2437, 1994.[Web of Science][Medline]
, in cultured rat mesangial cells. J Diabetes Complications 12: 328–336, 1998.[CrossRef][Web of Science][Medline]
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