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J Am Soc Nephrol 13:1595-1607, 2002
© 2002 American Society of Nephrology

Angiotensin II Increases Parathyroid Hormone-Related Protein (PTHrP) and the Type 1 PTH/PTHrP Receptor in the Kidney

Oscar Lorenzo*, Marta Ruiz-Ortega*, Pedro Esbrit{dagger}, Mönica Rupérez*, Arantxa Ortega{dagger}, Soledad Santos{dagger}, Julia Blanco{ddagger}, Luis Ortega{ddagger} and Jesus Egido*

*Laboratory of Vascular and Renal Research, and {dagger}Laboratory of Bone and Mineral Metabolism, Fundación Jiménez Díaz, Universidad Autónoma, Madrid, Spain; and {ddagger}Pathology Department, Hospital Clínico, Madrid, Spain.

Correspondence to Dr. Marta Ruiz-Ortega, Associate Investigator, Laboratory of Vascular and Renal Research, Avda.Reyes Católicos 2, Madrid 28040, Spain. Phone:34-91-5504800 ext. 3168; Fax: 34-91-5494764; E-mail:mruizo{at}fjd.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Angiotensin II (AngII) participates in the pathogenesis of kidney damage. Parathyroid hormone (PTH)–related protein (PTHrP), a vasodilator and mitogenic agent, is upregulated during renal injury. The aim of this study was to investigate the potential relation between AngII and PTHrP system in the kidney. Different methods were used to find that both rat mesangial and mouse tubuloepithelial cells express PTHrP and the type 1 PTH/PTHrP receptor (PTH1R). In these cells, AngII increased PTHrP mRNA and protein production. In contrast, PTH1R mRNA was increased in mesangial cells and downregulated in tubular cells, but its protein levels were unmodified in both cells. AT1 antagonist, but not AT2, abolished AngII effects on PTHrP/PTH1R. The in vivo effect of AngII was further investigated by systemic infusion (a low dose of 50 ng/kg per min) into normal rats. In controls, PTHrP immunostaining was mainly detected in renal tubules. In AngII-infused rats, PTHrP staining increased in renal tubules and appeared in the glomerulus and the renal vessels. After AngII infusion, PTHR1 staining was markedly increased in all these renal structures at day 3 but remained elevated only in tubules at day 7. The AT1 antagonist, but not the AT2, significantly diminished AngII-induced PTHrP and PTHR1 overexpression in the renal tissue, associated with a decrease in tubular damage and fibrosis. The results indicate that AngII regulates renal PTHrP/PTH1R system via AT1 receptors. These findings demonstrate that PTHrP upregulation occurs in association with the mechanisms of AngII-induced kidney injury.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Parathyroid hormone-related protein (PTHrP) is a widespread factor that appears to act in an autocrine/paracrine fashion to induce a variety of physiologic effects (1). The N-terminal region of PTHrP, interacting with the type 1 PTH/PTHrP receptor (PTH1R), displays hypotensive and inotropic cardiac effects, and has been proposed to act as a regulatory factor of systemic and renal hemodynamics (2,3). PTHrP is also mitogenic for both tubular and mesangial cells, and it is upregulated in the rat kidney after renal injury (4–10). A current hypothesis suggests that PTHrP might have a role(s) in the mechanisms associated with renal injury and/or repair (11).

Activation of the renal renin-angiotensin system (RAS) has been described during kidney injury (12). Angiotensin II (AngII), the main effector peptide of RAS, is a renal growth factor that plays an active role in the progression of kidney damage (12–15). This vasoactive peptide causes hyperplasia/hypertrophy of mesangial cells, tubular cells, and renal interstitial fibroblasts and regulates the expression of various factors involved in inflammation and renal fibrosis (12–17). Recent evidence suggests the interaction between RAS and PTHrP. AngII rapidly and transiently induces PTHrP mRNA in rat vascular smooth muscle cells (VSMC) (18). In addition, systemic hypertension induced by combined salt loading and AngII infusion in rats was associated with PTHrP overexpression in both the aorta and heart (19). In two rat models of tubular damage, an upregulation of both PTHrP and angiotensin-converting enzyme (ACE) genes was observed, suggesting that renal AngII generation could participate in the mechanisms leading to PTHrP overproduction (9,10). However, whether AngII might directly regulate the PTHrP/PTH1R system in the kidney has not yet been examined.

In this study, we found that both rat mesangial and mouse tubuloepithelial cells MCT express PTHrP and PTH1R. Our findings demonstrate that AngII, via AT1 receptors, increases PTHrP in these cells in vitro, and in glomeruli, different nephron segments, and renal vasculature in the rat kidney in vivo, related to changes in the PTH1R. These data indicate that AngII-induced renal damage is associated with PTHrP upregulation throughout the renal parenchyma.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Design
Systemic infusion of AngII (solved in saline) in female Wistar rats was performed subcutaneously by osmotic minipumps (Alza, Palo Alto, CA) at 50 ng/kg per min, as described (20). Animals were sacrificed at days 3 and 7 (n = 8 rats in each group). Tissue samples were immediately removed and further processed for histologic studies. To determine the AngII receptor subtype involved, some animals (n = 6 rats) were treated with the AT1 antagonist, losartan (kindly provided by Merck Sharp and Dome, Madrid, Spain), 10 mg/Kg per d in the drinking water, or the AT2 antagonist, PD123319 (Sigma, St. Louis, MO), 30 mg/kg per min subcutaneously by osmotic minipumps (n = 4 rats), starting 24 h before AngII infusion. These doses have previously demonstrated to cause an effective blockade of each receptor (20). Control groups were also studied: saline-infused, untreated, or treated with AT antagonist. Systolic arterial BP was measured in conscious, restrained rats by the tail-cuff sphygmomanometer (NARCO Biosystems, Houston, TX). The BP value for each rat was calculated as the mean of three separate measurements at each session.

Cell Cultures
Mesangial cells were cultured from isolated rat glomeruli by sequential sieving and differential centrifugation, as described previously (21). Cells were grown in RPMI 1640 medium, pH 7.4, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/L glutamine, and 10% fetal bovine serum (FBS) at 37°C in 5% CO2. These cells were characterized by phase-contrast microscopy, positive staining for desmin and vimentin, and negative staining for keratin and factor VIII antigen, excluding epithelial and endothelial contamination, respectively (21). Rat aortic VSMC were cultured in Dulbecco’s modified Eagle’s medium with 10% FBS, as reported (22). These cells were characterized by phase-contrast microscopy and immunofluorescence staining for smooth muscle {alpha}-actin and express AT1 and AT2 receptors (22). The murine proximal tubuloepithelial cells MCT, kindly supplied by Dr. E. Neilson (University of Pennsylvania, Philadelphia, PA), were grown in RPMI 1640 with 10% FBS and antibiotics. These cells display a proliferative response to PTHrP (23) and have AT1 and AT2 receptors (20). After confluence, cells were FBS-starved for 48 h before the experiments.

Reverse Transcription-PCR (RT-PCR)
Cell total RNA was isolated using a standard method with Trizol reagent (Life Technologies-BRL, Grand Island, NY). RT-PCR (Access RT-PCR System; Promega, Madison, WI) was carried out with total RNA (30 to 100 ng) and specific primers: 5'-TGCAGCGGAGACTGGTTCAG-3' (sense) and 5'-CCTCGTCGTCTGACCCAAA-3' (antisense), yielding a 266-bp product, corresponding to nucleotides 35 to 301 in rat PTHrP cDNA (9,10); and 5'-CATTGTGGCAGATCCAGATGC-3' (sense), and 5'-AGTCTAGCCCCCTAGTGCC-3' (antisense), yielding a 495-bp product corresponding to nucleotides 1376–1870 in a region of the cDNA encoding the C-terminal intracellular tail of rat PTH1R (24). [{alpha}32P]dCTP (0.5 µCi, 3000 Ci/mmol; New England Nuclear, Zaventem, Belgium) was added to the reaction mixture (10 µl), which was incubated for 45 min at 48°C and 2 min at 95°C, followed by 30 cycles of 1 min at 94°C, 1 min at 60°C, and 2 min at 68°C, with a final extension of 7 min at 68°C. Preliminary experiments established that these conditions provided submaximal cDNA amplifications. Control experiments were done with RNA samples but without avian myeloblastosis virus reverse transcriptase. The PCR products were analyzed on 6% polyacrylamide gels, which were dried, and then exposed to X-OMAT AS films (Eastman Kodak, Rochester, NY). Values obtained after densitometric scanning (ImageQuant; Molecular Dynamics, Sunnyvale, CA) of the different PCR products were normalized against those of the corresponding glyceraldehyde 3-phosphate dehydrogenase (G3PDH) product (a constitutive control) (9,10).

RNase Protection Assay
RNase protection analyses were performed as described previously (10), using 20 to 40 µg of total RNA and the following RNA probes: an anti-sense PTHrP probe that corresponds to a 343-bp fragment of the rat PTHrP gene coding region cloned into the pBluescript plasmid; a 481-bp anti-sense RNA probe corresponding to nucleotides 151 to 631 in the coding region of the rat PTH1R gene cloned into the PCR-II plasmid (Invitrogen, Groningen, The Netherlands); two 349- and 283-bp antisense cRNA probes for mouse PTHrP and the PTH1R, respectively. The plasmids for preparing these probes were kindly provided by Dr. A. F. Stewart (University of Pittsburgh, Pittsburgh, PA). As an internal standard, an anti-sense cRNA probe corresponding to a 115-bp fragment of the human 28S rRNA gene (Ambion, Austin, TX) was used. The probes were labeled with [{alpha}32P]UTP, using the Riboprobe-in vitro-Transcription-System (Promega), and were then hybridized to cell total RNA in 80% formamide, 40 mmol/L piperazine-N,N'-bis(2-ethanosulfonic acid), pH 6.7, and 400 mmol/L NaCl at 55°C overnight. Samples were sequentially treated with RNase A (40 µg/ml), RNase T1 (2 µg/ml), and proteinase K (60 µg/ml). RNA hybrids were then phenol/chloroform extracted before ethanol precipitation. Protected fragments were fractionated on QuickPoint Pre-Cast gels (Novex, San Diego, CA) and analyzed by autoradiography.

Northern Blot Analyses
Northern blot analyses were carried out as described (21). Briefly, 40 µg of cell total RNA was denatured and electrophoresed on 1% agarose-formaldehyde gels and then transferred to nylon membranes (Genescreen, New England Nuclear). Membranes were hybridized with the [{alpha}32P]-labeled complementary RNA probes described above before being exposed to autoradiographic films. G3PDH was used as control of RNA loading.

Western Blot Analyses
Mesangial cells and VSMC were homogenized in lysis buffer (50 mmol/L TrisHCl, 150 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 0.2% Triton X-100, 0.3% NP-40, 0.1 mmol/L PMSF, and 1 µg/ml pepstatin A), and proteins were separated on 12.5% polyacrylamide-SDS gels under reducing conditions. After electrophoresis, samples were transferred to PVDF membranes (Millipore, Bedford, MA). The membranes were then blocked in 10 mmol/L Tris pH 7.5, 0.4 mol/L NaCl, containing 0.1% Tween-20, 1% bovine serum albumin (BSA), and 5% dry skimmed milk for 30 min at 37°C before being incubated overnight at 4°C in the same buffer with 2500-fold dilution of rabbit polyclonal anti-PTHrP antiserum C6 recognizing a C-terminal epitope in the intact PTHrP molecule (25) or 5 µg/ml of affinity-purified anti-PTH1R antibody Ab-VII (Babco, Richmond, CA) recognizing the N-terminal extracellular domain of the human PTH1R. {alpha}-Tubuline (1:5000 dilution) was also detected with a specific monoclonal antibody (Sigma) as a constitutive control. Detection was performed by incubation with a peroxidase-conjugated secondary antibody (The Binding Site, Birmingham, UK) and developed using enhanced chemiluminescence (ECL, Amersham, Buckinghamshire, UK).

Histologic Studies
The kidney samples were studied by hematoxylin/eosin and Masson staining and examined by light microscopy. The localization of PTHrP and PTH1R was performed by immunohistochemistry and immunocytochemistry using specific antibodies: a rabbit polyclonal anti-PTHrP antiserum C6 (100-fold dilution) or affinity-purified anti-PTH1R antibody Ab-VII (5 µg/ml), which detects the PTH1R in rat kidney cortex (9,10). For in vivo studies, paraffin-embedded renal tissue sections (4 µm) were mounted on poly-L-lysine-coated slides. The slides were deparaffinized with xylene and then rehydrated with graded ethanol concentrations. For in vitro studies, mesangial cells and VSMC grown on chamber slides (Nalge Nunc, Naperville, IL) were fixed in methanol/acetone at -20°C. Immunohistochemistry was then carried out as follows. Endogenous peroxidase was blocked by incubating in 3% H2O2/methanol (1:1) for 30 min. The slides were subsequently incubated with 1.5% swine serum and 1% BSA in phosphate-buffered saline (PBS) for 1 h at 37°C to reduce nonspecific staining and overnight at 4°C with either antiserum C6 or antibody Ab-VII in PBS containing 1% BSA. The slides were subsequently incubated with swine-biotinylated anti-rabbit IgG in PBS with 1% BSA for 30 min and the avidin-biotin-peroxidase complex (Vectastain Elite; Vector, Burlingame, CA) for 30 min. Positive staining was developed with 3,3'-diaminobenzidine (Dako, Bucks, UK). The tissue sections and mesangial cells and VSMC were counterstained with hematoxylin. Negative controls without the primary antibody were included in each experiment.

Morphology and immunostaining were scored by semiquantitative determination as previously reported (20) and graded as follows: 0, no staining; 1+, mild staining; 2+, moderate staining; 3+, marked staining. Identification of different cell types was based on topographical criteria commonly used by our group (20,21). The mean number of positive cells per glomerular cross-section was determined by evaluating 10 to 15 glomeruli. The whole interstitium was examined from each animal, separately evaluating proximal, distal, and collecting ducts. For renal vessel evaluation, only arteries were scored (including arterioles, interlobular, and larger arteries altogether, but not veins). The immunohistochemistry experiments were performed from three to five kidney sections from each experimental animal that were stained and analyzed semiquantitatively to obtain a mean score per animal. In all cases, evaluations were performed by two independent observers in a blinded fashion, and the mean score value was then calculated for each rat. Tubular damage was defined as flattening of epithelium, lumen increase, vacuolization, desquamations, necrosis, and loss of brush border in proximal tubuli; glomerular sclerosis was defined as ischemic glomeruli and increase in mesangial matrix, as described previously (20). For aorta, the quantification was done by computer analyses, as described (26). The arterial cross-sections stained with the antibodies were digitized via an Olympus microscope (BH-2; Olympus, Tokyo, Japan) connected to a CCD video camera; value ranging from 0 to 255 was assigned to each pixel, and automatic analysis was performed. Results are expressed as immunostained area.

Statistical Analyses
Results are expressed as mean ± SEM. Statistical significance was assessed using the t test or ANOVA when appropriate. Differences were considered significant when P < 0.05.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rat Mesangial Cells and MCT Cells Express PTHrP and the PTH1R
In this study, we evaluated the presence of PTHrP and the PTH1R in serum-deprived rat mesangial cells by using different mRNA and protein assays. Rat VSMC were used as positive control cells (18,27). RT-PCR showed similar PTHrP and PTH1R transcripts in both mesangial cells and VSMC (Figure 1A). The presence of PTHrP mRNA in mesangial cells was confirmed by Northern blot analyses, as shown by a band of 1.2-kb (Figure 1B) as described previously in VSMC (27). Using this method, no PTH1R signal was found in mesangial cells (not shown). However, the presence of PTH1R transcript was confirmed in these cells by RNase protection assay (Figure 1C). Presence of the PTHrP/PTH1R system system was also assessed in MCT cells. We found that MCT cells express PTHrP mRNA, by using either RT-PCR or Northern blot (Figure 1, A and B), and the PTH1R mRNA, by RT-PCR and RNAse protection assay (Figure 1, A and C).



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  		Figure 1. Rat glomerular mesangial cells and mouse tubuloepithelial cells (MCT) express parathyroid hormone (PTH)–related protein (PTHrP) and the PTH/PTHrP receptor (PTH1R) mRNA. Total RNA was isolated from serum-starved mesangial cells (MC), MCT, and rat vascular smooth muscle cells (VSMC) (positive control cells). (A) Using reverse transcriptase–PCR (RT-PCR), renal cells were found to express PTHrP and the PTH1R transcripts similar to those found in VSMC. Similar results were obtained by using Northern blot analysis to evaluate PTHrP mRNA (B) or RNAse protection assay for assessing PTH1R mRNA (C), as described in Materials and Methods. As a constitutive control, 28S mRNA is also shown (C). The size of the corresponding products is indicated in each case. The figure represents the results from 2 to 4 independent experiments.

 
We further evaluated the presence of PTHrP and the PTH1R in mesangial cells by Western blot analyses. Using the anti-PTHrP antiserum C6, we found a band of approximately 18 kD, consistent with the presence of the entire PTHrP molecule (28) (Figure 2A), and a band of approximately 90 kD with the anti-PTH1R antibody VII in these cells (Figure 2A). Similar results were found in VSMC extracts (Figure 2A). Moreover, diffuse PTHrP staining was localized by immunocytochemistry in the cytosol, whereas PTH1R staining showed distinct clusters in both mesangial cells and VSMC (Figure 2B). A similar pattern for the latter stemming was observed in rat Walker 256 tumor cells (24). Negative controls (incubation without primary antibody) are also included in Figure 2B. Taken together, these data clearly demonstrate that mesangial cells express PTHrP and the PTH1R.



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  		Figure 2. PTHrP and the PTH1R are detected by Western blot analyses (A) and immunocytochemistry (B) in rat mesangial cells (MC). (A) Protein extracts from MC were analyzed by Western blot with either antiserum C6 (PTHrP) or Ab-VII antibody (PTH1R). Only single bands corresponding to an apparent molecular weight of approximately 18 kD or approximately 90 kD, respectively, were detected. (B) Immunostaining of MC with the antiserum C6 shows a diffuse cytoplasmic pattern, whereas distinct clusters of immunostaining were observed with the Ab-VII antibody. As positive controls, rat VSMC were used in each case. A control tissue sample incubated without primary antibody is also shown. Magnification, x100.

 
AngII Induces PTHrP Overexpression via AT1 in Rat Mesangial Cells
We next investigated whether AngII stimulated PTHrP expression in growth-arrested rat mesangial cells in vitro. By RT-PCR and Northern blot analyses, we observed that AngII increased PTHrP mRNA within 1 to 3 h, normalizing at 6 h, in these cells (Figure 3, A and B). A similar time-dependent PTHrP mRNA response to AngII was found in VSMC (Figure 3C), which is consistent with previous findings (18). In mesangial cells, we observed that the stimulatory effect of AngII was dose-dependent, so that the response with 10-7 mol/L of this peptide was about fourfold over control at 3 h (by RT-PCR: P < 0.05; n = 3, Figure 3B). Moreover, AngII increased PTHrP protein production in these cells as early as 6 h, representing about threefold over control, and remained elevated for at least 48 h, as shown by Western blot analyses (Figure 4A).



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  		Figure 3. Angiotensin II (AngII) upregulates PTHrP mRNA expression in rat MC. Quiescent cells were treated with 10-7 mol/L AngII for various time periods. At 3 h, some cell cultures were pretreated with either the AT1 antagonist, losartan (10-6 mol/L), or the AT2 antagonist, PD123319 (10-5 mol/L), before stimulation with AngII at different concentrations. Northern blot analyses (A) and RT-PCR analyses (B and C) shows that AngII upregulates PTHrP mRNA expression in rat mesangial cells via AT1 receptors (A and B) in a similar manner as in VSMC (positive control cells) (C). In each case, the upper panels show representative autoradiograms and the lower panels display the corresponding densitometric values as mean ± SEM from four different experiments. G3PDH was used as an internal control. *P < 0.05 versus control;#P < 0.05 versus AngII.

 


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  		Figure 4. AngII increases PTHrP protein content in rat MC. (A) Quiescent cells were treated with 10-7 mol/L AngII for variable time periods. (B) Cells were pretreated with losartan or PD123319, as described in the legend to Figure 3, before AngII stimulation for 24 h. PTHrP was analyzed by Western blot, using antiserum C6 (upper panel). {alpha}-Tubuline was used as an internal loading control (middle panel). In the lower panel, bar graphs of mean ± SEM of five independent experiments are shown. *P < 0.05 versus control; # P < 0.05 versus AngII.

 
AngII elicits its cellular responses through binding to two specific receptors, AT1 and AT2 (29), and thus we examined the receptor subtype involved in these processes. Pretreatment of mesangial cells with the AT1 antagonist losartan (10-6 mol/L) for 1 h caused a significant diminution in AngII-induced PTHrP at both mRNA (Figure 3, A and B) and protein levels (Figure 4B). Meanwhile, the AT2 antagonist, PD123319 (10-5 mol/L) had no effect. In VSMC, AngII increased PTHrP mRNA (Figure 3C) and its protein levels (not shown) also via AT1.

We assessed whether AngII might also modulate the PTH1R expression in rat mesangial cells. Stimulation with AngII at 10-7 mol/L from 1 h to 18 h increased PTH1R mRNA slightly but not significantly, as shown by RT-PCR and RNAse protection assay (Figure 5, A and B). In contrast, AngII did not modify PTH1R protein levels, at least until 48 h of incubation (Figure 5C).



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  		Figure 5. AngII increases the PTH1R mRNA and protein expression in rat mesangial cells. Quiescent cells were stimulated with 10-7 mol/L AngII for variable time periods. Total RNA was isolated, and the PTH1R mRNA levels were then determined by RT-PCR and RNAse protection assay. (A) A representative autoradiogram of RT-PCR is shown in the upper panels, and bar graphs corresponding to mean ± SEM of five independent experiments are shown in the lower panels. G3PDH was used as internal control. Panel B shows a representative RNAse protection assay of AngII-treated cells analyzed as a pool of five independent experiments. 28S was used as internal control. (C) The PTH1R protein levels in cell protein extracts isolated after AngII treatment were analyzed by Western blot with the Ab-VII antibody. {alpha}-Tubuline was used as an internal loading control (middle panel). In the lower panel, bar graphs of mean ± SEM of three independent experiments are shown.

 
Effect of AngII on PTHrP and the PTH1R in MCT Cells
AngII stimulated PTHrP mRNA in MCT cells analyzed by RNAse protection assay (Figure 6A); a maximal response occurred within 1 to 3 h. Preincubation with AT1 or AT2 antagonists showed that PTHrP mRNA regulation occurred via AT1, because only Losartan abolished it (Figure 6A). AngII, also through AT1, downregulated PTH1R mRNA levels at 1 to 6 h, reaching those of controls at 24 h, as shown by RNase protection assay (Figure 6B) and RT-PCT (Figure 6C). PTH1R protein levels were not modified by AngII stimulation at the times studied (up to 48 h), as shown by Western blot analyses (Figure 6D).



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  		Figure 6. Effect of AngII on PTHrP and PTH1R levels in MCT cells. Quiescent cells were treated with 10-7 mol/L AngII for variable time periods, and total RNA was isolated. (A) PTHrP mRNA analyzed by RNAse protection assay. *P < 0.05 versus control; # P < 0.05 versus AngII. The PTH1R mRNA levels were determined byRNAse protection assay (B) and RT-PCR (C). Representative autoradiograms are shown in the upper panels, and bar graphs corresponding to mean ± SEM of three independent experiments are shown in the lower panels. 28S or G3PDH were used as internal controls. (D) The PTH1R protein levels were determined at 6, 24, and 48 h after AngII treatment by Western blot with Ab-VII antibody (upper panel). {alpha}-Tubuline was used as an internal loading control (middle panel). In the lower panel, bar graphs of mean of three independent experiments are shown.

 
AngII Infusion into Normal Rats Induces Renal PTHrP Upregulation through AT1 Receptor Activation
Systemic infusion of AngII for 3 d into normal rats caused a slight elevation in BP within the normotensive range (121 ± 9 mmHg compared with 100 ± 2 in controls; P < 0.05; n = 10). At this time period, the majority of the glomeruli showed a normal appearance, but there were mild tubular lesions. At day 7 of AngII infusion, tubular atrophy and fibrosis appeared in tubulointerstitial areas (Figure 7), which is consistent with previous findings (20,31). We evaluated PTHrP expression in the kidney of these animals using immunohistochemistry. In controls, PTHrP immunostaining was predominantly found in the renal tubules (Figure 8, A and B). After infusion of AngII for 3 d, PTHrP staining markedly increased in proximal, distal, and collecting tubuli and appeared in glomerular epithelial and endocapillary cells (representing endothelial and mesangial cells and occasionally macrophages in the tuft) (Figure 8A). PTHrP positivity also increased in VSMC of renal arteries in these AngII-infused animals (Figure 8A). At day 7 after AngII infusion, the increase in PTHrP staining remained in the renal tubuli, but was somewhat lower in the glomeruli, and decreased to normal values in the renal vessels (Figure 8B). The semiquantitative score, determined as described in Materials and Methods, is shown in Figure 8C. Moreover, PTHrP overstaining in the renal tissue was significantly correlated with tubular injury (r = 0.9; P < 0.05; n = 8).



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  		Figure 7. Morphologic lesions in the kidney of AngII-infused rats. Semiquantitative score of tubular injury was evaluated as described in Materials and Methods. Rats were infused with AngII (50 ng/kg per min) for 3 to 7 d (n = 8 in each group). Some animals were also treated with either losartan (10 mg/kg per d) (n = 6) or PD123319 (30 mg/kg per d) (n = 4) starting 24 h before AngII infusion. Results are mean ± SEM.*P < 0.05 versus control; # P < 0.05 versus AngII infusion.

 


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  		Figure 8. AngII infusion induces PTHrP upregulation in the rat kidney via AT1 receptors. Rats were infused with AngII, pretreated or not with either losartan or PD123319, as described in the legend to Figure 7. PTHrP immunostaining was performed with the antiserum C6. (A) In control rats, PTHrP staining was almost undetectable in the glomerulus and renal arteries and was weak in the renal tubules. In AngII-infused rats at day 3, PTHrP immunostaining was clearly shown in the glomerulus and localized to the podocytes (arrows) and to endocapillary cells in the mesangial area (arrowhead). PTHrP positivity also dramatically increased in the renal arteries (mainly VSMC). In addition, there was a marked increase in the cytosolic staining in the renal tubules after AngII infusion. (B) PTHrP staining remained increased in both the glomerulus and tubules at day 7 after AngII infusion. Treatment with losartan, but not with PD123319, abolished the AngII-induced increase in PTHrP staining throughout the renal parenchyma. Figure shows the negative control of the immunostaining, as described in Materials and Methods. (C) Semiquantitative score of PTHrP immunostaining in renal tissue of AngII-infused rats. Results are means ± SEM of 4 to 8 animals per group. *P < 0.05 versus control; # P < 0.05 versus AngII infusion. Magnifications: x400 in A; x200 in B.

 
Administration of losartan at 10 mg/kg per d, a dose that normalized the slight increase in BP (99 ± 2 mmHg; P < 0.05 versus AngII alone at day 3; n = 6) and the increased tubular damage in AngII-infused rats (Figure 7) (20), abrogated the increase in PTHrP immunostaining in these animals (Figure 8B). In contrast, PD123319 at 30 mg/kg per min, which was shown to decrease the inflammatory cell infiltration in the kidney of these AngII-infused rats (20), did not affect BP (123 ± 16 mmHg; P < 0.05 versus controls; n = 4), fibrosis and tubular damage (Figure 7), or the maximal increase in renal PTHrP immunostaining in these animals (Figure 8).

AngII Infusion Increases the PTH1R in the Rat Kidney by AT1 Receptor Activation
Systemic infusion of AngII into normal rats for 3 d significantly increased PTH1R immunostaining in the cortical tubules in both basolateral and apical membranes, in the glomerulus in epithelial and endocapillary cells, and in the renal arteries, mainly in VSMC (Figure 9). In renal tubules, PTH1R staining remained elevated, at least up to day 7 after AngII infusion (Figure 9). Losartan, but not PD123319, treatment prevented the increase in PTH1R staining in renal tissue (Figure 9).



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  		Figure 9. AngII infusion increases the PTH1Rin the rat kidney via AT1 receptors. Rats were infused with AngII, pretreated or not with either losartan or PD123319, as described in the legend to Figure 7. PTH1R staining was performed with the anti-PTH1R antibody Ab-VII. (A) In control rats, there was a weak PTH1R immunostaining in all renal structures. In AngII-infused rats at day 3, PTH1R staining increased in the glomerulus and renal tubules and in the renal arteries. In these animals, glomerular podocytes (arrows) and endocapillary cells in the mesangial area (arrowhead) show intense positivity. In addition, in the renal tubules, PTH1R staining was increased in both apical (A) and basolateral (B) membranes. In the renal arteries, increased PTH1R was present mainly in VSMC. (B) PTHrP staining remained increased only in the renal tubules at day 7 after AngII infusion. Treatment with losartan, but not with PD123319, abolished the AngII-induced increase in renal PTH1R immunostaining. The figure shows the negative control of the immunostaining, as described in Materials and Methods. (C) Semiquantitative score of PTH1R immunostaining in the kidney of AngII-infused rats. Results are means ± SEM of 4 to 8 animals per group. *P < 0.05 versus control; # P < 0.05 versus AngII infusion. Magnifications: x400 in A; x200 in B.

 
AngII Infusion Increases PTHrP and the PTH1R in the Rat Aorta
AngII infusion for 3 and 7 d was associated with an increased staining for both PTHrP and the PTH1R in the rat aorta, mainly in VSMC, which was decreased by losartan (Figure 10).



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  		Figure 10. AngII infusion increases PTHrP and the PTH1R in the rat aorta via AT1 receptors. At day 7 after AngII infusion, a marked increase in PTHrP and PTH1R immunostaining—localized mainly in VSMC—was observed in the rat aorta, which decreased by losartan pretreatment. (B) PTHrP and the PTH1R staining in the aorta of AngII-infused rats determined as described in Materials and Methods. Results are mean ± SEM of 4 to 8 animals per group. *P < 0.05 versus control; # P < 0.05 versus AngII infusion. Magnifications: x100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We and others have previously found that renal proximal tubule cells in vitro produce and secrete PTHrP (5,7). In addition, we recently reported that mesangial cells in rats with protein overload-induced tubulointerstitial damage show PTHrP immunostaining (9). In this study, using several mRNA and protein analyses procedures, we have shown that cultured rat mesangial cells and mouse tubuloepithelial MCT cells express PTHrP and the PTH1R. The latter provides a rationale for the previously reported mitogenic effects of PTHrP in both cell types (4,23). Previous reports also indicate that rat and human mesangial cells proliferate in response to PTHrP (4,8). However, one of these studies and another previous failed to detect the PTH1R in these cells in both rat and humans by either Northern blot analysis, in situ hybridization, or RT-PCR with primers amplifying a region of this receptor cDNA encoding the N-terminal extracellular domain (8,31). Our results herein clearly demonstrate that PTH1R is present in mesangial cells from normal rats. Previous failure to find this receptor in these cells might be due to use of different methods to detect PTH1R mRNA. In this regard in the present study, we only found the PTH1R signal in rat mesangial cells by using RT-PCR or RNAse protection assay but not by Northern blot analysis.

The role of PTHrP in renal pathophysiology is poorly understood. Previous in vitro and in vivo data indicate that PTHrP may act as a growth factor for renal cells (4,5,7,8,10,23). High levels of PTHrP mRNA were observed in developing tubules and glomerulus during kidney maturation and in response to renal damage (4,6,9,10,32). Interestingly, an increase in renal PTHrP expression, associated with increased ACE mRNA and/or AngII immunostaining, was found to occur in rats with tubular injury induced by either protein overload or folic acid injection (9,10). Consistent with these early findings, we presently found that systemic infusion of AngII into normal rats dramatically increased renal PTHrP immunostaining. Thus, although in the normal rat kidney, PTHrP positivity was present mainly in the renal tubules, which is consistent with previous observations (2,4,6,9,10), PTHrP staining was found to appear in the glomerulus and to increase in the renal tubules as early as at day 3 in AngII-infused rats. Moreover, this staining remained elevated at day 7, when both renal tubular damage and fibrosis were evident. In this regard, PTHrP overexpression in both renal tubules and glomerulus has also shown to occur in several rat models of tubulointerstitial injury (4,9,10). Moreover, in rats with protein overload-induced tubular damage, elevated ACE was associated with PTHrP reexpression in the glomerulus, in both mesangial and epithelial cells (9), as found to occur in response to AngII infusion. Collectively, these findings support that renal PTHrP upregulation appears to be related to the mechanisms associated with AngII-induced kidney injury.

In this study, we also found that AngII infusion increased the PTH1R immunostaining throughout the renal parenchyma. This is in contrast to previous findings in other models of kidney injury in which downregulation of the PTH1R, as shown by a dramatic decreased immunostaining with the same antibody as that used herein, was associated with PTHrP overexpression in the rat kidney (9,10). Moreover, a decreased density of the PTH1R has recently been found to occur in renal cortical membranes from spontaneously hypertensive rats (SHR) whose renal arteries have elevated PTHrP content (33–35). In the present study, PTH1R mRNA was slightly increased in rat primary mesangial cells, whereas it is downregulated in MCT cells, after AngII treatment. In contrast, PTH1R protein levels remained were unaltered for 48 h after AngII stimulation in both cell types. Of interest, in vitro data in various cell types, such as VSMC and the renal epithelial cell line OK, have shown that both homologous and heterologous downregulation of the PTH1R mainly involve complex posttranscriptional mechanisms, such as its intracellular processing and/or coupling (27,36). Whether such a scenario might be related to the effects of AngII on the PTH1R in vivo is presently unknown. In any event, the present findings strongly suggest that AngII-induced renal injury occurs associated to upregulation of PTH1R.

Previous in vitro data from other investigators and the present results demonstrate that AngII stimulates PTHrP in VSMC from normotensive rat aorta (18,37–39). Moreover, systemic hypertension induced in Sprague-Dawley rats by combined infusion of a high dose of AngII and a high salt diet for 12 d increased PTHrP gene expression in the aorta (19). In addition, PTHrP mRNA levels are elevated in various vascular beds, including renal vessels of adult SHR compared with those in age-matched Wistar-Kyoto controls (33,38,39). Mechanical stretch, which would be increased by systemic hypertension, is a potent stimulus for AngII-induced PTHrP expression in VSMC from rat aorta (37,39); therefore, the putative role of AngII on PTHrP overexpression in the aforementioned rat models was difficult to assess. The present results indicate that, in the absence of high BP, AngII increases PTHrP and PTH1R staining both in the aorta and renal arteries and strongly support that AngII can directly affect the PTHrP/PTH1R system in vivo.

The AngII receptor subtype involved in renal injury is not completely defined. In experimental models of renal injury, AT1 antagonists decrease proteinuria, cell proliferation, matrix accumulation, and production of several growth factors (14). However, AT2 receptors seem to play an important role in the inflammatory process (15,20,40). Our present in vitro and in vivo data indicate that the AngII effects on the renal PTHrP/PTH1R system are likely mediated by AT1. In AngII-induced renal injury, we have observed that the AT1 antagonist losartan, but not the AT2 antagonist PD123319, diminished tubular damage and fibrosis, along with renal PTHrP/PTH1R overexpression; a correlation was found between these beneficial effects of AT1 blockade and changes in the PTHrP/PTH1R system.

We recently demonstrated that losartan decreased renal activation of the transcription factors AP-1 and nuclear factor-{kappa}B (NF-{kappa}B) induced by AngII infusion into rats in both the glomerulus and tubules (20). Although a putative interaction between AngII and the renal PTHrP/PTH1R system is unlikely to occur through NF-{kappa}B activation, such an interaction might involve the AP-1 complex. In renal cells, AngII upregulates several genes controlled by AP-1, including c-fos, transforming growth factor–{beta}, and matrix proteins (14). This transcription factor regulates PTHrP gene expression in a variety of cells (1); AngII might therefore stimulate renal PTHrP expression through the AP-1 pathway. In addition, PTHrP itself induces c-fos and activates AP-1 signaling in various cell types by interacting with the PTH1R (41,42). Moreover, protein kinase A and cAMP response element-binding protein (CREB) as well as protein kinase C activation appear to mediate at least some of the effects of PTHrP on cell proliferation and/or cell differentiation in various cell types, including renal cells (1,2,5,23,24,41). A recent study has also shown that PTHrP increases mitogen-activated protein kinase (MAPK) and Ras activity associated with its osteogenic features (43). Interestingly, AngII also activates CREB and MAPK via AT1, which are related to its effects on renal cell proliferation and fibrosis (14,20). Collectively, these data suggest that activation of the PTHrP/PTH1R system could recapitulate some of the AngII-induced effects via AT1 in the kidney, such as those on cell proliferation and fibrosis, by mechanisms involving different intracellular mediators.

PTHrP is also now emerging as a key regulatory factor of VSMC proliferation in vascular beds. Thus, exogenous PTHrP is anti-mitogenic in these cells, whereas endogenous PTHrP is a potent proliferative factor by a mechanism involving its nuclear translocation (33,44). This situation seems to be reversed in the SHR model (33). Interestingly, the PTH1R can be internalized into the nucleus of renal cells (45). The putative contribution of these PTHrP/PTH1R features to the renal effects of this protein are presently unknown.

Complete understanding of the true PTHrP action(s) in the kidney in normal and pathologic conditions requires further study. However, our present findings provide novel insights that support the notion that the PTHrP/PTH1R system has an important and complex role in the mechanisms associated with AngII-induced renal injury.


   Acknowledgments
 
These studies have been supported by grants from the Fondo de Investigación Sanitaria (FIS 99/0425 and 01/3130), Comunidad Autónoma de Madrid (CAM 08.4/0017/2000 and 08.6/0038/2000), Ministerio de Ciencia y Tecnología (MCYT; PB98–0700-C02–02), Sociedad Española de Nefrologia, and Fundación Renal Iñigo Alvarez de Toledo. We thank Dr. A. F. Stewart (University of Pittsburgh, Pittsburgh, PA) for the critical reading of the manuscript. MR and AO are fellows of the Fundación Conchita Rábago. OL is a fellow of FIS. SS is a fellow of MCYT. Color reproduction of figures was supported by a grant from FISS.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication August 21, 2001. Accepted for publication February 23, 2002.




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