Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney

Matthias Simon1, John G. Maresh4, Stephen E. Harris2, James D. Hernandez1, Mazen Arar3, Merle S. Olson4, and Hanna E. Abboud1

1 Division of Nephrology and 2 Division of Endocrinology, Department of Medicine and 3 Division of Pediatric Nephrology and 4 Department of Biochemistry, University of Texas Health Science Center, and Audie L. Murphy Memorial Veterans Affairs Hospital San Antonio, Texas 78284


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BMP-7, a member of the bone morphogenic protein subfamily (BMPs) of the transforming growth factor-beta superfamily of secreted growth factors, is abundantly expressed in the fetal kidney. The precise role of this protein in renal physiology or pathology is unknown. A cDNA that encodes rat BMP-7 was cloned and used as a probe to localize BMP-7 mRNA expression by in situ hybridization in the adult rat kidney. The highest expression of BMP-7 mRNA could be seen in tubules of the outer medulla. In glomeruli, a few cells, mainly located at the periphery of the glomerular tuft, showed specific and strong signals. Also, high BMP-7 mRNA expression could be localized to the adventitia of renal arteries, as well as to the epithelial cell layer of the renal pelvis and the ureter. Preliminary evidence suggests that BMP-7 enhances recovery when infused into rats with ischemia-induced acute renal failure. We examined BMP-7 mRNA expression in kidneys with acute renal failure induced by unilateral renal artery clamping. BMP-7 mRNA abundance as analyzed by solution hybridization was reduced in ischemic kidneys after 6 and 16 h of reperfusion compared with the contralateral kidney. In situ hybridization in ischemic kidneys showed a marked decrease of BMP-7 mRNA in the outer medulla and in glomeruli. Utilizing rat metanephric mesenchymal cells in culture, we also demonstrate that BMP-7 induces epithelial cell differentiation. Taken together, these data suggest that BMP-7 is important in both stimulating and maintaining a healthy differentiated epithelial cell phenotype.

transforming growth factor-beta superfamily; cytokines; growth factors; differentiation; metanephric mesenchyme


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BONE MORPHOGENIC PROTEINS (BMPs) are members of a unique subfamily of the transforming growth factor-beta (TGF-beta ) superfamily of secreted growth factors (22). The relationship between members of the family is based on amino acid sequence homology of a highly conserved seven-cysteine domain in the COOH-terminal region of the proteins. In addition, there appears to be a functional similarity, since all members of the TGF-beta superfamily have been implicated in organ development (19, 22, 30, 41). BMPs were isolated from bone matrix and demonstrated to induce ectopic bone formation in various tissues in vivo (27, 33, 34, 40). Expression in the kidney or a potential role in renal biology has been shown so far for BMP-2, -3, -4, -5, -7, and -9 (3, 10, 13, 15, 16, 20, 21, 25, 31, 32, 37).

Studies in mice revealed that the BMP-7 gene is highly expressed in the kidney. BMP-7 mRNA is abundantly expressed during mouse renal organogenesis. It localizes to the ureteric bud tips, in the comma- and S-shaped bodies, and in premature glomeruli (10, 18). Recently, two independent groups reported that BMP-7-deficient mice lack condensation of the renal mesenchyme around the ureteric bud tips and mesenchymal-to-epithelial differentiation with subsequent absence of nephrons (7, 17). This identified BMP-7 as an early inducer of nephrogenesis. More recently, using organ culture of mouse metanephric rudiments, Vukicevic et al. (38) demonstrated inhibition of mesenchymal condensation and epithelial differentiation using BMP-7 antisense oligonucleotides or anti-BMP-7 antibodies. These experiments suggest an important role for BMP-7 in fetal renal epithelial cell differentiation. Tubular epithelial cells have been shown to undergo a dedifferentiation process in renal ischemia. Growth factors that function as inducers of cell differentiation or growth during fetal organogenesis have been shown to afford a variable degree of protection in models of acute renal ischemia (8, 9). Several preliminary in vivo studies using animal models of acute and chronic renal failure demonstrated that the administration of BMP-7 improves renal function and preserves, to a variable degree, renal epithelial cell morphology (6, 14, 35, 36).

To better understand the potential role of BMP-7 in the adult kidney, we cloned a rat BMP-7 cDNA sequence and used it as a probe to localize BMP-7 gene expression by in situ hybridization in normal rat kidney and in rat kidneys with acute renal failure induced by ischemia/reperfusion. Furthermore, we explored the role of BMP-7 in the differentiation of metanephric mesenchymal cells into epithelial cells.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of rat BMP-7 cDNA. Poly(A)+ RNA from both fetal and adult rat kidney RNA was used to prepare cDNA using Superscript (Moloney murine leukemia virus) reverse polymerase and oligo(dT) priming. Two 30-cycle rounds of PCR amplification of the cDNA using nested primers with sequences conserved amongst human, mouse and dog were performed within a rat sequence corresponding to bp 424-1399 of the human sequence. The sense primer sequence corresponding to bp 424-444 was 5'-AGGGCTTCTCCTACCCCTACA-3'. The external and internal antisense primers were 5'-CGGACCACCATGTTTCTGTATTTC-3' and 5'-GGAGCTGTCATCGAAGTAGAGGAC-3', respectively. The PCR product was subcloned into pBluescript using TA cloning. Sequencing and comparison with previously published human and mouse sequences revealed 89% and 95% nucleotide identity, respectively, within the uniquely determined 937-bp cloned region.

Renal ischemia model. Male Sprague-Dawley rats weighing 225-250 g were used. Rats were fed ad libitum and housed in an animal facility at 21°C with a 12:12-h light-dark cycle for ~1 wk. Anesthesia was induced by injection of pentobarbital sodium at 100 mg/kg body wt ip. Kidneys were accessed via a mid-abdominal incision. The left renal artery was identified, freed by blunt dissection, and occluded using the loop-clamp technique. Core body temperature was maintained at 37°C by placing the animal on a homeothermic table, and the abdominal viscera were covered with saline-soaked gauze. After a clamping period of 60 min, the loop was removed, and the kidney was carefully observed to ensure total reperfusion as judged visually. If reperfusion was incomplete, then the experiment was terminated and the animal was killed. Kidneys were removed under anesthesia 3, 6, and 16 h after total reperfusion, cut in 3-5 mm coronal sections, frozen in liquid nitrogen, and stored at -70°C. For in situ hybridization, one section was fixed in freshly prepared, buffered 4% paraformaldehyde overnight and processed further in a paraffin-embedding automat using a standard protocol, supplied by the manufacturer.

RNA isolation and analysis by RNase protection assay. Total RNA was isolated from kidney tissue by the modified method of Chomczynski and Sacchi (4) using RNAzol. An RNase protection assay was used to analyze BMP-7 mRNA expression. The DNA template was constructed by subcloning a 300-bp EcoR I fragment of the rat BMP-7 cDNA (bp 668-968) into pGEM-3Zf(+). To generate the single-stranded 32P-labeled RNA antisense probe, the plasmid was linearized with Xho I and transcribed with T7 RNA polymerase using the T7/Sp6 transcription kit and 50 µCi of [32P]UTP for each transcription reaction under conditions described by the manufacturer. As a control gene, a 90-bp Pst I/Rsa I fragment of human ribosomal RNA (36B4) in pGEM-3Zf(+) was cut with Rsa I and transcribed with T7 RNA polymerase. The labeled RNA (1 × 106 cpm) was hybridized at 50°C to 20 µg of total RNA prepared from rat kidney tissue. After hybridization, the samples were digested with RNase A (50 mg/ml) and RNase T1 (2 mg/ml) followed by the addition of proteinase K to inactivate the remaining RNase. After multiple extractions in phenol-chloroform, the samples were precipitated in ethanol, redissolved in loading buffer containing 90% formamide, and separated on a 6% denaturing polyacrylamide-urea gel. Gels were exposed to Kodak X-Omat film with intensifying screens at -70°C.

In situ hybridization. The technique was performed as described earlier (28). In brief, 2- to 3-µm paraffin sections were mounted on charged glass slides and stored in airtight boxes at -70°C with desiccant up to 4 wk without a change in results. Riboprobes were generated as described for the RNase protection assay. Some of the experiments were repeated using a pGEM3(Zf+) plasmid containing a 469-bp cDNA insert of murine BMP-7 (bp 673-1141) to generate antisense transcripts for in situ hybridization, with similar results (17). In vitro transcription was carried out using a commercial kit and 200 µCi [35S]UTP for each transcription reaction under conditions described by the manufacturer. The probes were run on a 6% denaturing polyacrylamide-urea gel and visualized by autoradiography to ensure full-length transcripts. For in situ hybridization, [35S]mRNAs were diluted to 2 × 104 to 5 × 105 cpm/ml in hybridization buffer containing 2 mM EDTA (pH 7.5), 20 mM Tris (pH 7.5), 0.6 M NaCl, 2× Denhardt's solution, 20% dextran sulfate, 0.1 mg/ml tRNA, and 0.2 M dithiothreitol. After deparaffinization, kidney sections were digested with 20 mg/ml proteinase K in PBS for 10 min. Sections were postfixed for 5 min in 4% paraformaldehyde and acetylated using 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min. A volume of 25-50 µl of hybridization mixture was placed on each section and covered with a siliconized glass coverslip. Hybridization was performed in moist chambers at 50°C for 16 h. Coverslips were removed by washing in 4× saline sodium citrate (SSC) and 2× SSC for 10 min each at 37°C. Slides were then washed in 0.5× SSC for 15 min at 37°C followed by a washing step in 0.5× SSC for 30 min at 60°C. After rinsing the slides in NTE buffer (10 mM Tris, 10 mM NaCl, and 1 mM EDTA) for 15 min at 37°C, RNase A was added at a concentration of 20 mg/ml and incubated for 30 min at 37°C. Slides were washed in NTE for 15 min, 0.5× SSC for 30 min at 37°C and in 0.5× SSC for 15 min and 0.1× SSC for 15 min at room temperature and dehydrated. Sections were dried for 1 h at room temperature, and emulsion autoradiography was performed using Kodak NTB2 emulsion. The coated slides were exposed at 4°C for 10-14 days. Slides were developed in Kodak D19, fixed in Kodak Unifix, and counterstained with hematoxylin and eosin.

Culture of metanephric mesenchymal cells. Metanephric mesenchymal cell cultures were prepared in Dr. Mazen Arar's laboratory as described by Herzlinger with some modification (11). Briefly, 13-day gestation embryonic kidney rudiments were isolated, and the ureteric bud was freed surgically. The remaining mesenchyme was incubated in 0.2% collagenase for 5 min and then transferred to DMEM media with 10% fetal calf serum. Metanephric mesenchymal cells were then dissociated mechanically by gentle aspiration through a pipette and plated in DMEM containing 10% serum. Cells were grown at 37°C in 5% CO2. Immunohistochemical analysis using the avidin-biotin complex technique was performed as described previously (2).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Localization of BMP-7 mRNA expression in the adult rat kidney. A low-power overview in dark-field microscopy of a kidney section after in situ hybridization with BMP-7 antisense RNA shows the renal expression of BMP-7 as a layer of bright silver grains (Fig. 1A). Strong BMP-7 expression can be seen in a stripe pattern throughout the medulla with the highest signal expressed in the outer medulla (om). The cortical tubulointerstitium (c) exhibits weak and scattered BMP-7 hybridization, which is specific compared with the nonspecific signal using sense hybridization (Fig. 1B). High-power bright-field microscopy of the outer medulla reveals the BMP-7 signal over the tubules as black silver grains (Fig. 1C). This expression is highly specific compared with the sense hybridization signal (Fig. 1D). Also, the tubules of the inner medulla showed specific BMP-7 expression (Fig. 1E), although this signal was much weaker compared with the outer medulla.


View larger version (152K):
[in this window]
[in a new window]
 
Fig. 1.   A and B: dark-field microscopy of kidney medulla at low-power magnification (×40, green filter). A: hybridization signal with bone morphogenetic protein-7 (BMP-7) antisense probe is seen as a layer of intense bright silver grains in a stripe pattern throughout the medulla particularly in the outer part (om). Only weak scattered BMP-7 expression is seen in the cortical tubulointerstitium (c). Moderate BMP-7 signal can be seen also in the inner medulla (im). B: hybridization with sense probe shows a weak nonspecific background. C-F: bright-field microscopy of outer medulla at high-power magnification (×400). C: specific hybridization signal with BMP-7 antisense probe is seen as black grains over epithelial cells in tubules of outer medulla. D: hybridization with sense probe shows only weak nonspecific hybridization. E: hybridization with BMP-7 antisense probe demonstrates widespread signal involving almost all tubules in inner medulla. This signal is much less intense but still specific compared with sense hybridization (F).

A strong and specific hybridization signal for BMP-7 mRNA expression could be seen in glomeruli in a peripheral pattern suggesting expression in glomerular visceral epithelial cells. Also, some parietal epithelial cells of the Bowman's capsule show a positive signal (Fig. 2A) compared with nonspecific signal using control sense probe (Fig. 2B). Also, there is a diffuse but specific signal in the cortical tubulointerstitium surrounding the glomerulus.


View larger version (129K):
[in this window]
[in a new window]
 
Fig. 2.   A: hybridization with antisense BMP-7 probe demonstrating a strong and specific signal that localizes in a peripheral pattern over many glomerular cells (arrowheads). In addition, parietal epithelial cells of the Bowman's capsule show positive signal (double arrowheads, top right). B: hybridization with control sense probe demonstrating minimal background signal. C: hybridization with antisense BMP-7 probe. In bright-field microscopy, BMP-7 mRNA localizes to adventitia of renal arteries. Smooth muscle layer and endothelial cells are negative. Inset: dark-field microscopy of a segment of vessel wall demonstrating intense signal of bright silver grains. D: Hybridization with sense probe demonstrates a weak nonspecific signal. Inset: dark-field microscopy of vessel wall segment background. E and F: a strong and highly specific signal is also seen over the epithelial cell layer of renal pelvis and ureter adjacent to papilla. Smooth muscle layer does not express BMP-7. Magnifications: A-F, ×360; insets in C and D, ×90, green filter.

Strong BMP-7 mRNA expression was seen surrounding renal arteries (Fig. 2C and inset). At higher power, this signal clearly could be localized to the adventitia of the arteries (Fig. 2C), whereas the smooth muscle layer and the endothelium of the artery showed only nonspecific background signal (Fig. 2D). The epithelial cell layer of the renal pelvis and the ureter was found to be another site of high BMP-7 mRNA expression (Fig. 2, E and F). The smooth muscle layer of the pelvis and ureter again were negative.

BMP-7 mRNA abundance is diminished in ischemic kidneys. Whole kidney BMP-7 mRNA was analyzed quantitatively using an RNase protection assay (Fig. 3). Kidneys subjected to ischemia/reperfusion injury following clamping the renal artery for 60 min showed no change in mRNA abundance after 3 h of reperfusion. However, whole kidney BMP-7 mRNA abundance was decreased after 6 h and even more so after 16 h of reperfusion.


View larger version (54K):
[in this window]
[in a new window]
 
Fig. 3.   Analysis of BMP-7 mRNA in normal and ischemic rat kidney by RNase protection. Kidney tissue was homogenized, and total RNA was extracted. A 32P-labeled antisense RNA was transcribed from a 300-bp rat BMP-7 cDNA fragment-containing plasmid. Twenty micrograms of total kidney RNA was hybridized with the antisense RNA overnight at 50°C. Nonhybridized excess RNA was digested with RNase A and RNase T1 followed by proteinase K digestion. [32P]RNA:RNA hybrids were separated on a denaturing 6% PAGE gel and visualized using autoradiography. The BMP-7 mRNA abundance was diminished after 6 h and more so after 16 h of reperfusion following 60 min of renal ischemia induced by unilateral clamping of the renal artery. Simultaneous hybridization for ribosomal RNA (36B4) shows equal loading of the RNA samples.

BMP-7 mRNA expression is decreased in the outer medulla and glomeruli. The in situ hybridization for BMP-7 mRNA expression in the ischemic kidneys after 16 h of reperfusion showed a diminished signal over the medulla (dark-field microscopy) (Fig. 4B). The overall expression of BMP-7 mRNA was nearly undetectable particularly in the outer medulla (high-power bright-field) (Fig. 4C). Tubules in the outer medulla showed signs of injury with loss of brush-border membranes, detachment of tubular cells from their basement membrane, and loss of cells due to necrosis. Some cells were still viable and attached to the basement membrane; however, no or only weak expression of BMP-7 mRNA could be detected (Fig. 4D).


View larger version (102K):
[in this window]
[in a new window]
 
Fig. 4.   A-D: In situ hybridization for BMP-7 mRNA in normal kidney compared with ischemic kidney. A: dark-field microscopy of normal kidney medulla in low-power mode. B: dark-field of ischemic kidney medulla shows a less intense signal over the outer medulla without the stripe pattern seen in the control kidney. C: high-power magnification of outer medulla of normal kidney. D: high-power magnification of outer medulla of ischemic kidney shows ischemia-related pathology. There is loss of brush-border membranes, detachment of tubular cells from their basement membrane, and loss of cells due to necrosis indicated by reduction of the number of nuclei. Overall expression of BMP-7 mRNA is diminished to a great extent. Of note is that even in tubular cells that are still viable and attached to their basement membrane, the BMP-7 mRNA signal is lost. Magnification for A-D, ×360.

Expression of BMP-7 was also diminished in glomeruli of kidneys subjected to ischemia and 16 h of reperfusion. The peripheral glomerular pattern seen in the control kidneys remained detectable, but to a much lesser degree (Fig. 5B). Also, the expression in the cortical tubulointerstitium surrounding the glomeruli was weaker after ischemia/reperfusion compared with the control kidneys (Fig. 5A).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5.   A-C: dark-field microscopy showing in situ hybridization for BMP-7 mRNA (×360, green filter). A: BMP-7 mRNA expression over peripheral cells of normal kidney glomerulus (arrowheads). B: BMP-7 mRNA expression over glomerulus of ischemic kidney is diminished. Note also that BMP-7 mRNA expression on cortical tubular epithelial cells is much less intense in ischemic kidney compared with normal kidney. C: control sense hybridization.

The expression of BMP-7 mRNA in the adventitia of renal arteries and in the epithelial cell layer of the renal pelvis and the ureter was not affected by renal ischemia/reperfusion (data not shown).

BMP-7 induces differentiation of metanephric mesenchymal cells into epithelial cells. Metanephric mesenchymal cells maintained in culture retain their phenotypic characteristics including fibroblastic morphology and expression of alpha -smooth muscle actin and vimentin. Upon treatment of confluent cells with 250 ng/ml BMP-7, foci of condensed rounded polyhedral cells develop in the cultures. These condensations of cells appear as early as two days after treatment with BMP-7. The positive staining for cytokeratin is consistent with epithelial cell phenotype (Fig. 6).


View larger version (112K):
[in this window]
[in a new window]
 
Fig. 6.   A: metanephric mesenchymal cells maintained in serum-free medium for 24 h exhibited elongated fibroblastic morphology. B: cells treated with 250 ng/ml BMP-7 for 6 days develop condensation of rounded epithelial-like cells. C: cells maintained in serum-free medium stain negative for cytokeratin. D: positive cytokeratin staining in condensation of cells upon treatment with BMP-7. Magnification, ×75.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of the BMP-7 gene and screening for its expression in mice revealed the kidney as the organ with highest BMP-7 mRNA content (26). Also, BMP-7 mRNA has been shown to be expressed throughout renal development including the earliest stages of metanephric maturation (10, 18, 39). These reports suggest a potential role for BMP-7 in the adult kidney. Our data characterize the distribution of BMP-7 mRNA in the adult rat kidney. Strong expression is found in tubular epithelial cells of the outer medulla and in glomerular epithelial cells. Thus the pattern of BMP-7 expression in the adult kidney is similar to the expression pattern during various stages of fetal kidney development, demonstrating BMP-7 expression in differentiating epithelial cells and structures (10, 18, 39). Moreover, our results show that the abundance of BMP-7 mRNA in the intact kidney during ischemia is significantly diminished compared with the contralateral kidney. This decrease in BMP-7 mRNA abundance is prominent at two major sites, e.g., the outer medullary tubules and glomerular epithelial cells. The decrease in BMP-7 mRNA abundance in the outer medulla might be due at least in part to cell detachment and necrosis following ischemia. However, despite the marked loss of renal function that occurs in the ischemia/reperfusion model, the number of epithelial cells that undergo necrosis and detachment cannot account for this decrease in mRNA abundance. Equal amounts of mRNA were loaded as confirmed by the inclusion of 36B4, a ribosomal RNA, as a housekeeping gene. In addition, tubular epithelial cells in the outer medulla of the ischemic kidney that appear normal and remain attached to their basement membrane also have diminished BMP-7 mRNA expression by in situ hybridization. These results suggest that renal ischemia leads to a decrease in BMP-7 mRNA abundance specifically in these two structures. Two key studies point to an important role for BMP-7 in inducing and/or maintaining epithelial cell differentiation. First, there is severe renal dysorganogenesis in the BMP-7-deficient mouse with an absence of nephron formation (7, 17). Second, recombinant BMP-7 protein is capable of inducing epithelial differentiation with subsequent nephron formation in metanephric organ culture. Moreover, the induction of epithelial differentiation of the metanephric mesenchyme in organ culture by spinal cord tissue can be blocked completely by BMP-7 antisense oligonucleotides or anti-BMP-7 antibodies (38). Our data utilizing metanephric mesenchymal cells in culture confirm these observations. The addition of BMP-7 to the cells results in differentiation of a subpopulation of these cells into epithelial cells that express cytokeratin. Also, the data in BMP-7-deficient mice suggest that BMP-7, perhaps in concert with other factors, is necessary for renal organogenesis (17). Potential mechanisms of action of BMP-7 in addition to differentiation include effects on proliferation and/or prevention of apoptosis prior to mesenchymal epithelial differentiation. In the adult kidney, epithelial cells already possess a fully differentiated and highly specialized phenotype. Renal injury results in morphological and functional changes in epithelial cells (23, 29). Tubular epithelial cells are particularly susceptible and appear to dedifferentiate during renal ischemia.

The diminished expression of BMP-7 in ischemic kidneys is consistent with a recent report (1) demonstrating a marked decrease in the expression of BMP-7 mRNA and protein in the medulla of ischemic kidneys and with several reports showing a beneficial effect of BMP-7 on renal structure and function. The studies reported a beneficial effect of recombinant BMP-7 infused in rat models of acute and chronic renal injury. Two preliminary studies published as abstracts reported an increase in glomerular filtration rate and renal blood flow and a decrease in serum creatinine and blood urea nitrogen in rats that received BMP-7 injections (14, 36). Another study demonstrated that BMP-7 infusion preserves the integrity of vascular smooth muscle cells and maintains actin expression in alpha -smooth muscle cells (35). In this study, BMP-7 decreased endothelial expression of the intercellular adhesion molecule ICAM, evidence for suppression of the inflammatory response associated with ischemia/reperfusion injury. In the five-sixths nephrectomy model, there is an increase in BMP-7 mRNA expression in response to the injection of recombinant BMP-7 associated with partial preservation of renal function and structure (6). Taken together, the results of these studies and the high constitutive expression of BMP-7 in the adult kidney and its decreased expression in renal ischemia shown in this study suggest that BMP-7 may play a role in adult kidney physiology as well pathophysiology. It is unclear whether the decrease in BMP-7 in ischemic renal epithelial cells contributes in some way to the reported injury of these cells. However, it is most likely that BMP-7 functions to maintain a fully differentiated, healthy epithelial cell phenotype in the adult kidney. The structural and functional improvements that follow BMP-7 administration and our data supporting epithelial cell differentiation of metanephric mesenchymal cells confirm this contention. Interestingly, BMP-7 has been shown to stimulate proliferation of primary smooth muscle cell cultures, whereas in established smooth muscle cell cultures, BMP-7 inhibits cell growth and maintains the expression of markers that are characteristic of fully differentiated smooth muscle cell phenotype (5).

BMP-7 mRNA was also expressed normally in two other anatomical sites that deserve comment. The biological relevance of the expression of BMP-7 mRNA in the adventitia of renal arteries remains to be determined. The abundance of BMP-7 mRNA in the adventitia of renal arteries and on the epithelial cell layer of the pelvis and the ureter was not affected by the ischemia. However, there is evidence that this family of proteins may be involved in the biology of vascular wall. BMP-2, another member of this family of proteins, is expressed in atherosclerotic plaques. In vivo BMP-2 inhibits smooth muscle cell proliferation in a rat carotid artery balloon injury model (24). Similar inhibitory effects of BMP-2 on cell proliferation are seen in cultured smooth muscle cells and in mesangial cells (unpublished data) (24).

BMP-7 expression was also high and specific in the epithelial cell layer of the renal pelvis and the ureter. This pattern of expression fits well with the original observation of Huggins (12) in 1931, who demonstrated that urinary tract epithelium induces new bone formation in adjacent tissue after surgical approximation. It is possible that BMP-7 may act to protect urogenital epithelium from the constant exposure to urinary constituents. Further studies are needed to evaluate whether a loss of BMP-7 is involved in pathological conditions of the ureter and the bladder, such as urinary tract obstruction, interstitial cystitis or carcinogenesis.

In summary, this study demonstrates the constitutive expression of BMP-7 mRNA in the adult kidney. Specific BMP-7 mRNA expression is localized to tubular and glomerular epithelial cells, the adventitia of renal arteries, and the epithelium of the renal pelvis and ureter. Renal ischemia/reperfusion injury decreases BMP-7 expression specifically in the epithelium of outer medullary tubules and glomerular epithelial cells. We hypothesize that BMP-7 functions to maintain epithelial cell differentiation in normal adult kidneys. A decrease of its expression in ischemia may be involved in pathobiological manifestations of ischemia/reperfusion injury.


    ACKNOWLEDGEMENTS

We thank Merna Gonzales and Sergio Garcia for technical help and Janet Ortiz for typing the manuscript.


    FOOTNOTES

This study was supported in part by the Department of Veterans Affairs Medical Research Service and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-33665 and DK-43988 (to H. E. Abboud) and DK-19473 (to M. S. Olson) and by Grants-in-Aid from the American Heart Association-Texas Affiliate (to M. Arar). M. Simon was a recipient of a research fellowship of the Deutsche Forschungsgemeinschaft and is currently a recipient of a fellowship of the National Kidney Foundation. J. D. Hernandez was supported by a National Institutes of Health Minority Supplement Award.

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. §1734 solely to indicate this fact.

Address for reprint requests: H. E. Abboud, Division of Nephrology, Dept. of Medicine, Univ. of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284-7882 (E-mail: abboud{at}uthscsa.edu).

Received 21 July 1998; accepted in final form 8 October 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Almanzar, M. M., K. S. Frazier, P. H. Dube, A. I. Piqueras, W. K. Jones, M. F. Charette, and A. L. Paredes. Osteogenic protein-1 mRNA expression is selectively modulated after acute ischemic renal injury. J. Am. Soc. Nephrol. 9: 1456-1463, 1998[Abstract].

2.   Barnes, J. L., and H. A. Abboud. Temporal expression of autocrine growth factors corresponds to morphological features of mesangial proliferation in habu snake venom-induced glomerulonephritis. Am. J. Pathol. 143: 1366-1376, 1993[Abstract].

3.   Chevaile, A., B. C. Santos, S. Thies, and S. R. Gullans. BMP-9, a new member of the TGF-beta superfamily, is expressed in the kidney and inhibits proliferation of mesangial and MDCK cells (Abstract). J. Am. Soc. Nephrol. 7: 1654, 1996.

4.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

5.   Dorai, H., A. Shepard, J. Maliakal, H. Oppermann, S. Vukicevic, and T. K. Sampath. Osteogenic protein-1 (OP-1/BMP-7) modulates smooth muscle cell growth and maintains the expression of cell phenotype and protects against cell injury mediated by nephrotoxic and inflammatory agents in vitro (Abstract). J. Am. Soc. Nephrol. 8: 297, 1997.

6.   Dube, P., K. Frazier, M. Charette, and A. Paredes. Osteogenic protein-1 (rhOP-1) treatment induces tubular regeneration in the acute phase of the rat remnant kidney model (RRKM) (Abstract). J. Am. Soc. Nephrol. 8: 614, 1997.

7.   Dudley, A. T., K. M. Lyons, and E. J. Robertson. A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev. 9: 2795-2807, 1995[Abstract].

8.   Edelstein, C. L., H. Ling, A. Wangsiripaisan, and R. W. Schrier. Emerging therapies for acute renal failure. Am. J. Kidney Dis. 30: S89-S95, 1997[Medline].

9.   Harris, R. C. Growth factors and cytokines in acute renal failure. Adv. Ren. Replace. Ther. 4: 43-53, 1997[Medline].

10.   Helder, M. N., E. Özkaynak, K. T. Sampath, F. P. Luyten, V. Latin, H. Oppermann, and S. Vukicevic. Expression pattern of osteogenic protein-1 (bone morphogenetic protein-7) in human and mouse development. J. Histochem. Cytochem. 43: 1035-1044, 1995[Abstract/Free Full Text].

11.   Herzlinger, D., C. Koseki, T. Mikawa, and Q. Al-Awqati. Metanephric mesenchyme contains multipotent stem cells whose fate is restricted after induction. Development 114: 565-572, 1992[Abstract].

12.   Huggins, C. B. The formation of bone under the influence of epithelium of the urinary tract. Arch. Surg. 22: 377-408, 1931.

13.   Jones, C. M., K. M. Lyons, and B. L. Hogan. Involvement of Bone Morphogenetic Protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development 111: 531-542, 1991[Abstract].

14.   Kelly, K. J., and J. V. Bonventre. Osteogenic protein-1 protects the rat kidney from ischemic injury (Abstract). J. Am. Soc. Nephrol. 7: 1827, 1996.

15.   Kim, Y.-S., S. Harris, J. Wozney, and H. E. Abboud. Effect of bone morphogenetic protein-2 (BMP-2) on mesangial cell growth and migration: role of protein kinase C (Abstract). J. Am. Soc. Nephrol. 7: 1660, 1996.

16.   Knittel, T., P. Fellmer, L. Muller, and G. Ramadori. Bone morphogenetic protein-6 is expressed in nonparenchymal liver cells and upregulated by transforming growth factor-beta 1. Exp. Cell Res. 232: 263-269, 1997[Medline].

17.   Luo, G., C. Hofmann, A. L. Bronckers, M. Sohocki, A. Bradley, and G. Karsenty. BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. Genes Dev. 9: 2808-2820, 1995[Abstract].

18.   Lyons, K. M., B. L. Hogan, and E. J. Robertson. Colocalization of BMP 7 and BMP 2 RNAs suggests that these factors cooperatively mediate tissue interactions during murine development. Mech. Dev. 50: 71-83, 1995[Medline].

19.   Lyons, K. M., C. M. Jones, and B. L. Hogan. The DVR gene family in embryonic development. Trends Genet. 7: 408-412, 1991[Medline].

20.   Lyons, K. M., R. W. Pelton, and B. L. Hogan. Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). Development 109: 833-844, 1990[Abstract].

21.   Lyons, K. M., R. W. Pelton, and B. L. Hogan. Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development. Genes Dev. 3: 1657-1668, 1989[Abstract].

22.   Massaque, J. The transforming growth factor-beta family. Annu. Rev. Cell Biol. 6: 597-641, 1990.

23.   Molitoris, B. A. Cellular basis of ischemic acute renal failure. In: Acute Renal Failure (3rd ed.), edited by J. M. Lazarus, and B. M. Brenner. New York: Churchill Livingstone, 1993, p. 1-32.

24.   Nakaoka, T., K. Gonda, T. Ogita, Y. Otawara-Hamamoto, F. Okabe, Y. Kira, K. Harii, K. Miyazono, Y. Takuwa, and T. Fujita. Inhibition of rat vascular smooth muscle proliferation in vitro and in vivo by bone morphogenetic protein-2. J. Clin. Invest. 100: 2824-2832, 1997[Abstract/Free Full Text].

25.   Özkaynak, E., P. N. Schnegelsberg, D. F. Jin, G. M. Clifford, F. D. Warren, E. A. Drier, and H. Oppermann. Osteogenic protein-2. A new member of the transforming growth factor-beta superfamily expressed early in embryogenesis. J. Biol. Chem. 267: 25220-25227, 1992[Abstract/Free Full Text].

26.   Özkaynak, E., P. N. Schnegelsberg, and H. Oppermann. Murine osteogenic protein (OP-1): high levels of mRNA in kidney. Biochem. Biophys. Res. Commun. 179: 116-123, 1991[Medline].

27.   Sampath, T. K., and A. H. Reddi. Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc. Natl. Acad. Sci. USA 78: 7599-7603, 1981[Abstract].

28.   Simon, M., H.-J. Gröne, O. Jöhren, J. Kullmer, K. H. Plate, W. Risau, and E. Fuchs. Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F240-F250, 1995[Abstract/Free Full Text].

29.   Solez, K. The morphology of acute renal failure. In: Acute Renal Failure, edited by J. M. Lazarus, and B. M. Brenner. New York: Churchill Livingstone, 1993, p. 33-52.

30.   Sporn, M. B., and A. B. Roberts. Transforming growth factor-beta: recent progress and new challenges. J. Cell Biol. 119: 1017-1021, 1992[Medline].

31.   Suzuki, A., S. Nishimatsu, K. Murakami, and N. Ueno. Differential expression of Xenopus BMPs in early embryos and tissues. Zoolog. Sci. 10: 175-178, 1993[Medline].

32.   Takahashi, H., and T. Ikeda. Transcripts for two members of the transforming growth factor-beta superfamily BMP-3 and BMP-7 are expressed in developing rat embryos. Dev. Dyn. 207: 439-449, 1996[Medline].

33.   Urist, M. R. Bone: formation by autoinduction. Science 150: 893-899, 1965[Medline].

34.   Urist, M. R., Y. K. Huo, A. G. Brownell, W. M. Hohl, J. Buyske, A. Lietze, P. Tempst, M. Hunkapiller, and R. J. DeLange. Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography. Proc. Natl. Acad. Sci. USA 81: 371-375, 1984[Abstract].

35.   Vukicevic, S., V. Basic, D. Rogic, N. Basic, M.-S. Shih, A. Shepard, D. Jin, B. Dattatreyamurty, W. Jones, H. Dorai, S. Ryan, D. Griffiths, J. Maliakal, M. Jelic, M. Pastorcic, A. Stavljcnic, and T. K. Sampath. Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat. J. Clin. Invest. 102: 202-214, 1998[Abstract/Free Full Text].

36.   Vukicevic, S., M. Grgic, A. Stavijenic, and T. K. Sampath. Recombinant human OP-1 (BMP-7) prevents rapid loss of glomerular function and improves mortality associated with chronic renal failure (Abstract). J. Am. Soc. Nephrol. 7: 1867, 1996.

37.   Vukicevic, S., M. N. Helder, and F. P. Luyten. Developing human lung and kidney are major sites for synthesis of bone morphogenetic protein-3 (osteogenin). J. Histochem. Cytochem. 42: 869-875, 1994[Abstract/Free Full Text].

38.   Vukicevic, S., J. B. Kopp, F. P. Luyten, and T. K. Sampath. Induction of nephrogenic mesenchyme by osteogenic protein 1 (bone morphogenetic protein 7). Proc. Natl. Acad. Sci. USA 93: 9021-9026, 1996[Abstract/Free Full Text].

39.   Vukicevic, S., V. Latin, P. Chen, R. Batorsky, A. H. Reddi, and T. K. Sampath. Localization of osteogenic protein-1 (bone morphogenetic protein-7) during human embryonic development: high affinity binding to basement membranes. Biochem. Biophys. Res. Commun. 198: 693-700, 1994[Medline].

40.   Wozney, J. M., V. Rosen, A. J. Celeste, L. M. Mitsock, M. J. Whitters, R. W. Kriz, R. M. Hewick, and E. A. Wang. Novel regulators of bone formation: molecular clones and activities. Science 242: 1528-1534, 1988[Medline].

41.   Ying, S. Y. Inhibins, activins, and follistatins. J. Steroid Biochem. 33: 705-713, 1989[Medline].


Am J Physiol Renal Physiol 276(3):F382-F389
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society