Contributions of angiotensin II and tumor necrosis factor-alpha to the development of renal fibrosis

Guangjie Guo1, Jeremiah Morrissey1,2, Ruth McCracken1, Timothy Tolley1, Helen Liapis3, and Saulo Klahr1

1 Departments of Internal Medicine, 2 Cell Biology and Physiology, and 3 Pathology, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri 63110-1092


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Angiotensin II upregulates tumor necrosis factor-alpha (TNF-alpha ) in the rat kidney with unilateral ureteral obstruction (UUO). In a mouse model of UUO, we found that tubulointerstitial fibrosis is blunted when the TNF-alpha receptor, TNFR1, is functionally knocked out. In this study, we used mutant mice with UUO in which the angiotensin II receptor AT1a or the TNF-alpha receptors TNFR1 and TNFR2 were knocked out to elucidate interactions between the two systems. The contribution of both systems to renal fibrosis was assessed by treating TNFR1/TNFR2-double knockout (KO) mice with an angiotensin-converting enzyme inhibitor, enalapril. The increased interstitial volume (Vvint) in the C57BI/6 wild-type mouse was decreased in the AT1a KO from 32.8 ± 4.0 to 21.0 ± 3.7% (P < 0.005) or in the TNFR1/TNFR2 KO to 22.3 ± 2.1% (P < 0.005). The Vvint of the TNFR1/TNFR2 KO was further decreased to 15.2 ± 3.7% (P < 0.01) by enalapril compared with no treatment. The induction of TNF-alpha mRNA and transforming growth factor-beta 1 (TGF-beta 1) mRNA in the kidney with UUO was significantly blunted in the AT1a or TNFR1/TNFR2 KO mice compared with the wild-type mice. Treatment of the TNFR1/TNFR2 KO mouse with enalapril reduced both TNF-alpha and TGF-beta 1 mRNA and their proteins to near normal levels. Also, alpha -smooth muscle actin expression and myofibroblast proliferation were significantly inhibited in the AT1a or TNFR1/TNFR2 KO mice, and they were further inhibited in enalapril-treated TNFR1/TNFR2 KO mice. Incapacitating the angiotensin II or the TNF-alpha systems individually leads to partial blunting of fibrosis. Incapacitating both systems, by using a combination of genetic and pharmacological means, further inhibited interstitial fibrosis and tubule atrophy in obstructive nephropathy.

interstitial volume; myofibroblast; tubulointerstitial fibrosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A WIDE VARIETY OF RENAL DISEASES, regardless of etiology, lead to fibrosis of the tubulointerstitium compartment (4, 18). An increased intrarenal production of angiotensin II is widely considered the major causative factor that initiates and subsequently impels the progression of tubulointerstitial fibrosis (14, 15). We have used a rodent model of unilateral ureteral obstruction (UUO) to study the molecular and cellular mechanisms underlying renal fibrosis to develop therapeutic approaches to decrease or halt the progression of renal disease. Previous studies by our laboratory (9, 13) and those by others (2) have clearly documented that angiotensin II affects both renal epithelial cells and renal fibroblasts, primarily through the AT1 receptor. Additional studies indicate that the effects of angiotensin II, through the angiotensin II type 2 (AT2) receptor, also contribute to the development and progression of renal fibrosis (16, 17).

Angiotensin II promotes the synthesis of several cytokines and growth factors that, in turn, contribute to the progression of renal fibrosis (14, 15). In a previous study, we found that tumor necrosis factor-alpha (TNF-alpha ) expression is increased in a rat model of UUO (12). The induction of TNF-alpha mRNA at early times (4 h) was sensitive to angiotensin-converting enzyme (ACE) inhibition, but this sensitivity was lost at latter times (5 days) (12). In a recent study, by using a mouse model of UUO, we have shown that the effects of TNF-alpha , related to renal fibrosis, are mediated through both the TNFR1 and TNFR2 receptors, with the TNFR1 receptor playing a predominating role (6).

In the present study, we utilized a combination of genetic knockout and "pharmacological knockout" in the mouse model of UUO to examine the pathways involved in renal fibrosis. Mice in which both the TNFR1 and TNFR2 receptors had been genetically eliminated were treated with enalapril to functionally eliminate angiotensin II-mediated effects. The impact of this combined genetic-pharmacological approach was then assessed to determine whether factors other than angiotensin II and TNF-alpha affect the initiation and progression of renal fibrosis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and experimental protocols. Mice in which both of the TNF-alpha receptors (TNFR1 and TNFR2) or the AT1a receptor had been genetically knocked out, and C57BI/6 background mice were used in these experiments. Experimental protocols were approved by the Animal Care Committee of Washington University School of Medicine. Wild-type C57BI/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME), whereas the double-knockout mice were a product of our own breeding program. Breeder mice in which the AT1a receptor had been genetically knocked out were a generous gift from Iekuni Ichikawa. These mice were further bred into the C57BI/6 background. All animals underwent surgical procedures designed to produce UUO, as described previously (6, 9, 13). All animals were fed a standard rodent chow and had free access to water. Some animals received 200 mg/l of enalapril in the drinking water for the duration of the UUO. Animals were killed under pentobarbital anesthesia after 5 days of ureteral obstruction, and the kidneys were perfused with an ice-cold balanced salt solution to remove bloodborne cells. The kidneys were quickly removed, a 2-mm coronal slice was placed in the fixative Histo choice (Amresco, Solon, OH), and the remaining cortex was rapidly dissected and homogenized to prepare total RNA (6).

Morphometric analysis of the interstitial volume, collagen IV, and alpha -smooth muscle actin matrix score and electron microscopy. A standard point-counting method (6, 9, 13) was used to quantitate the volume of the renal interstitium. The relative volume of the renal cortical interstitium (Vvint) was determined on sections by using an antibody to collagen IV to stain fibers in tubular basement membrane (TBM), glomeruli, and the interstitial space. The matrix score for alpha -SMA expression in the renal cortical interstitium was determined by procedures well established in our laboratory, as described previously (6, 9). Ten separate, nonoverlapping microscopic fields of each kidney section were averaged to yield the score of each kidney. The scores for five to nine separate animals for each genotype and treatment modality were then averaged.

For electron microscopy, 1-mm strips of kidney were immersed in buffered 2% glutaraldehyde. The tissue was postfixed in osmium tetroxide. Sections were stained with uranyl acetate and lead citrate and examined with a Phillips CM10 electron microscope.

Messenger RNA quantitation. Total RNA (2 or 4 ug) was used to prepare cDNA as in earlier studies (6, 9, 13). The amount of mRNA for TNF-alpha and transforming growth factor-beta 1 (TGF-beta 1) was determined by using primers and protocols described previously for reverse transcription, followed by PCR of the cDNA (11, 12). To quantitate PCR products and to confirm the integrity of the RNA preparation, we coamplified a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in companion tubes. The PCR products of GAPDH, TGF-beta 1, and TNF-alpha were electrophoresed in the same gel to eliminate gel-to-gel or film-to-film variance. Dideoxy-chain termination sequencing of the PCR products confirmed that indeed TNF-alpha , TGF-beta 1, and GAPDH mRNA were amplified.

ELISA assays for TNF-alpha and TGF-beta 1. Portions of each kidney pole comprising cortex were rapidly homogenized in 4 vol of ice-cold buffer containing 10 mM HEPES, pH 7.9, 0.1 mM EGTA, 1 mM dithiothreitol, and a protease inhibitor cocktail for mammalian tissues (P8340, Sigma, St. Louis, MO). The homogenate was centrifuged at 3,000 g for 15 min at 4°C. The supernatants were stored at -70°C until time of assay. On thawing of the protein, content (Bio-Rad assays, Bio-Rad, Richmond, CA) was adjusted to 1.5 mg/ml with additional homogenization buffer.

The amount of TGF-beta 1 protein present in the mouse kidney cortex was determined with an Opt EIA kit (catalog no. 559119) obtained from BD Pharmingen (San Diego, CA). This kit is capable of measuring mouse TGF-beta 1. Preliminary experiments with acidification of the sample to activate potential latent TGF-beta 1 indicated that >90% of the TGF-beta 1 immunoreactivity was in the active and not the latent form.

The amount of TNF-alpha protein present in the mouse kidney cortex was determined with an Opt EIA kit (catalog no. 2698KI) also obtained from BD Pharmigen. This kit directly measures mouse TNF-alpha . Each sample was determined in duplicate, and the average value of each kidney was, in turn, used to calculate the kidney level of TNF-alpha or TGF-beta 1.

Statistics. Data, shown as means ± SD, were analyzed by the unpaired t-test. Comparisons between values for the contralateral and obstructed kidneys were performed by using an paired t-test. ANOVA was employed to determine the significance of the Vvint, collagen IV, or alpha -SMA matrix score, TNF-alpha or TGF-beta 1 mRNA.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Interstitial volume. Ligation of the ureter leads to a progressive interstitial fibrosis of the obstructed kidney that develops in a matter of days. The Vvint of the kidney increases due to a combination of fibroblast proliferation, monocyte/macrophage infiltration, and excessive matrix protein deposition (3, 13). This is illustrated in Fig. 1, which depicts sections of renal cortex that have been evaluated for collagen IV by immunohistochemical means. In general, collagen IV is found in the basement membrane surrounding tubules, peritubular capillaries, and Bowman's capsule (Fig. 1, contralateral). The immunohistochemical location of collagen IV was indistinguishable in the contralateral kidneys of the C57BI/6 wild-type, the AT1a knockout, or the TNFR1 and TNFR2 double knockout mice (not shown). When a standardized point grid was superimposed over digitized images of magnified renal cortex that was processed for the location of collagen IV, it was seen that obstruction of the ureter caused a significant increase in the number of points (circled in Fig. 1) that appear over the interstitium (Fig. 1, contralateral C57BI/6 vs. obstructed C57BI/6). Ureteric obstruction of the kidney of the TNFR1 and TNFR2 double knockout mouse in the C57BI/6 background also produced an increase in the collagen IV appearing in the interstitium, but not, however, to the extent of that seen in the wild-type mouse. Enalapril treatment of the double knockout mice slightly, but significantly, decreased the amount of collagen IV found in the interstitium after 5 days of UUO. These results and those of the AT1a knockout mice are summarized in Fig. 2. Obstruction of the ureter led to a 470% increase of the Vvint of the wild-type mouse kidney (Fig. 2). Enalapril treatment of wild-type C57BI/6 mice blunted this increase of the interstitial volume to ~300%. The absence of a functional AT1a receptor in C57BI/6 mice also had an ameliorative effect on the increase in the Vvint, and ACE inhibitor treatment had no significant effect in the AT1a knockout mice (Fig. 2). Functional knockout of both TNF-alpha receptors was also associated with a blunted increase in the Vvint. Enalapril treatment of the TNFR1 and TNFR2 double knockout mice led to a further reduction (P < 0.01) of the Vvint, compared with no treatment. The Vvint increased only 180% above that seen in the contralateral kidneys (P < 0.01). This residual increase in the Vvint in the face of pharmacological knockout of angiotensin II formation and genetic knockout of TNF-alpha action was significant, compared with the contralateral kidney (P < 0.03).


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Fig. 1.   Collagen IV matrix score of renal cortex of mice after 5 days of unilateral ureteral obstruction (UUO). Sections of renal cortex of C57BI/6 wild-type or tumor necrosis factor-alpha receptor (TNFR1) and TNFR2 knockout mice that had been treated or not treated with enalapril were used for immunohistochemical location of collagen type IV.



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Fig. 2.   Interstitial volume of the kidney cortex of mice after 5 days of UUO. The interstitial volume (Vvint) of C57BI/6 (n = 9), angiotensin II receptor (AT1a) knockout (n = 6), and TNFR1 and TNFR2 knockout (n = 7) mice that had been treated or not treated with enalapril is shown. A horizontal line depicting the Vvint of all of the TNFR1 and TNFR2 knockout contralateral kidneys is depicted, whereas the Vvint of the kidneys with an obstructed ureter are shown as bars.

alpha -SMA matrix score. Another index of interstitial fibrosis of the tubulointerstitium in renal disease is the differentiation of the fibroblast to a myofibroblast (3, 9). This may be quantitiated by the immunohistochemical determination of alpha -SMA. Figure 3 depicts the same microscopic sections of kidney as those seen in Fig. 1. These sections had been double stained for collagen IV (Fig. 1) and for alpha -SMA (Fig. 3). In the normal kidney (not shown) or the contralateral kidney of mice with UUO, alpha -SMA is largely, if not exclusively, confined to the arteries and small vessels. In the kidney with an obstructed ureter, the alpha -SMA is also present in the widened interstitium (Fig. 3, obstructed panels) as well as in the blood vessels. It is seen that there is less alpha -SMA in the interstitial space of the kidney in the sections obtained from the TNFR1 and TNFR2 double knockout mouse and even less when these mice were treated with enalapril. The results of several individual animals of each genotype and treatment are summarized in Fig. 4. The mean alpha -SMA score of the contralateral kidneys was indistinguishable from kidneys of normal mice and averaged 0.12 ± 0.04 (Fig. 4). Obstruction of the ureter led to a significant increase in the alpha -SMA score to 2.42 ± 0.17 (n = 9) in the kidney cortex of the wild-type C57BI/6 mice. Enalapril treatment decreased the alpha -SMA matrix score by ~40% to 1.44 ± 0.19. As was seen with the Vvint, the absence of a functional AT1a receptor and ACE inhibitor treatment also significantly decreased the matrix score from that of the wild-type animals. There was no difference in the renal cortical matrix score of the AT1a knockout mouse of animals treated or not treated with enalapril (n = 6). Absence of functional TNFR1 and TNFR2 receptors also led to a significant reduction in the alpha -SMA matrix score of the ureteral obstructed kidneys. Enalapril treatment of these double knockout animals, however, led to a further reduction in the matrix score for alpha -SMA to 0.81 ± 0.12, which is significant (P < 0.01) compared with the score of 1.35 ± 0.20 in the nine untreated animals (n = 7 for each). This residual matrix score in the ACE inhibitor-treated double knockout mouse was still significantly greater (P < 0.01) compared with the contralateral kidney (Fig. 4).


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Fig. 3.   alpha -Smooth muscle actin (alpha -SMA) matrix score of renal cortex of mice after 5 days of UUO. Sections of renal cortex of C57BI/6 wild-type or TNFR1 and TNFR2 knockout mice that had been treated or not treated with enalapril were used for immunohistochemical location of alpha -SMA.



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Fig. 4.   alpha -SMA score of the kidney cortex of mice after 5 days of UUO. The matrix score of C57BI/6 (n = 9), AT1a knockout (n = 6), and TNFR1 and TNFR2 knockout (n = 7) mice that had been treated or not treated with enalapril is shown. A thick horizontal line depicting the score of all of the TNFR1 and TNFR2 knockout contralateral kidneys is depicted, whereas the matrix score of the kidneys with an obstructed ureter are shown as bars.

Electron microscopy. Figure 1 shows that in the contralateral kidney there is a close juxtaposition of the tubules and the peritubular capillaries. This close apposition of capillaries to the tubule is disrupted in the kidney with an obstructed ureter by cellular elements and by collagen IV now appearing as a matrix protein (Fig. 1). In a previous study, we showed that collagen types I and III are also deposited in the widened interstitium, adding to the matrix proteins that disrupt the interstitium, (11). Electron microscopic examination of the renal cortex of the contralateral unobstructed kidney also demonstrates abutment of the basement membranes of proximal tubules and the close proximity of peritubular capillaries to these tubules (Fig. 5A). Peritubular capillaries are surrounded by their own basement membranes (small arrows in Fig. 5A). An occasional fibroblast is present in the interstitial space outside the basement membranes (large arrow in Fig. 5A). The cortical interstitium of the kidney with an obstructed ureter is widened and contains an occasional trilobed inflammatory cell (Fig. 5B) along with several fibroblast-like cells (Fig. 5, B and C). The peritubular capillary (small arrows, Fig. 5, B and C) are still surrounded by their basement membrane but are separated from the tubules. In the mouse model of UUO in the C57BI/6 strain, there are few inflammatory cells in the interstitium at 5 days of ureter obstruction (Morrissey J, unpublished observations). A survey of eight electron microscopic fields (×4,400-7,700) of contralateral kidneys and 22 fields of the same power from kidneys with an obstructed ureter indicates that in our mouse model, the major cellular elements occupying the interstitium at 5 days of UUO are fibroblasts. From Fig. 3, it is apparent that these fibroblasts contain alpha -SMA and have differentiated to myofibroblasts.



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Fig. 5.   Electron micrographs of renal cortex. A: contralateral unobstructed kidney of the C57BI/6 mouse. B and C: kidney with an obstructed ureter of the C57BI/6 mouse. All magnification ×7,700.

Examination of the interstitium of the kidney by light microcopy indicates that there was a proportionate reduction in the amount of cellular elements (subsequently characterized by electron microscopy as predominantly fibroblasts) and noncellular matrix exemplified as collagen IV. This proportionate reduction was independent of the animal genotype.

TNF-alpha and TGF-beta 1 mRNA levels. Angiotensin II initiates an increase in the renal production of several cytokines and growth factors, which then contribute to the progression of renal fibrosis. Principal factors in the progression of renal disease are TNF-alpha and TGF-beta 1 (6, 14, 15). In general, the amount of GAPDH mRNA measured in the total RNA purified from the renal cortex was invariant regardless of the genotype, treatment of the mouse, or kidney of the mouse (not shown). Also, in general, the level of either TNF-alpha or TGF-beta 1 mRNA in the contralateral kidney of the mice was invariant (not shown) but consistent with the relative level shown in the second lane from the left for both TNF-alpha or TGF-beta 1 mRNA. Depending on the genotype or treatment, the level of TNF-alpha or TGF-beta 1 mRNA varied in the kidney with an obstructed ureter (Figs. 6 and 7). This is summarized for several animals in each group in Fig. 6 for TNF-alpha or in Fig. 7 for TGF-beta 1 mRNA. The salient feature of the combination of TNF-alpha receptor genetic knockout and angiotensin II "pharmacological knockout" is that the amount of TNF-alpha or TGF-beta 1 mRNA is statistically indistinguishable from the levels seen in the contralateral kidneys (Figs. 6 and 7). Importantly, the levels of TGF-beta 1 mRNA were not increased in the kidney of the AT1a knockout animals with an obstructed ureter (Fig. 7).


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Fig. 6.   Relative ratio of TNF-alpha to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the renal cortex of mice after 5 days of UUO. The relative mRNA level found in the kidney with an obstructed ureter of animals treated or not treated is depicted as a bar for each genotype (n = 4 each). The ratio for each contralateral kidney of the TNFR1 and TNFR2 knockout animals (n = 8 total) is depicted as a thick horizontal line.



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Fig. 7.   Relative ratio of TGF-beta 1 to GAPDH mRNA in the renal cortex of mice after 5 days of UUO. The relative mRNA level found in the kidney with an obstructed ureter of animals treated or not treated is depicted as a bar for each genotype (n = 4 each). The ratio for each contralateral kidney of the TNFR1 and TNFR2 knockout animals (n = 8 total) is depicted as a thick horizontal line.

TNF-alpha and TGF-beta 1 protein levels. The amount of TNF-alpha and TGF-beta 1 protein measured by ELISA methods (Table 1) generally reflected the mRNA levels seen in Figs. 6 and 7. With an intact TNF-alpha signaling system, enalapril treatment inhibited TNF-alpha production by ~24-25% in the wild-type or AT1a knockout mice. With knockout of the TNF-alpha receptors, however, enalapril was ~93% effective above baseline at inhibiting TNF-alpha production in the kidney with an obstructed ureter, compared with the contralateral unobstructed kidneys. The basal level of TNF-alpha in the contralateral kidneys was independent of genotype and enalapril treatment. This suggests that inactivation of both systems is required to substantially reduce TNF-alpha production.

                              
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Table 1.   Quantitation of TNF-alpha and TGFbeta 1 in mouse kidney cortex

The production of TGF-beta 1 appears to be closely tied to the angiotensin II system (Table 1). Treatment with enalapril or inactivation of the AT1a receptor significantly decreased TGF-beta 1 protein levels. There was no statistical difference between the level of TGF-beta 1 expression in the enalapril-treated TNFR1/2-knockout mice and similarly treated AT1a knockout (P < 0.15) and wild-type C57BI/6 mice (P < 0.10).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we clearly show that both angiotensin II and TNF-alpha contribute to renal fibrosis in the mouse model of ureteral obstruction. These conclusions are based on both the genetic knockout of the TNF-alpha receptors and the AT1a receptor and the pharmacological knockout of angiotensin II generation.

In the presence of enalapril, in AT1a knockout mice, the contribution of angiotensin II through the AT2 receptor should be minimized. In the AT1a knockout mice treated with enalapril, there was a significant amount of TNF-alpha generation. In the TNFR1/2 knockout mouse, enalapril treatment significantly reduced TNF-alpha mRNA and protein. This suggests that in the absence of angiotensin II signaling, but in the presence of TNF-alpha signaling (AT1a knockout mouse), the TNF-alpha system is contributing to the increase in interstitial volume and alpha -SMA matrix score. This also suggests that the TNF-alpha system is somewhat independent of the angiotensin II system and contributes to renal fibrosis. When the angiotensin II system plus the TNF-alpha system are simultaneously incapacitated, both TNF-alpha and TGF-beta 1 levels are reduced to levels indistinguishable from the levels seen in the contralateral kidneys. The residual interstitial volume, collagen IV matrix score, and alpha -SMA matrix score present in enalapril-treated TNFR1/2 knockout mice is still significantly above that seen in the contralateral kidneys. The results seen in the AT1a knockout mice or mice treated with enalapril suggest that the TGF-beta 1 system is mainly driven by the angiotensin II system. The TNF-alpha system appears to be driven by both angiotensin II and by the presence of it's own intact receptors.

The dose of enalapril given is in excess of that thought to be necessary to inhibit angiotensin II formation (10, 19). Regardless of arguments of the effectiveness of enalapril to inhibit ACE activity and, consequently, angiotensin II concentration, the absence of an AT1a receptor in the knockout mice incapacitates the angiotensin II system signaling through this receptor. In the absence of AT1a signaling, TGF-beta 1 mRNA and protein are substantially decreased but TNF-alpha mRNA and protein are only modestly reduced.

It has been recognized that in some models of renal disease, higher doses of ACE inhibitors than that needed to blunt angiotensin II formation have an additional beneficial effect (10, 19). In the absence of functional TNF-alpha signaling systems, and in the presence of excessive ACE inhibitor treatment, there remains a significant residual of interstitial fibrosis. This suggests that other vasoactive/growth factor systems contribute, in part, to the initiation and progression of tubulointerstitial damage because both TGF-beta 1 and TNF-alpha mRNA and protein levels were decreased to amounts statistically indistinguishable from those levels found in the unobstructed contralateral kidney.

At this point we cannot say whether the myofibroblasts arise by proliferation of the occasional fibroblast seen in the interstitium (Fig. 5A) or by epithelial-mesenchymal transdifferentiation. From Figs. 1 and 5, B and C, it is also apparent that there are many areas of interstitium containing fibrillar protein. How these fibrillar protein depositions (presumably collagens) affect fluid and solute reabsorption per se is not known; however, their presence is presumed to decrease the efficiency of transport processes. Regardless of the genotype of the animal (i.e., AT1a or TNFR1/2 knockout), the proportion of myofibroblasts and matrix protein in the widened interstitium of the kidney with an obstructed ureter was essentially the same.

An increase in renal angiotensin II levels is thought to be a major factor in the initiation of a cascade of events leading to fibrotic changes in the kidney (14, 15). The initial increase in angiotensin II, in turn, causes upregulation of several vasoactive substances, cytokines, and growth factors (14, 15). This secondary increase in profibrotic substances probably fuels the progression of renal fibrosis. After angiotensin II initiates an increased production of other vasoactive substances or growth factors, a decrease in angiotensin II through pharmacological treatment may or may not be effective in slowing the progression of renal disease. This may lead to irreversible steps in renal fibrotic disease that are now refractory to ACE inhibitor treatment. In this study, we found that when the contributions of angiotensin II and TNF-alpha are minimized, if not entirely eliminated, there are other undefined factors that appear to contribute to renal fibrosis. Studies from our own laboratory have focused on the involvement of TNF-alpha (6, 14). Interestingly, in the AT1a receptor knockout mice, the induction of TGF-beta 1 mRNA or TGF-beta protein was significantly and substantially eliminated (Fig. 6 and Table 1). The model of renal disease resulting from UUO appears to be confined to the tubulointerstitial compartment of the kidney until late in the progression of disease (9). The lack of TGF-beta 1 mRNA induction in the AT1a knockout mouse is supported by the studies of Hisada and co-workers (7) using similar gene knockout mice in a model of antiglomerular basement membrane disease. This strongly suggests that the AT1b receptor that is present is not linked to pathological fibrosis with respect to induction of the profibrotic TGF-beta 1. However, the AT1b receptor may be linked to the induction of other profibrotic factors because hallmarks of disease such as alpha -SMA and increased interstitial volume were reduced by only about half. A previous study using knockout or transgenic mice for the angiotensinogen gene suggested that angiotensin II accounted for ~60% of renal fibrosis. (5).

A candidate system that has been suggested as a contributor to renal disease is the endothelin family. Originally described as vasoactive factors, endothelins appear to exacerbate renal damage in a model of passive Heymann nephritis (1, 20). In addition, overexpression of human endothelin-1 by transgenic mice was found to result in injury to both the glomerular and tubulointerstitial compartments of the kidney (8). A combination of angiotensin II and endothelin-1 blockade was recently suggested for renoprotection of patients who did not respond solely to ACE inhibitors (1).

In summary, these studies suggest that each of the different growth factor/cytokine systems contributes to the initiation and progression of renal disease that culminates in fibrosis. The angiotensin II system appears to be a major driving force in the development of fibrotic renal disease. The TNF-alpha system is also involved in fibrotic renal disease. About 20-30% of renal fibrosis appears to be driven by at least one other, if not more, renal growth factor/cytokine systems. This suggests that a multipharmacological approach is necessary to halt renal disease progression. Whereas some systems may be initially responsive to ACE inhibitor treatment, these systems may subsequently become refractory. This suggests that treatment may need to be tailored during the different stages of the disease.


    ACKNOWLEDGEMENTS

The assistance of Monica Waller and Rosalie Dustmann in the preparation of this manuscript is greatly appreciated.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Program Project Grant DK-09976.

Address for reprint requests and other correspondence: J. Morrissey, Washington Univ. School of Medicine at Barnes-Jewish Hospital (North Campus), 216 S. Kingshighway Blvd., St. Louis, MO 63110-1092 (E-mail:morrisse{at}imgate.wustl.edu).

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.

Received 31 May 2000; accepted in final form 18 December 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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