Macrophages and progressive renal disease in experimental hydronephrosis

Macrophages and Progressive Renal Disease in Experimental Hydronephrosis Jonathan

f?. Diamond,

MD

0 Many recent clinical and experimental studies have clearly demonstrated that one of the initial events taking place in the process of progresslve renal injury is monocytic infiltration of the glomerular and tubulointerstitial compartments. In this report, experimental data supporting the role of the infiltrating renal macrophage (Ma) as a mediator of interstitial fibrosis during the course of obstructive nephropathy will bs reviewed as it pertains to the unilateral ureteral obstrut%n model in the rat. The central pathobiologic theme drawn on data from this model is that fibrogenic cytokines, especially transforming growth factor+, are, in part, Me-derived and represent pivotal links between the initial postobstructive renal inflammation and the late development of renal scarring. The tubular epithelium, as a consequence of the mechanical disturbance produced by ureteral obstruction, may elaborate a host of Me chemoattractant moieties. Many substances can be released by these infiltrating Me; however, our studies have focused on transforming growth factor-/H. Transforming growth factor+ is an important regulator of extracellular matrix, through its direct effects and modulation of other growth factors to maintain matrix homeostasis. We propose that the markedly increased expression of transforming growth factor/?l following ureteral ligation, as detected by a number of laboratories, induces a profibrogenic state and initiates a cascade of dysregulatory events, including the upregulation of tissue inhibitors of metalloproteinase. Transforming growth factor-p1 also may serve as a potent stimulwr for the modulation of quiescent intersthial fibroblasts into myofibroblasts. From a therapeutic standpoint, targeting these early cellular and molecular events may be extremely important in interrupting the interstitfal fibrotic response to long-term obstructive uropathy. 0 19S by the National Kidney Foundation, Inc. INDEX WORDS: Obstructive

nephropathy;

macrophages;

cytokines;

M

ANY RECENT clinical and experimental studies have clearly demonstrated that one of the initial events taking place in the process of progressive renal injury is monocytic infiltration of the glomerular and tubulointerstitial compartments (for review see ref 1). These bone marrow-derived macrophages (Mos) release a potent armamentarium of proinflammatory mediators, including cytokines, growth factors, oxygen-free radicals, proteases, and extracellular matrix products, and have been implicated in a diverse number of nonimmune models of progressive renal disease, such as the experimental nephrotic syndrome produced by either puromytin aminonucleoside or doxorubicin, renal ablation (“remnant kidney”), and protein overload proteinuria. ’ In this report, experimental data supporting the role of the infiltrating renal MP, as a mediator of interstitial fibrosis during the course of obstructive nephropathy will be reviewed as it pertains to the unilateral ureteral obstruction (UUO) model in the rat. The central pathobiologic theme drawn on data from this model is that fibrogenic cytokines, especially transforming growth factor/? (TGF-p), are, in part, Me-derived and represent pivotal links between the initial postobstructive renal inflammation and the late development of renal scarring. We recently focused our attention on this American

Jouma/

of Kidney

Diseases,

Vol 26, No 1 (July),

interstiiial

fibrosis.

model, in sharp contradistinction to the abovementioned models,’ because ureteral obstruction is a nonproteinuric and nonhyperlipidemic disorder. Review of our experimental studies will demonstrate that there is yet another nonimmune stimulus (ie, the mechanical disturbance produced by urinary tract obstruction), which is capable of eliciting a florid Mo infiltration of the kidney and, if unrelieved, contributes to the casFrom the Departments of Medicine and Cellular and Molecular Physiology, Milton S. Hershey Medical Center ana’ the Pennsylvania State University College of Medicine, Hershey, PA. Received October 20,1994; accepted in revisedform January IO, 1995. The experimental studies referred to in this article were funded by a Baxter Healthcare (McGaw Park, IL) Extramural Research Grant and an American Heart Association (Pennsylvania Affiliate; Camp Hill, PA) Research Grant-in-Aid. Dr Diamond is a recipient of an Established Investigator Award of the American Heart Association (Dallas, TX) and the Alyce G. Spector Research Award from the Central Pennsylvania Kidney Foundation (Harrisburg, PA). Presented in part at the conference on Lipids, Hypertension, and Nutrition in Renal Disease, sponsored by The Avram Center for Kidney Diseases and The Long Island College Hospital, New York, NY, September 29-October 1. 1994. Address reprint requests to Jonathan R. Diamond, MD, Division of Nephrology. Milton S. Hershey Medical Center, PO Box 850, Hershey, PA 17033. 0 1995 by the National Kidney Foundation, Inc. 0272~6386/9X?601-0021$3.00/O 1995:

pp 133-140

133

JONATHAN

134

cade of events that ultimately leads to the development of interstitial fibrosis. Obstructive uropathy refers to a group of parenchymal and extraparenchymal renal disorders that exhibit an impaired flow of urine or tubular fluid as a consequence of structural or functional abnormalities in the urinary tract. The functional and morphologic derangements that develop as sequelae to urinary tract obstruction constitutes obstructive nephropathy. The seminal investigations into the pathobiology of obstructive nephropathy were pioneered by Klahr2 The acute hemodynamic abnormalities have been well described by his group2; however, the cellular and molecular aberrations,3-5 which mediate the development of interstitial fibrosis6 and, ultimately, renal failure, are only recently becoming evident. Recent evidence has demonstrated that the process of fibrosis represents an imbalance between extracellular matrix protein synthesis and degradation.7 In this review, experimental evidence supporting an MoTGF-P-renal fibrosis axis will be presented. CHEMOAlTFtACTANT SIGNALS AND MACROPHAGE INFlLTFtATlON IN OBSTRUCTIVE NEPHROPATHY

There is an increase in the interstitial Mo content at 4 to 12 hours after obstruction, which continues to increase rapidly thereafter.5*8*9 The signal responsible for the recruitment of leukocytes into the kidney after ureteral obstruction appears to be specific for Mos because polymorphonuclear leukocytes are not seen in the kidney after ureteral obstruction of less than 24 hours’ duration.8,9 Rovin et al” demonstrated that acute ureteral obstruction results in the release of a specific monocyte chemoattractant from the affected kidney. Initial analysis suggested that this released factor was a lipid in nature.” We recently observed5 that monocyte chemoattractant peptide-l (MCP-1) mRNA was detected between 12 and 96 hours in the obstructed rat kidney, but was absent in the contralateral unobstructed kidney specimens at all time points. In addition, apical tubular MCP-1 expression in the renal cortex at the time of immunolabeling was present from 12 through 96 hours after UUO in the obstructed kidney but not the contralateral unobstructed kidney specimen. This is not the first demonstration of renal tubular epithelial

R. DIAMOND

MCP-1 expression in vivo. Safirstein et al” noted epithelial immunolocalization for MCP-1 in renal tubules in a rat model of ischemic acute renal failure, which was associated with an interstitial Mo infiltrate. In studying the proinflammatory conditions in cultured human renal cortical epithelial cells, Schmouder et alI2 noted fourfold to fivefold increments in steady-state mRNA levels of MCP-1 as well as antigenic peptide in the conditioned media of these cells after cytokine stimulation. Thus, the cortical tubular epithelium appears to be a ready source of MCP-1 during acute inflammatory states. Other proteins with potential chemoattractant activity also may be involved in the Mp, infiltration of the renal interstitium. In a model of focal tubulointerstitial injury associated with angiotensin II infusion, Giachelli et alI3 recently observed a dramatic increase in the focal expression of osteopontin mRNA and protein in renal distal cortical tubules and surrounding Bowman’s capsules. Osteopontin is a highly acidic, secreted glycoprotein, also known as uropontin, eta- 1, secreted phosphoprotein I, and bone sialoprotein I.13 A number of inflammatory cytokines (eg, TGF-P, interleukin- 1, tumor necrosis factor, etc) have been shown to stimulate osteopontin synthesis in various cell types.13 Giachelli et alI3 noted that the elevated expression of osteopontin occurred early and was followed by a monocyte/Mo influx. The Mos localized almost exclusively to sites of tubular osteopontin, suggesting that inappropriate expression of osteopontin by kidney tubules might be an important chemoattractant mechanism in directing an inflammatory response, which would be necessary for the subsequent development of tubulointerstitial injury.13 Osteopontin also contains an arginine-glycine-aspartate cell adhesion sequence, and purified osteopontin has been shown to facilitate adhesion of cultured fibroblasts,13 which also might be important for the development of interstitial fibrosis. We, too, have recently observed’3a that beginning at 4 hours after UUO, there is marked upregulation of renal cortical osteopontin mRNA (Fig 1) and a focal pattern of immunolocalization of this peptide in the cortical tubular epithelium and in the parietal epithelium of Bowman’s capsule in obstructed kidneys only. Contralateral unobstructed kidneys only faintly exhibit the peptide

MACROPHAGES

123

IN EXPERIMENTAL

4 56

HYDRONEPHROSIS

769l9til213 1.6kb

Fig 1. Northern blot analysis of total cortical RNA from a sham-operated rat kidney (lane l), from obstructed kidneys (lanes 2 to 7), and from contralateral unobstructed kidney specimens of indiiklual rats at 4 hours after UUO (lanes 8 to 13). (Top) Total cortical RNA was probed for rat osteopontin (OP) using the 267 cDNA probe (kindly provided by Dr C. Giachelli, University of Washington, Seattle, WA). (Bottom) Intact 288 and 188 ribosomal RNA bands using ethidium bromide staining of the agarose gel. There is markedly increased expression of steady-state mRNA levels of OP in obstructed kidneys compared with either the contralateral unobstructed kidney specimens or the cortex of a normal age-matched rat kidney at this earfy 4-hour time interval post-UUO.

in Bowman’s capsule. At 96 hours after UUO, persistent upregulated expression of osteopontin mRNA continues, but the pattern of immunolocalization within the cortical tubular epithelium of obstructed kidneys is now diffuse (Fig 2). The factor(s) causing an obstructed kidney to release a chemoattractant for Mos remains unknown, as does the specific cellular source of

135

this factor, although our immunohistochemical findings of upregulated expression of MCP-l5 and osteopontin’3a as well as the findings of Giachelli et a1,13with regard to osteopontin expression in another model of tubulointerstitial injury, support a cortical tubule epithelial origin. It is possible that an increase in tubular pressure or membrane stretch in response to ureteral obstruction causes the tubular cells to release a chemoattractant. CORTICAL TRANSFORMING GROWTH FACTOR-P EXPRESSION IN OBSTRUCTIVE NEPHROPATHY

Elimination of the infiltrating Mo burden by prior whole body X-irradiation in rats with bilateral ureteral obstruction markedly attenuated a thromboxane Al-mediated cortical vasoconstriction in the postobstructed kidney.’ These data identified the infiltrating interstitial Mo as a potent source of vasoactive (ie, thromboxane AJ and, perhaps, other proinflammatory mediators. We, among others, have documented upregulation of TGF-P in a variety of models of renal disease. 14-‘*The sustained aberrant expression of renal TGF-P results in the pathologic accumulation of extracellular matrix material in both the glomerulus and interstitial compartments.‘4-‘* In the UUO model, Kaneto et al3 demonstrated that a twofold to threefold increase in TGF-PI mRNA occurred in obstructed kidneys relative

Fig 2. Composite photomicrograph of avidin biotinylated peroxidase immunolabeling for rat osteopontin using a mouse monoclonal IgG antibody, MPI-IIBlO (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) from the renal cortex of an obstructed kidney (A) and the contralateral unobstructed kidney (B) at 96 hours post-UUO. It is evident in panel A that there is pronounced immunolocalixation for osteopontin in the parietal epithelium of Bowman’s capsule as well as diise staining within the cortical tubular epithelium. In sharp contrast, the contralateral unobstructed kidney specimen only demonstrates faint immunolocalization of osteopontin in Bowman’s capsule. (Methyl green counterstain; magnification x 110.)

136

JONATHAN

to either normal control kidneys or contralateral unobstructed kidney specimens at 3,5,7, and 14 days after obstruction. Although a tubular cell origin for the increased mRNA level of TGF-P after obstruction was postulated, these investigators also commented that the Mo could be responsible. Walton et al4 detected a rapid upregulation of gene expression for TGF-Pl within the renal cortex of obstructed rats. However, the cell type exhibiting the increased TGF-P mRNA level was not determined. Recently, we noted highly significant 2.6-, 5%, and 7.0-fold increments in renal cortical TGF-,Bl mRNA levels at 12, 48, and 96 hours, respectively, in the obstructed kidney versus the contralateral unobstructed kidney specimen.5 Intracellular TGF-pl, on immunolabeling, was detected only in the obstructed kidneys of UUO rats at all three time points and was confined to peritubular cells of the renal interstitium. We have demonstrated” that previous whole body X-irradiation of rats with UUO produced a greater than twofold decrement in steady-state renal cortical TGF-P mRNA levels, further supporting our contention that TGF-,B is, in part, Mo derived in this model. In addition, a significant correlation between interstitial Met number and cortical TGF-P 1 n-RNA levels was noted,5 supporting the postulate that the Mo is a source of this peptide growth factor in the UUO model. Transforming growth factor-p is a peptide growth factor, secreted by many cell types, that has effects on extracellular matrix protein production? The profibrogenic effect of TGF-0 is achieved by a combination of inhibition of the degradation of matrix proteins by increased generation of proteinase inhibitors and by decreased expression of degradative proteins, such as collagenase.’ The net effect of TGF-/3 on extracellular matrix is one of accumulation.’ Furthermore, TGF-0 is a chemoattractant for fibroblasts and also stimulates fibroblast proliferation.’ MYOFIBROBLAST TRANSFORMATION OBSTRUCTIVE NEPHROPATHY

IN

Alpers et al*’ have identified accumulation of human renal cortical interstitial cells expressing smooth muscle actin at sites of chronic tubulointerstitial injury. Johnson et al” have noted that angiotensin II-infused rats developed focal tubulointerstitial injury that was evident at 14 days

R. DIAMOND

postinfusion. In this model,” the peritubular space possessed a mononuclear infiltrate and numerous interstitial cells that expressed alphasmooth muscle actin. Recently, we have observed myotibroblast transformation in the cortex of the obstructed rat kidney.” There was evidence of increased alpha-smooth muscle actin mRNA levels in cortical RNA, as well as increased immunolabeling of alpha-smooth muscle actin and desmin in the interstitium of only obstructed kidneys. Among the smooth muscle cell markers expressed by fibroblast populations undergoing differentiation into myofibroblasts in vivo and in vitro, alpha-smooth muscle actin is the most common, followed by desmin, while myosin is practically always absent.22*23The role of the myofibroblast in granulation tissue contraction and in fibrotic disorders is well established,% but the regulatory mechanisms of this maladaptive process are not yet fully clarified. Locally liberated cytokines can modulate the phenotypic features of fibroblasts and enhance the expression of smooth muscle markers.22-26 Several cytokines (ie, interleukin- 1, tumor necrosis factor, plateletderived growth factor), which may contribute to the development of granulation tissue, do not induce the appearance of the alpha-smooth muscle actin-rich myofibroblasts in vivo.“*25 However, accumulation of Mo clusters (induced by local granulccyte-macrophage colony-stimulating factor application) in rat subcutaneous tissueE resulted in abundant expression of alpha-smooth muscle actin by myofibroblasts. Moreover, in transgenic mice expressing granulocyte-macrophage colony-stimulating factor, fibrotic nodules developed in amas where Mos accumulated.24 Since granulocyte-macrophage colony-stimulating factor does not act directly on cuhured fibroblasts to induce alphasmooth muscle actin synthesis, Vyalov et aP concluded that granulocyte macrophage colony-stimulating factor-induced clustered Mos produce a factor(s) capable of stimulating myofibroblast transformation. Desmouliere et al% showed that TGF-pl induced alpha-smooth muscle actin expression in growing and quiescent fibroblasts, and that preincubation of the culture medium with TGF-PI neutralizing antibodies decreased it. TISSUE

INHIBITORS

OF METALLOPROTEINASE

The induction of a profibrogenic state following urinary tract obstruction involves not only

MACROPHAGES

IN EXPERIMENTAL

HYDRONEPHROSIS

the deposition of extracellular matrix but also the dysregulation of degradative proteases, which are involved in extracellular matrix processing and tissue remodeling. A misconception exists that the cellular and molecular perturbations stimulating fibrogenesis develop late after the onset of urinary tract obstruction. The extracellular matrix exists in a dynamic state of both synthesis and degradation. This process maintains the integrity of the interstitial space and is responsible for the regulation of a number of cellular events, including adhesion, migration, proliferation, and differentiation. Matrix metalloproteinases (MMPs) are a group of enzymes capable of degrading both the collagenous and noncollagenous components of the extracellular matrix, and thus are intimately involved in the regulation of tissue remodeling.27X28 These MMPs are secreted as zymogens and their activity is controlled, in part, through cleavage activation or by specific tissue inhibitor of metalloproteinase (TIMP) proteins.27~28 The family of mammalian TIMPs includes three proteins that have individual characteristics as well as some common features. Although both TIMP-1 and TIMP-2 are tight-binding inhibitors of all active MMPs, they also form specific complexes with latent 92-kd gelatinase B and 72-kd gelatinase A, respectively.29.30 Although little is known about the biochemistry of TIMP-3, an important aspect of this inhibitor is that it is found exclusively in the extracellular matrix, in contrast to both TIMP-1 and TIMP-2.“’ Tissue inhibitor of metalloproteinase-3, but not TIMP- 1 or TIMP-2, was recently shown to be abundantly expressed in normal adult mouse kidney.3’ Taken together, these observations suggest that TIMPs may be interchangeable in some of their actions, but that each type likely has additional specific physiologic roles. We recently delineated the kinetics of TIMP- 1, TIMP-2, and TIMF-3 mRNA expression within the renal cortex during the early phase of experimental hydronephrosis in the rat3* since TGF-P could contribute to the fibrosis process by upregulating TIMP expression.’ We postulated that alterations in the regulation of TIMP expression may contribute to the inception of the fibrogenic response within hours after ureteral obstruction commences, which, if uncorrected, may contribute to interstitial fibrosis after a protracted course.

137

There was a marked elevation of TIMP-1 mRNA expression following UUO, which was first noted at 12 hours after ureteral ligation.32 By 96 hours after UUO, there was a 30-fold increment in TIMP-1 mRNA in the obstructed kidneys compared with the contralateral unobstructed kidney or sham-operated rat specimens.32 In contradistinction to TIMP- 1, we noted a decrease in TIMP-3 mRNA levels at 12 hours after ureteral obstruction, which persisted at the 24-, 48-, and 96-hour time intervals.32 Gene expression of TIMP-2 remained at a relatively constant level during our entire period of study.32 Intriguingly, in a rabbit UUO model, Sharma et al6 observed that TIMP-2 mRNA expression appeared to be biphasic with peaks at both days 3 and 16 in post-UUO animals, but no change at day 7. This finding is the first in vivo demonstration in the UUO model of differential mRNA expression for these three members of the TIMP family of proteins. One profibrogenic effect of TGF-P is achieved by a combination of inhibiting normal extracellular matrix degradative proteases and upregulating TIMP expression.’ Transforming growth factor-01 also modulates TIMP-1, TIMP-2, and TIMP-3 expression in vitro3’.33 and specifically decreases the expression of collagenase and stromolysin,’ thereby placing this peptide growth factor as a pivotal moiety in extracellular matrix homeostasis. However, little is known about how growth factors may alter TIMP expression in vivo. In a variety of cultured cell and tissue types a consensus has emerged indicating that TIMP-I transcription is highly inducible by diverse cytokines, hormones, and miscellaneous stimuli.3’,33 In contrast TIMP-2 usually is expressed in a constitutive fashion.33 Our observations in this model of chronic interstitial injury following UUO are consistent with this paradigm. However, the results that we have obtained for expression of the new member of the TIMP family, TIMP-3, raise interesting questions about its participation in normal renal function and responses to injury. The TIMP-3 gene gives rise to a prominent 4.5kb transcript and exhibits a pattern of expression similar to that of TIMP-1 in mouse and human fibroblasts.33 Although it is highly inducible by TGF-Pl in vitro, key differences in gene expression have been noted, particularly with regard to

JONATHAN

138

opposite regulation of TIMP-3 (induction) compared with TIMP-1 (repression) by dexamethasone in fibroblasts, and the low level of expression of TIMP-3 in bone and high renal and brain expression relative to TIMP-1 in adult mice.” Our results demonstrating opposite pattern of expression of TIMP- 1 and TIMP-3 in experimental hydronephrosis confirm that the genes probably are separately controlled by tissue-specific mechanisms. In addition, it is important to note that the TIMP-3 protein resides in the extracellular matrix, as opposed to TIMP-1 and TIMP-2, which diffuse freely through interstitial spaces.33 Our data therefore lead us to suggest that TIMP3 is a specialized constituent of the renal extracellular matrix that may play an important role in the maintenance of normal tissue architecture through interactions with structural components of the matrix. Alternatively, along the lines of the complexes between TIMP-1 and TIMP-2 and the latent forms of gelatinases, it may interact with and sequester an as yet unidentified MMP that is required for extracellular matrix turnover.

R. DIAMOND

Finally, one can speculate that TIMP-3 is derived from tubular epithelium and that its downregulated expression post-UUO is an .injurious .response to the mechanical disturbance produced by ureteral obstruction. Thus, both the augmented expression of TIMP-1 and the reduced expression of TIMP-3 could contribute to fibrosis in the UUO model by changing the quantity and type of extracellular matrix. In situ hybridization studies have revealed that tissue localization of TIMP-1 transcripts is present at sites of active remodeling, where its expression significantly overlaps with that of TGF@l .34 Our previously documented increase in TGF-Pl expression from 12 to 96 hours in the obstructed kidneys versus the contralateral unobstructed kidney specimens’ support a relationship between this peptide growth factor and TIMP1 as a pathobiologic axis for accumulation of extracellular matrix constituents and the development of interstitial fibrosis. Intriguingly, Jones et al35 noted a strong correlation between interstitial MB accumulation and TIMP-1 expression in the Myofibroblast transformation

overprouucuon

MMPs I ECM

Fig 3. A putatiie schematic representation of pathobiologic events that develop following ureteral obstruction. In response to the intratubular mechanical disturbance generated by ureteral obstruction, Me chemoattractant moietiis are upregulated and released by the tubular epithelium. Consequently, bone marrow-derived peripheral blood monocytes infiltrate the interstitial space within hours of ureteral obstruction. In addition to vasoactive substances (ie, thromboxane A& the Me releases many peptlde growth factors. Cur studies have focused on TGF+l as a fibrogenic cytokine. Transforming growth factor-j31 upregulates a number of processes, including TlMP-1 expression, extracellular matrix (ECM) production by interstitial fibroblasts, and the modulation of interstitial flbroblasts into myofibroblasts. All these maladaptive processes contribute to the development of interstitial fibrosis.

MACROPHAGES

IN EXPERIMENTAL

HYDRONEPHROSIS

experimental nephrotic syndrome model produced by puromycin aminonucleoside. One can postulate that either the resident interstitial fibroblast or the infiltrating Mo can represent the responsible cell for TIMP-1 upregulation in our model. SUMMARY

As shown in Fig 3, our laboratory investigations have led us to postulate that the tubular epithelium, as a consequence of the mechanical disturbance produced by ureteral obstruction, elaborates a host of Mo chemoattractant moieties. The tubular epithelium also may participate in the dysregulated expression of TIMP moieties and MMPs as well. Many substances can be released by the infiltrating MB; however, our studies have focused on TGF-Pl. Transforming growth factor-p is an important regulator of extracellular matrix’ through its direct effects and modulation of other growth factors to maintain matrix homeostasis. Its reciprocal effect on extracellular matrix synthesis, downregulation of proteases, and promotion of protease inhibitors place it in a central role during the early development of postobstructive interstitial fibrosis. In addition, as shown in Fig 3, we postulate that TGF-/I is, in part, M@ derived. This contention is supported by data from the experimental nephrotic syndrome.“,‘8.35 In addition to the components depicted in Fig 3, we would like to note the recent elegant studies of Kaneto et al3 who provided compelling evidence that angiotensin II is important in promoting TGF-p production in the obstructed kidney, perhaps from the tubular epithelium, as well as mediating many profibrogenic effects leading to interstitial fibrosis. Furthermore, we propose that the markedly increased expression of TGF-P 1 following ureteral ligation, detected by a number of laboratories,3-5 induces a profibrogenic state and initiates a cascade of dysregulatory events, including the upregulation of TIMP- 1. The mRNA expression of TIMP-2 appears to be independently regulated from TIMP-1, and TGF-01 fails to modulate its mRNA level during the early phases of ureteral obstruction. Of interest, we observed a significant downregulation of renal cortical TIMP-3 mRNA levels in the obstructed kidneys; however, the

139

pathophysiologic significance of this finding remains to be elucidated. In addition, Me-derived TGF-Pl may serve as a potent stimulus for the modulation of quiescent interstitial fibroblasts into myofibroblasts,” as suggested by the elegant studies of Gabbiani and co11eagues.25,26Further studies in our laboratory are investigating the regulatory mechanisms of this process. Finally, from a therapeutic standpoint, targeting these early cellular and molecular events may be extremely important in interrupting the interstitial fibrotic response to longterm obstructive uropathy. REFERENCES 1. van Goor H, Ding G, Kees-Fohs D, Grond J, Schreiner GF, Diamond JR: Macrophages and renal disease. Lab Invest 7 1:456-464, 1994 2. Klahr S: New insights into the consequences and mechanisms of renal impairment in obstructive nepbropathy. Am J Kidney Dis 29:689-699, 1992 3. Kaneto H, Morrissey J, Klahr S: Increased expression of TGF-01 mRNA in the obstructed kidney of rats with unilateral ureteral ligation. Kidney Int 44:313-321, 1993 4. Walton G, Buttyan R, Garcia-Montes E, Olsson C, Hensle T, Sawczuk I: Renal growth factor expression during the early phase of experimental hydronephrosis. J Ural 148:510514, 1992 5. Diamond JR, Kees-Fobs D, Ding G, Frye J, Restrepo NC: Macrophages, monocyte chemoattractant peptide-l and transforming growth factor-p1 in experimental hydronephrosis. Am J Physiol 266:F926-F933, 1994 6. Sharma AK, Mauer SM, Kim Y, Michael A: Interstitial fibrosis in obstructive nephropathy. Kidney Int 44:774-788, 1993 7. Massague JI: The transforming growth factor-p family. Ann Biochem 6:597-641, 1990 8. Harris KPG, Schreiner GF, Purkerson ML, Klahr S: Effect of leukocyte depletion on the post-obstructed rat kidney. Kidney Int 36:210-215, 1989 9. Schreiner GF, Harris KPG, Purkerson ML, Klahr S: Immunological aspects of acute ureteral obstruction. Kidney Int 34:487-493, 1988 10. Rovin BH, Harris KPG, Morrison A, Klahr S, Schreiner GF: Renal cortical release of a specific macrophage chemoattractant in response to ureteral obstruction. Lab Invest 63:213-220, 1990 11. Safirstein R, Megyesi J, Saggi SJ, Price PM, Poon M, Rollins BJ, Taubman MB: Expression of cytokine-like genes JE and KC is increased during renal ischemia. Am J Physiol 261:F1095-F1101, 1991 12. Schmouder RL, Strieter RM, Kunkel SL: Interferongamma regulation of human renal cortical epithelial cellderived monocyte chemoattractant peptide- 1. Kidney Int 44:43-49, 1993 13. Giachelli CM, Pichler R, Lombardi D, Denhardt DT, Alpers CE, Schwartz SM. Johnson RJ: Osteopontin expres-

JONATHAN

140

sion in angiotensin II-induced tubulointerstitial nephritis. Kidney lnt 45:515-524, 1994 l3a. Diamond JR, Kees-Folts D, Ricardo SD, Pruzuak A, Eufemio M: Early and persistent up-regulated expression of renal cortical osteopontin in experimental hydronephrosis. Am J Pathol 146: 1995 (in press) 14. Okuda S, Languino LR, Ruoslahti E, Border WA: Elevated expression of transforming growth factor-p and proteoglycan production in experimental glomerulonephritis. Possible role in expansion of the mesangial extracellular matrix. J Clin Invest 86:453-462, 1990 15. Fukui M, Nakamura T, Ebihara I, Nagaoka I, Tomino Y, Koide H: Low protein diet attenuates increased gene expression of platelet-derived growth factor and transforming growth factor-p in experimental glomerular sclerosis. J Lab Clin Med 121:224-234, 1993 16. Yamamoto T, Noble NA, Miller DE, Border WA: Sustained expression of TGF-PI underlies development of progressive kidney fibrosis. Kidney lnt 45916-927, 1994 17. Ding G, Pesek-Diamond I, Diamond JR: Cholesterol, macrophages and the gene expression of TGF-81 and fibronectin during nephrosis. Am J Physiol 264:F577-F584, 1993 18. Eddy AA: Protein restriction reduces transforming growth factor-/3 and interstitial fibrosis in nephrotic syndrome. Am J Physiol 266:F884-F893, 1994 19. Diamond JR, van Goor H, Ding G, Englemyer E: Myofibroblasts in experimental hydronephrosis. Am J Pathol 146:121-129, 1995 20. Alpers CE, Hudkins KL, Floege J, Johnson RI: Human renal cortical interstitial cells with some features of smooth muscle cells participate in tubulointerstitial and crescentic glomerular injury. J Am Sot Nephrol 5:201-210, 1994 21. Johnson RJ, Alpers CE, Yoshimura A, Lombardi D, Pritzl P, Floege J, Schwartz SM: Renal injury from angiotensin II-mediated hypertension. Hypertension 19464-474, 1992 22. Desmouliere A, Rubbia-Brandt L, Abdiu A, Walz T, Macierira-Coelho A, Gabbiani G: Alpha-smooth muscle actin is expressed in a subpopulation of cultured fibroblasts and is modulated by gamma interferon. Exp Cell Res 20164-73, 1992 23. Rubbia-Brandt L, Sappino AP, Gabbiani G: Locally applied GM-CSF induces alpha-smooth muscle actin con-

R. DIAMOND

taining fibroblasts. Virchows Arch B Cell Pathol 60173-82, 1991 24. Gabbiani G: The biology of the fibroblast. Kidney lnt 41:530-532, 1992 25. Vyalov S, Desmouliere A, Gabbiani G: GM-CSF-induced granulation tissue formation: Relationships between macrophage and myofibroblast accumulation. Virchows Arch B Cell Pathol 63:23 l-239, 1993 26. Desmouliere A, Geinoz H, Gabbiani F, Gabbiani G: TGF-PI induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 120:103-l I, 1993 27. Matrisian LM: The matrix-degrading metalloproteinases. Bioessays 14:455-463, 1992 28. Murphy G, Reynolds JJ: Current views of collagen degradation. Bioessays 2:55-60, 1985 29. Stetler-Stevenson WG, Krutzsch HC, Liotta LA: Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metalloproteinase family. J Biol Chem 264:1737417378, 1989 30. Stetler-Stevenson WG, Brown PD, Onisto M, Levy AT, Liotta LA: Tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA expression in tumor cell lines and human tumor tissues. J Biol Chem 265: 13933-13938, 1990 31. Leco KJ, Khokha R, Pavioff N, Hawkes S, Edwards DR: Tissue inhibitor of metalloproteinase-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues. J Biol Chem 269:9352-9360, 1994 32. Englemyer E,

van Goor H, Edward DR, Diamond JR: Differential mRNA expression of TIMP- I, -2, and -3 during experimental hydronephrosis. J Am Sot Nephrol 5:16751683, 1995 33. Edwards

DR, Murphy G, Reynolds JJ, Whitham SE, Docherty JP, Angel P, Heath JK: Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 6: 1899-1904, 1987 34. Nomura S, Hogan BL, Wills AJ, Heath JK, Edwards DR: Developmental expression of tissue inhibitor of metalloproteinases (TIMP). RNA 105:575-583, 1989 35. Jones CL, Buch S, Post M, McCulloch L, Liu E, Eddy AA: Renal extracellular matrix accumulation in acute puromycin nephrosis in rats. Am J Path01 141:1381-1396, 1992