Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy

Renal fibrosis is the hallmark of progressive renal disease of virtually any etiology. The model of unilateral ureteral obstruction (UUO) in the rodent generates progressive renal fibrosis. Surgically created UUO can be experimentally manipulated with respect to timing, severity, and duration, while reversal of the obstruction permits the study of recovery. The use of genetically engineered mice has greatly expanded the utility of the model in studying molecular mechanisms underlying the renal response to UUO. Ureteral obstruction results in marked renal hemodynamic and metabolic changes, followed by tubular injury and cell death by apoptosis or necrosis, with interstitial macrophage infiltration. Proliferation of interstitial fibroblasts with myofibroblast transformation leads to excess deposition of the extracellular matrix and renal fibrosis. Phenotypic transition of resident renal tubular cells, endothelial cells, and pericytes has also been implicated in this process. Technical aspects of the UUO model are discussed in this review, including the importance of rodent species or strain, the age of the animal, surgical procedures, and histological methods. The UUO model is likely to reveal useful biomarkers of progression of renal disease, as well as new therapies, which are desperately needed to allow intervention before the establishment of irreversible renal injury. Renal fibrosis is the hallmark of progressive renal disease of virtually any etiology. The model of unilateral ureteral obstruction (UUO) in the rodent generates progressive renal fibrosis. Surgically created UUO can be experimentally manipulated with respect to timing, severity, and duration, while reversal of the obstruction permits the study of recovery. The use of genetically engineered mice has greatly expanded the utility of the model in studying molecular mechanisms underlying the renal response to UUO. Ureteral obstruction results in marked renal hemodynamic and metabolic changes, followed by tubular injury and cell death by apoptosis or necrosis, with interstitial macrophage infiltration. Proliferation of interstitial fibroblasts with myofibroblast transformation leads to excess deposition of the extracellular matrix and renal fibrosis. Phenotypic transition of resident renal tubular cells, endothelial cells, and pericytes has also been implicated in this process. Technical aspects of the UUO model are discussed in this review, including the importance of rodent species or strain, the age of the animal, surgical procedures, and histological methods. The UUO model is likely to reveal useful biomarkers of progression of renal disease, as well as new therapies, which are desperately needed to allow intervention before the establishment of irreversible renal injury. Renal fibrosis is regarded as the final common pathway for most forms of progressive renal disease, and involves glomerular sclerosis and/or interstitial fibrosis. Because most renal disorders (whether glomerular or interstitial, congenital or acquired) lead to renal fibrosis, there is great interest in identifying underlying factors to prevent or reverse the changes. For over 50 years, surgical renal ablation in a variety of animal species has been used to model progressive renal disorders. A seminal paper published in 1981 emphasized the role of glomerular hyperfiltration in the initiation of lesions in remnant glomeruli in rats.1.Hostetter T.H. Olson J.L. Rennke H.G. et al.Hyperfiltration in remnant nephrons: a potentially adverse response to renal ablation.Am J Physiol. 1981; 241: F85-F93PubMed Google Scholar Subsequent studies showed that while renal ablation leads to glomerulosclerosis in rats, there is little apparent glomerular injury in C57BL/6 mice (the most commonly used ‘wild-type’ experimental mice).2.Ma L.J. Fogo A.B. Model of robust induction of glomerulosclerosis in mice: Importance of genetic background.Kidney Int. 2003; 64: 350-355Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar However, other mouse strains (129/Sv and Swiss-Webster mice) develop significant glomerular sclerosis,2.Ma L.J. Fogo A.B. Model of robust induction of glomerulosclerosis in mice: Importance of genetic background.Kidney Int. 2003; 64: 350-355Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar underscoring the effect of genetic background on the renal response to renal ablation. While the focus of investigation remained centered on the glomerulus for many years, attention turned also to tubular and interstitial injury, with models of ischemia/reperfusion, immune injury, and nephrotoxins.3.Schnaper H.W. Kopp J.B. Why kidneys fail: Report from an American Society of Nephrology Advances in Research Conference.J Am Soc Nephol. 2006; 17: 1777-1781Crossref PubMed Scopus (11) Google Scholar Clinical studies have also demonstrated that progression of renal insufficiency is much better correlated with renal interstitial fibrosis than with glomerular pathology.4.Schainuck L.I. Striker G.E. Cutler R.E. et al.Structural-functional correlations in renal disease. Part II: The correlations.Hum Pathol. 1970; 1: 631-641Abstract Full Text PDF PubMed Scopus (424) Google Scholar In the 1970s, unilateral ureteral obstruction (UUO) in the rabbit was shown to result in proliferation of renal interstitial fibroblasts and their transformation into myofibroblasts.5.Nagle R.B. Bulger R.E. Cutler R.E. et al.Unilateral obstructive nephropathy in the rabbit:I. Early morphologic, physiologic, and histochemical changes.Lab Invest. 1973; 28: 456-467PubMed Google Scholar Subsequent studies in the rabbit showed increased interstitial collagens I, III and IV, fibronectin, and heparin sulfate proteoglycan.6.Sharma A.K. Mauer S.M. Kim Y. et al.Interstitial fibrosis in obstructive nephropathy.Kidney Int. 1993; 44: 774-788Abstract Full Text PDF PubMed Scopus (150) Google Scholar Since then, animal models of UUO have been expanded and refined to elucidate the pathogenesis of obstructive nephropathy as well as mechanisms responsible for progressive renal fibrosis.7.Klahr S. Morrissey J. Obstructive nephropathy and renal fibrosis.Am J Physiol. 2002; 283: F861-F875Crossref PubMed Scopus (460) Google Scholar, 8.Vaughan E.D. Marion D. Poppas D.P. et al.Pathophysiology of unilateral ureteral obstruction: Studies from Charlottesville to New York.J Urol. 2004; 172: 2563-2569Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar In contrast to adults, in whom diabetes and hypertension are the major etiologies of renal failure, congenital urinary tract obstruction is the most important identifiable cause of renal failure in infants and children.9.Seikaly M.G. Ho P.L. Emmett L. et al.Chronic renal insufficiency in children: The 2001 annual report of the NAPRTCS.Pediatr Nephrol. 2003; 18: 796-804Crossref PubMed Scopus (139) Google Scholar The development of animal models of renal fibrosis presumes an understanding of the disorder under investigation. Unfortunately, there is no general agreement regarding a strict definition of renal fibrosis, which underscores the need for ongoing investigation of a process central to most progressive renal disease. A number of terms have been applied to the pathological process of renal accumulation of collagen or extracellular matrix: fibrosis, sclerosis, and scarring.10.Kriz W. LeHir M. Pathways to nephron loss starting from glomerular diseases--Insights from animal models.Kidney Int. 2005; 67: 404-419Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar This process appears to represent a maladaptive response to injury, which optimally should result in healing of the wound.3.Schnaper H.W. Kopp J.B. Why kidneys fail: Report from an American Society of Nephrology Advances in Research Conference.J Am Soc Nephol. 2006; 17: 1777-1781Crossref PubMed Scopus (11) Google Scholar, 11.Eddy A.A. Can renal fibrosis be reversed?.Pediatr Nephrol. 2005; 20: 1369-1375Crossref PubMed Scopus (39) Google Scholar If the insult is prolonged (as with chronic UUO), the outcome is irreversible renal injury. If the insult is removed (by relief of obstruction), or if the fibrotic response is blocked (by inhibition of gene expression, protein production, or receptor response), renal injury may be prevented or reversed. As early studies of the renal consequences of UUO were performed in the rabbit and dog, the preponderance of current studies are based on the rat and mouse. Since 1950, there have been over 10,000 publications involving ureteral obstruction, nearly 1000 of which address fibrosis (Medline). The advantages of using UUO as a model of renal fibrosis include the absence of an exogenous toxin, the lack of a ‘uremic’ environment, and the availability of the contralateral kidney as a control. Using the contralateral kidney as a control, however, does not take into account its cellular, metabolic, and functional renal compensatory changes in response to UUO.12.Chevalier R.L. Counterbalance in functional adaptation to ureteral obstruction during development.Pediatr Nephrol. 1990; 4: 442-444Crossref PubMed Scopus (15) Google Scholar For this reason, comparison of renal changes resulting from UUO should be compared with those in sham-operated animals (unless comparing a therapeutic intervention on the obstructed kidney). Contralateral nephrectomy in animals subjected to partial UUO reveals the effects of reduced renal mass superimposed on the obstructive injury. Surgical models of UUO have the added advantage of allowing variation in the severity, timing, and duration of obstruction, as well as the opportunity to study recovery following relief of the obstruction. Complete UUO initiates a rapid sequence of events in the obstructed kidney, leading within 24 h to reduced renal blood flow and glomerular filtration rate.8.Vaughan E.D. Marion D. Poppas D.P. et al.Pathophysiology of unilateral ureteral obstruction: Studies from Charlottesville to New York.J Urol. 2004; 172: 2563-2569Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar This is followed within several days by hydronephrosis, interstitial inflammatory infiltration (macrophages), and tubular cell death attributable to apoptosis and necrosis. Tubular epithelial cell death is caused by a number of stressors resulting from UUO, including ischemia, hypoxia, oxidant injury, and axial strain caused by tubular dilatation. There appears to be a close association between progressive fibrosis and tubular cell death.13.Docherty N.G. O'Sullivan O.E. Healy D.A. et al.Evidence that inhibition of tubular cell apoptosis protects against renal damage and development of fibrosis following ureteric obstruction.Am J Physiol Renal Physiol. 2006; 290 (Jan.F4-13, /1): F4-F13Crossref PubMed Scopus (130) Google Scholar Following complete UUO in the rat or mouse, the progression to a severely hydronephrotic kidney with marked loss of renal parenchyma takes place over 1–2 weeks, with more severe fibrosis in the neonate than the adult (JJ Minor, KA Gordon, MSF, BAT, RLC, unpublished data). Because most cases of clinical congenital obstructive nephropathy involve partial, rather than complete obstruction, models of partial UUO have been developed in the neonatal rat and mouse.14.Thornhill B.A. Burt L.A. Chen C. et al.Variable chronic partial ureteral obstruction in the neonatal rat: A new model of ureteropelvic junction obstruction.Kidney Int. 2005; 67: 42-52Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 15.Thornhill B.A. Forbes M.S. Marcinko E.S. et al.Glomerulotubular disconnection in neonatal mice after relief of partial ureteral obstruction.Kidney Int. 2007; 72: 1103-1112Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar These models are also useful in the study of the pathogenesis of renal fibrosis, because the lesions develop more slowly and may be better suited to the study of therapeutic manipulations.16.Chen C.O. Park M.H. Forbes M.S. et al.Angiotensin converting enzyme inhibition aggravates renal interstitial injury resulting from partial unilateral ureteral obstruction in the neonatal rat.Am J Physiol Renal Physiol. 2007; 292: F946-F955Crossref PubMed Scopus (29) Google Scholar The surgical procedure for creating an animal model of UUO is relatively straightforward, if performed as a single operation in an adult rat. Morbidity and mortality will be reduced by using a temperature-controlled operating table heated to body temperature, with the animal anesthetized with isoflurane/oxygen and with the use of a high-quality binocular microscope to visualize the operating field. This approach is particularly important in mice (especially in the neonatal period), or if the obstruction is to be relieved or reversed by a subsequent operation. If this be the case, meticulous technique is essential to avoid adhesions and tissue damage, and establishment of ureteral patency must be documented at the time of study.14.Thornhill B.A. Burt L.A. Chen C. et al.Variable chronic partial ureteral obstruction in the neonatal rat: A new model of ureteropelvic junction obstruction.Kidney Int. 2005; 67: 42-52Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 15.Thornhill B.A. Forbes M.S. Marcinko E.S. et al.Glomerulotubular disconnection in neonatal mice after relief of partial ureteral obstruction.Kidney Int. 2007; 72: 1103-1112Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Ligation of the ureter is the technique used most frequently. However, fistulae can form around the ligature, allowing urine to bypass the obstruction (unpublished observations). Adhesions forming around the ligature can increase the difficulty in removing the ligature if recovery is to be examined: this is a greater problem with adult than neonatal animals. Small vascular clips can be placed around the ureter, but this approach can injure the ureter, or allow urine to pass if not closed correctly. A piece of silastic tubing can be folded perpendicularly across the ureter to create an obstruction, or the tubing can be slit and fitted around the ureter longitudinally, forming a sleeve and creating partial obstruction. Partial UUO can also be created by inserting the ureter in a surgically created slit in the underlying psoas muscle. However, this technique leads to variable (often very mild) degrees of partial obstruction.17.Josephson S. Robertson B. Claesson G. et al.Experimental obstructive hydronephrosis in newborn rats. I. Surgical technique and long-term morphologic effects.Invest Urol. 1980; 17: 478-483PubMed Google Scholar A more reproducible method of creating variable, reversible partial UUO has been developed in the neonatal mouse.15.Thornhill B.A. Forbes M.S. Marcinko E.S. et al.Glomerulotubular disconnection in neonatal mice after relief of partial ureteral obstruction.Kidney Int. 2007; 72: 1103-1112Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar This involves the placement of a fine ligature around the ureter and a piece of stainless steel wire of known diameter, which has been placed parallel to the ureter. After ligation, the wire is slipped out, leaving a partial obstruction with the desired luminal diameter. This ligature can be removed at various intervals, allowing the study of recovery of the lesions. Glomerular filtrate rate can be measured following relief of UUO in the rat or mouse, using standard clearance techniques.18.Shi Y.M. Li C.L. Thomsen K. et al.Neonatal ureteral obstruction alters expression of renal sodium transporters and aquaporin water channels.Kidney Int. 2004; 66: 203-215Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 19.Cochrane A.L. Kett M.M. Samuel C.S. et al.Renal structural and functional repair in a mouse model of reversal of ureteral obstruction.J Am Soc Nephrol. 2005; 16: 3623-3630Crossref PubMed Scopus (122) Google Scholar A novel model of UUO has been described in the adult mouse subjected to contralateral nephrectomy at the time of ureteral reimplantation following 10 days of complete UUO.20.Tapmeier T.T. Brown K.L. Tang Z. et al.Reimplantation of the ureter after unilateral ureteral obstruction provides a model that allows functional evaluation.Kidney Int. 2008; 73: 885-889Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar While this model allows serial measurement of blood urea nitrogen as a reflection of glomerular filitrate rate in the postobstructed kidney, the development of significant proteinuria suggests that secondary glomerular injury contributes to the lesions. Tubular function can be measured in rats following the release of UUO or bilateral ureteral obstruction, and can be correlated with the expression of renal epithelial sodium channel or aquaporins.21.Li C. Wang W. Norregaard R. et al.Altered expression of epithelial sodium channel in rats with bilateral or unilateral ureteral obstruction.Am J Physiol. 2007; 293: F333-F341Google Scholar, 22.Li C. Wang W. Kwon T.H. et al.Downregulation of AQP1, -2, and -3 after ureteral obstruction is associated with a long-term urine-concentrating defect.Am J Physiol. 2001; 281: F163-F171Crossref PubMed Google Scholar As described below, the UUO model is well suited to the discovery of new biomarkers which predate irreversible injury and functional changes. The development of reproducible animal models of UUO, particularly in mice, requires an experienced animal surgeon. The use of mutant strains of mice increases the possibility of greater susceptibility to intraoperative or postoperative mortality compared with wild-type controls. Operation on neonatal animals requires that the pups be accepted by the mother, and nursed successfully. It is important to monitor the daily weight gain of each animal, to assure that overall growth is adequate; kidneys should also be weighed at the time of harvest, to document parenchymal growth. Renal fibrosis can be quantitated by digital morphometry of renal collagen distribution using tissue sections stained with Mallory trichrome (Figure 1a) or picrosirius red (Figure 1b). Types I, III, and IV collagen fibrils are identified by these methods,23.Hironaka K. Sakaida I. Uchida K. et al.Correlation between stellate cell activation and serum fibrosis markers in choline-deficient L-amino acid-defined diet-induced rat liver fibrosis.Dig Dis Sci. 2000; 45: 1935-1943Crossref PubMed Scopus (22) Google Scholar with Type IV constituting the majority of tubular basement membrane. The aniline blue of Mallory trichrome may also stain tubular casts, however (Figure 1a), and picrosirius red is better discriminated by image analysis software programs (Figure 1b). Moreover, the correlation with tissue hydroxyproline content of collagen identified by picrosirius is superior to collagen stained with trichrome.23.Hironaka K. Sakaida I. Uchida K. et al.Correlation between stellate cell activation and serum fibrosis markers in choline-deficient L-amino acid-defined diet-induced rat liver fibrosis.Dig Dis Sci. 2000; 45: 1935-1943Crossref PubMed Scopus (22) Google Scholar It is important to use an unbiased sampling approach: image analysis software can be used to measure randomly selected fields on the tissue section. In addition, total renal collagen content can be quantitated, although this does not directly address its distribution among renal compartments. Because of the importance of the number of glomeruli per kidney as a determinant of progression of renal disease, an unbiased rigorous approach has been developed: the disector technique.24.Nyengaard J.R. Stereological methods and their application in kidney research.J Am Soc Nephol. 1999; 10: 1100-1123PubMed Google Scholar Comparison of this time-consuming approach with simply counting all glomeruli in a single mid-polar planar section of a whole kidney showed excellent correlation in both rats and mice.25.Chevalier R.L. Thornhill B.A. Chang A.Y. et al.Recovery from release of ureteral obstruction in the rat: relationship to nephrogenesis.Kidney Int. 2002; 61: 2033-2043Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 26.Dziarmaga A. Eccles M. Goodyer P. Suppression of ureteric bud apoptosis rescues nephron endowment and adult renal function in Pax2 mutant mice.J Am Soc Nephol. 2006; 17: 1568-1575Crossref PubMed Scopus (54) Google Scholar The latter technique is therefore more practical for most rodent models of UUO. As discussed below, animal models have revealed an extraordinary complexity in the renal cellular response to UUO, and there is significant disagreement regarding the pathogenic implications of fibrosis. On one hand, renal interstitial matrix accumulation is regarded as a primary cause of peritubular capillary obliteration, tubular atrophy, and progressive renal insufficiency.27.Eddy A.A. Molecular basis of renal fibrosis.Pediatr Nephrol. 2000; 15: 290-301Crossref PubMed Scopus (532) Google Scholar The alternate view is that renal inflammation leads to glomerular and tubular damage of some nephrons, which in turn leads to injury to remaining nephrons, with interstitial fibrosis representing a secondary manifestation of disease.10.Kriz W. LeHir M. Pathways to nephron loss starting from glomerular diseases--Insights from animal models.Kidney Int. 2005; 67: 404-419Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar Recent studies have revealed major pathways leading to the development of renal interstitial fibrosis following UUO (Figure 2): (1) interstitial infiltration by macrophages (Figure 1c), which produce cytokines responsible for tubular apoptosis and fibroblast proliferation and activation; (2) tubular cell death by apoptosis and necrosis (Figure 1d), leading to the formation of atubular glomeruli and tubular atrophy; (3) phenotypic transition of resident renal cells. Chronic UUO activates the renin–angiotensin system, with production of reactive oxygen species and nuclear factor-κ B, which promotes macrophage infiltration28.Tashiro K. Tamada S. Kuwabara N. et al.Attenuation of renal fibrosis by proteasome inhibition in rat obstructive nephropathy: possible role of nuclear factor kappaB.Int J Mol Med. 2003; 12: 587-592PubMed Google Scholar and renal tubular apoptosis and interstitial fibrosis in rats.29.Miyajima A. Kosaka T. Seta K. et al.Novel nuclear factor kappaB activation inhibitor prevents inflammatory injury in unilateral ureteral obstruction.J Urol. 2003; 169: 1559-1563Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Classically activated macrophages can generate tumor necrosis factor-α, which mediates proapoptotic signaling and renal tubular cell apoptosis following UUO.30.Misseri R. Meldrum D.R. Dinarello C.A. et al.TNF-alpha mediates obstruction-induced renal tubular cell apoptosis and proapoptotic signaling.Am J Physiol. 2004; 288: F406-F411Google Scholar By contrast, alternatively activated macrophages generate anti-inflammatory cytokines, and induce cell survival and proliferation.31.Duffield J.S. The inflammatory macrophage: a story of Jekyll and Hyde.Clin Sci. 2003; 104: 27-38Crossref PubMed Scopus (360) Google Scholar In addition to the differentiation of infiltrating hematopoietic stem cells and proliferation of resident interstitial fibroblasts, tubular cells can undergo epithelial–mesenchymal transition,32.Iwano M. Plieth D. Danoff T.M. et al.Evidence that fibroblasts derive from epithelium during tissue fibrosis.J Clin Invest. 2002; 110: 341-350Crossref PubMed Scopus (1612) Google Scholar by which process epithelial cells acquire mesenchymal characteristics and invade the interstitium to contribute to the deposition of extracellular matrix (Figure 2). Similarly, endothelial cells can undergo endothelial–mesenchymal transition,33.Zeisberg E.M. Potenta S.E. Sugimoto H. et al.Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.J Am Soc Nephrol. 2008; 19: 2282-2287Crossref PubMed Scopus (615) Google Scholar or can undergo apoptosis, leading to capillary loss and consequent renal ischemia and hypoxia.34.Ohashi R. Shimizu A. Masuda Y. et al.Peritubular capillary regression during the progression of experimental obstructive nephropathy.J Am Soc Nephrol. 2002; 13: 1795-1805Crossref PubMed Scopus (138) Google Scholar In addition to tubular cells and endothelial cells, pericytes can also differentiate into myofibroblasts,35.Lin S.L. Kisseleva T. Brenner D.A. et al.Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.Am J Pathol. 2008; 173: 1617-1627Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar which express α-smooth muscle actin (Figure 1e), and are major contributors to interstitial extracellular matrix. The importance of endothelial cells and pericytes as a major source of renal collagen-producing cells following UUO has only recently been recognized, which shifts attention from the tubular epithelial cell to the renal vasculature as a focus for renal fibrotic injury.33.Zeisberg E.M. Potenta S.E. Sugimoto H. et al.Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.J Am Soc Nephrol. 2008; 19: 2282-2287Crossref PubMed Scopus (615) Google Scholar, 35.Lin S.L. Kisseleva T. Brenner D.A. et al.Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.Am J Pathol. 2008; 173: 1617-1627Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar Significant advances in understanding the process of epithelial–mesenchymal transition followed the development of an antibody to fibroblast-specific protein-1 (Figure 1f), which permitted the tracking of the lineage of cells undergoing phenotypic transition.36.Strutz F. Okada H. Lo C.W. et al.Identification and characterization of a fibroblast marker: FSP1.J Cell Biol. 1995; 130: 393-405Crossref PubMed Scopus (834) Google Scholar However, it should be recognized that this antibody binds also to a number of cell types, including endothelial cells (Figure 1f) and macrophages.35.Lin S.L. Kisseleva T. Brenner D.A. et al.Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.Am J Pathol. 2008; 173: 1617-1627Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar This problem can be addressed with the application of molecular lineage tracing techniques.33.Zeisberg E.M. Potenta S.E. Sugimoto H. et al.Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.J Am Soc Nephrol. 2008; 19: 2282-2287Crossref PubMed Scopus (615) Google Scholar, 35.Lin S.L. Kisseleva T. Brenner D.A. et al.Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.Am J Pathol. 2008; 173: 1617-1627Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar The use of immunohistochemistry to localize individual cell types can be combined with digital morphometry to quantitate the parameters and to make statistical comparisons. In addition, lectins (or other segment-specific markers) can be used to identify tubular segments, permitting further refinement of localization of cell types. Certain lectins, such as derived from Lotus tetragonolobus (which labels mouse proximal tubules) (Figure 1g), permit identification of tubular cell remnants after significant tubular injury and fragmentation resulting from UUO (Figure 1h).15.Thornhill B.A. Forbes M.S. Marcinko E.S. et al.Glomerulotubular disconnection in neonatal mice after relief of partial ureteral obstruction.Kidney Int. 2007; 72: 1103-1112Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar However, Lotus lectin can also bind to intercalated cells of collecting ducts and elastic tissue of arterioles.37.Schulte B.A. Spicer S.S. Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates.Am J Anat. 1983; 168: 345-362Crossref PubMed Scopus (99) Google Scholar Thus, as with the tissue identification of collagen, apoptosis, and fibroblasts, the accurate identification of tubular segments requires an experienced microscopist to interpret the localization of the marker in the context of renal morphology. Peritubular capillaries can be identified by immunohistochemistry using antibody to platelet endothelial cell adhesion molecule-1. As shown in Figures 1i and j, UUO in the neonatal mouse leads to decreased peritubular capillary density in the renal medulla. In the past decade, many important pathways responsible for renal fibrosis have been elucidated, using mice subjected to gene deletion or increased expression (either transgenic or delivery of the gene). These have been reviewed recently, summarizing the data leading to the elucidation of molecular pathways.38.Bascands J.L. Schanstra J.P. Obstructive nephropathy: insights from genetically engineered animals.Kidney Int. 2005; 68: 925-937Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 39.Chevalier R.L. Obstructive nephropathy: towards biomarker discovery and gene therapy.Nat Clin Prac Nephrol. 2006; 2: 157-168Crossref PubMed Scopus (147) Google Scholar A number of studies point to endogenous renal angiotensin II as a central mediator of the renal response to UUO, including inflammation, apoptosis, and interstitial fibrosis.40.Chevalier R.L. Cachat F. Role of angiotensin II in chronic ureteral obstruction.in: Wolf G. The Renin-Angiotensin System and Progression of Renal Diseases. 1 ed. Karger, Basel2001: 250-260Crossref Scopus (19) Google Scholar In neonatal mice with 0, 1, 2, or 4 functional copies of the angiotensinogen gene subjected to UUO, a linear relationship existed between the number of gene copies and the severity of renal fibrosis.41.Fern R.J. Yesko C.M. Thornhill B.A. et al.Reduced angiotensinogen expre