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• W2043343174 abstract "Renal interstitial fibrosis contributes to the progression of most chronic kidney diseases and is an important pathologic feature of urinary tract obstruction. To study the origin of this fibrosis, we used a fetal non-human primate model of unilateral ureteric obstruction focusing on the role of medullary collecting duct (CD) changes. Obstruction at 70 days gestation (full term approximately 165 days) results in cystic dysplasia with significant medullary changes including loss of the epithelial phenotype and gain of a mesenchymal phenotype. These changes were associated with disruption of the epithelial basement membrane and concomitant migration of transitioning cells presumed responsible for the observed peritubular collars of fibrous tissue. There was an abundance of cells that co-expressed the intercalated cell marker carbonic anhydrase II and smooth muscle actin. These cells migrated through the basement membrane and were significantly reduced in obstructed ducts with peritubular collars. Our studies suggest that fetal urinary tract obstruction results in a CD epithelial–mesenchymal transition contributing to the interstitial changes associated with poor prognosis. This seems restricted to the intercalated cells, which contribute to the expansion of the principal cell population and the formation of peritubular collars, but are depleted in progressive injury. Renal interstitial fibrosis contributes to the progression of most chronic kidney diseases and is an important pathologic feature of urinary tract obstruction. To study the origin of this fibrosis, we used a fetal non-human primate model of unilateral ureteric obstruction focusing on the role of medullary collecting duct (CD) changes. Obstruction at 70 days gestation (full term approximately 165 days) results in cystic dysplasia with significant medullary changes including loss of the epithelial phenotype and gain of a mesenchymal phenotype. These changes were associated with disruption of the epithelial basement membrane and concomitant migration of transitioning cells presumed responsible for the observed peritubular collars of fibrous tissue. There was an abundance of cells that co-expressed the intercalated cell marker carbonic anhydrase II and smooth muscle actin. These cells migrated through the basement membrane and were significantly reduced in obstructed ducts with peritubular collars. Our studies suggest that fetal urinary tract obstruction results in a CD epithelial–mesenchymal transition contributing to the interstitial changes associated with poor prognosis. This seems restricted to the intercalated cells, which contribute to the expansion of the principal cell population and the formation of peritubular collars, but are depleted in progressive injury. Progressive organ damage due to tissue scarring and the development of fibrosis is a paradigm shared by numerous human diseases including chronic kidney disease. The postnatal obstructed kidney has been the focus of recent attention and experimentally has become an important model of renal fibrosis.1.Cachat F. Lange-Sperandio B. Chang A.Y. et al.Ureteral obstruction in neonatal mice elicits segment-specific tubular cell responses leading to nephron loss.Kidney Int. 2003; 63: 564-575Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 2.Chevalier R.L. Goyal S. Wolstenholme J.T. et al.Obstructive nephropathy in the neonatal rat is attenuated by epidermal growth factor.Kidney Int. 1998; 54: 38-47Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 3.Matsell D.G. Tarantal A.F. Experimental models of fetal obstructive nephropathy.Pediatr Nephrol. 2002; 17: 470-476Crossref PubMed Scopus (46) Google Scholar, 4.Yabuki A. Maeda M. Matsumoto M. et al.SAMP1/Sku as a murine model for tubulointerstitial nephritis: a study using unilateral ureteral obstruction.Exp Anim. 2005; 54: 53-60Crossref PubMed Scopus (10) Google Scholar, 5.Yang J. Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar Developmental anomalies and associated fetal obstructive nephropathies are the most important causes of childhood kidney failure. Congenital urinary tract obstruction results in an alteration in normal patterns of gene and protein expression responsible for nephrogenesis and nephron endowment, an alteration in the expression of vasoactive and renotrophic factors responsible for the establishment and maintenance of normal glomerular filtration, and the induction of dysplasia, including concomitant mesenchymal reaction and renal fibrosis.6.Chevalier R.L. Molecular and cellular pathophysiology of obstructive nephropathy.Pediatr Nephrol. 1999; 13: 612-619Crossref PubMed Scopus (103) Google Scholar Postnatal renal outcome is directly linked both to nephron endowment and to the severity of mesenchymal fibrosis. Although the role of obstruction and fibrosis in the postnatal kidney has been well studied, the role of obstruction and fibrosis in these congenital disorders has not been explored. Prior publications from our group have reported on the fetal non-human primate model of obstructive renal dysplasia, demonstrating that ultrasound-guided unilateral ureteric obstruction during the early second trimester results in renal pathology characteristic of human fetal obstructive renal disease.3.Matsell D.G. Tarantal A.F. Experimental models of fetal obstructive nephropathy.Pediatr Nephrol. 2002; 17: 470-476Crossref PubMed Scopus (46) Google Scholar, 7.Matsell D.G. Mok A. Tarantal A.F. Altered primate glomerular development due to in utero urinary tract obstruction.Kidney Int. 2002; 61: 1263-1269Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 8.Tarantal A.F. Han V.K. Cochrum K.C. et al.Fetal rhesus monkey model of obstructive renal dysplasia.Kidney Int. 2001; 59: 446-456Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar We have highlighted the disruption of normal nephrogenesis, the defects in branching morphogenesis, and the decrease in glomerular endowment. In addition to these features, we have also demonstrated marked medullary changes, including architectural disorganization, expansion of the medullary interstitium, and disruption of the collecting duct (CD) epithelium, normally consisting of principal and intercalated cells. The intent of this study was to test the hypothesis that the CD epithelium's response to congenital urinary tract obstruction includes mesenchymal transformation, which then contributes to the development of progressive fibrosis. We examined both obstructed and contralateral kidneys of experimental animals at approximately 150 days gestation (near term) and similarly aged control kidneys. As in previous reports,8.Tarantal A.F. Han V.K. Cochrum K.C. et al.Fetal rhesus monkey model of obstructive renal dysplasia.Kidney Int. 2001; 59: 446-456Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar unilateral ureteric obstruction with alginate beads at 70 days gestation (early second trimester) successfully induced renal dysplasia, with distortion of the renal architecture, including altered glomerular development with a decrease in glomerular number and the development of glomerular cysts. In particular, we observed dilatation and cystic transformation of medullary CDs, the formation of peritubular smooth muscle collars, and the expansion of the medullary interstitium (Figure 1). We observed corresponding increases in medullary vimentin (Vim) and alpha smooth muscle actin (SMA) immunoreactivity in both the renal interstitium and in the peritubular cellular collars with E-cadherin expression decreased in the medullary CD epithelium. To quantify the histopathological changes, we extracted RNA from dissected medullary and cortical tissue from obstructed, contralateral, and control fetal kidneys and performed quantitative PCR analysis. As expected, mRNA changes corresponded to the changes in immunohistochemistry with a significant increase in Vim, decrease in E-cadherin, and no change in transforming growth factor-β. Of note, these differences were more pronounced in medullary than in cortical samples (Figure 2). In the control kidney, medullary CDs were identified by the expression of principal cell-specific water channel aquaporin-2 (AQP2)9.Deen P.M. Verdijk M.A. Knoers N.V. et al.Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine.Science. 1994; 264: 92-95Crossref PubMed Scopus (718) Google Scholar, 10.Fushimi K. Uchida S. Hara Y. et al.Cloning and expression of apical membrane water channel of rat kidney collecting tubule.Nature. 1993; 361: 549-552Crossref PubMed Scopus (829) Google Scholar in a majority of cells (Figure 3). This late gestation CD epithelium expresses the expected basolateral adherens junction protein E-cadherin and the apical structural protein cytokeratin (CK), characteristic of a differentiated epithelial cell layer. Notably, and as expected, the epithelia of normal unobstructed CDs did not show any evidence of Vim immunoreactivity. However, and at variance with previous reports in adult human kidney biopsy specimens11.Rastaldi M.P. Ferrario F. Giardino L. et al.Epithelial–mesenchymal transition of tubular epithelial cells in human renal biopsies.Kidney Int. 2002; 62: 137-146Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar and postnatal mouse kidneys, we identified cells in the CDs of control kidneys that exhibited diffuse SMA immunoreactivity that, to our knowledge, have not been observed in normal postnatal kidneys. This was unexpected and surprising, since SMA expression implies a mesenchymal or less differentiated phenotype. This SMA expression colocalized with the expression of the intercalated cell marker, CAII,12.Brown D. Kumpulainen T. Roth J. et al.Immunohistochemical localization of carbonic anhydrase in postnatal and adult rat kidney.Am J Physiol. 1983; 245: F110-F118PubMed Google Scholar, 13.Kim J. Tisher C.C. Linser P.J. et al.Ultrastructural localization of carbonic anhydrase II in subpopulations of intercalated cells of the rat kidney.J Am Soc Nephrol. 1990; 1: 245-256PubMed Google Scholar and was mutually exclusive with AQP2. Furthermore, and unlike the columnar epithelium that constitutes the majority of the CD, these cells display a pyramidal morphology with a widened basolateral and narrowed apical surface. Fetal kidneys obstructed at 70 days gestation and studied near term contained both normal and segmentally dysplastic medullary elements. In particular, CDs in affected segments of the inner medulla exhibited significant dilatation. In addition, dramatic expansion of the adjacent interstitial compartment was observed in regions of the more severely dilated ducts. Obstructed CDs also exhibited an increase in the presence of peritubular SMA+ cells ranging from localized expression adjacent to one or two CD epithelial cells to the formation of dense peritubular collars fully surrounding the duct (Figure 4). Typically, though not exclusively, more severely dilated ducts exhibited more fully developed peritubular collars. Since this was the most predominant and consistent feature of fetal urinary tract obstruction, we used the degree of peritubular collars formation to characterize the severity of CD injury. In conjunction with the prominent dilation of CDs and adjacent peritubular collar formation observed following obstruction, these kidneys also displayed a temporal and spatial sequence of phenotypic changes that suggests the occurrence of epithelial–mesenchymal transition (EMT) (Figure 5). In particular, the epithelia of obstructed CDs exhibited disruption of E-cadherin, whereby its localization at the interepithelial junctions became less distinct with a corresponding increase in diffuse cytoplasmic immunoreactivity. Furthermore, localized regions of the epithelium exhibited a complete loss of E-cadherin, presumably highlighting sites of active EMT. Expression of the principal cell marker AQP2 also appeared to be downregulated and exhibited altered cellular localization when compared to both adjacent undilated CDs and the CDs of normal kidneys. In contrast, Vim expression, which is absent from control epithelium, was acquired in the basolateral aspect of CD cells adjacent to the formation of peritubular SMA collars. This Vim expression is seen throughout the obstructed CD in both AQP2+ and SMA+/CAII+ cell populations, but probably represents divergent responses between migrating and non-migrating cells. Like Vim, expression of fibroblast surface protein was minimal in the control CD, but markedly increased in the obstructed duct epithelium. As expected, interstitial fibroblasts expressed fibroblast surface protein. These results suggest a loss of epithelial and a gain of mesenchymal characteristics in the obstructed CD epithelium. Among the phenotypic changes observed within the dilated CD epithelia, an apparent increase in the absolute number of epithelial cells was noted. This observation suggests that proliferation may be occurring in these CDs to maintain an intact epithelial layer when dilation occurs. Immunohistochemical staining for Ki-67, a cell cycle protein-indicating cell proliferation,14.Scholzen T. Gerdes J. The Ki-67 protein: from the known and the unknown.J Cell Physiol. 2000; 182: 311-322Crossref PubMed Scopus (3214) Google Scholar revealed increased nuclear expression in selected obstructed CDs versus control specimens. The expression of Ki-67 in the CD epithelium was restricted to the AQP2+ principal population and was not seen in SMA+/CAII+ cells of either obstructed or control kidneys. Increased proliferative events were also observed in forming peritubular collars surrounding obstructed CDs (Figure 6). Based on these findings, the inner medullary CD of the fetal kidney appears to be composed of at least two populations of epithelial cells. Similar to the adult kidney, the predominant population of cells is the AQP2+ principal cell population comprising approximately 80% of epithelium. These studies indicate that the remaining 20% of the fetal CD is composed of SMA+/CAII+ cells that may represent an immature intercalated cell population, a putative population of CD epithelial progenitors, or a combination of the two. Given the unexpected expression of SMA, and therefore of SMA+/CAII+ cells in the CD epithelium, and to explore the possibility that these cells are undergoing EMT, differences in the numbers of this subpopulation of cells between obstructed and control kidneys were compared. Importantly, in the obstructed ducts of the near-term fetal kidneys, we observed a decrease in the number of SMA+/CAII+ CD epithelial cells when compared to both adjacent, unaffected ducts in the same kidney and to ducts of contralateral kidneys and controls of comparable gestational age. This was confirmed by cell counts (Figure 7), which showed that there was a significant depletion of this cell population from approximately 17 to 12% in mild and moderately obstructed ducts, and to 8% in severely affected ducts with completely formed peritubular collars. By TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling) analysis, this depletion was not associated with epithelial cell apoptosis. These results reveal a loss of SMA+/CAII+ cells in CDs during obstruction that was statistically significant (P<0.01) when compared with control CDs. Given the unexpected finding of SMA+ cells in the fetal CD and their disappearance with injury, and to further support the possibility that the SMA+/CAII+ cells were undergoing EMT, the ability of these cells to cross the CD basement membrane was explored. As previously suggested,5.Yang J. Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 15.Ng Y.Y. Huang T.P. Yang W.C. et al.Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats.Kidney Int. 1998; 54: 864-876Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar EMT includes the mesenchymal expansion of the interstitium and the development of peritubular SMA collars, and therefore requires the migration of the transitioning cells through the basement membrane and into the interstitial space. Of note in mild to moderately obstructed CDs, there was an observable disruption in type IV collagen (Coll IV) expression of the basement membrane, specifically at the site of SMA collar formation. In addition to the loss of basement membrane integrity, co-localization experiments also demonstrated migration of SMA+/CAII+ cells through the plane of the CD basement membrane and into the SMA+ peritubular collars (Figure 8). Observation of these migratory events is uncommon in the near-term obstructed kidney, indicating that only a small fraction of total CD epithelium is undergoing EMT at any given moment. Although persisting early in transition, expression of CAII and other epithelial characteristics including E-cadherin appear to be lost soon after migration, as these cells acquire their final myofibroblastic phenotype. In this study, we have used a clinically relevant model of fetal urinary tract obstruction to define the relative contribution of medullary injury to progressive renal fibrosis and dysplasia. As described, significant medullary disruption occurs as a result of ureteric obstruction with segmental alteration of the medullary architecture featuring dilation and cystic transformation of medullary CDs, the formation of peritubular smooth muscle collars, and the expansion of the medullary interstitial compartment. These changes closely resemble the changes seen in human fetal urinary tract obstruction and fetal renal dysplasia,11.Rastaldi M.P. Ferrario F. Giardino L. et al.Epithelial–mesenchymal transition of tubular epithelial cells in human renal biopsies.Kidney Int. 2002; 62: 137-146Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 16.Grisaru S. Cano-Gauci D. Tee J. et al.Glypican-3 modulates BMP- and FGF-mediated effects during renal branching morphogenesis.Dev Biol. 2001; 231: 31-46Crossref PubMed Scopus (144) Google Scholar, 17.Shibata S. Nagata M. Pathogenesis of human renal dysplasia: an alternative scenario to the major theories.Pediatr Int. 2003; 45: 605-609Crossref PubMed Scopus (14) Google Scholar, 18.Woolf A.S. Price K.L. Scambler P.J. et al.Evolving concepts in human renal dysplasia.J Am Soc Nephrol. 2004; 15: 998-1007Crossref PubMed Scopus (133) Google Scholar and are highly reproducible in this model. To date, studies of the effects of urinary tract obstruction have utilized, almost exclusively, postnatal models of the rodent kidney.1.Cachat F. Lange-Sperandio B. Chang A.Y. et al.Ureteral obstruction in neonatal mice elicits segment-specific tubular cell responses leading to nephron loss.Kidney Int. 2003; 63: 564-575Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 2.Chevalier R.L. Goyal S. Wolstenholme J.T. et al.Obstructive nephropathy in the neonatal rat is attenuated by epidermal growth factor.Kidney Int. 1998; 54: 38-47Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 3.Matsell D.G. Tarantal A.F. Experimental models of fetal obstructive nephropathy.Pediatr Nephrol. 2002; 17: 470-476Crossref PubMed Scopus (46) Google Scholar, 4.Yabuki A. Maeda M. Matsumoto M. et al.SAMP1/Sku as a murine model for tubulointerstitial nephritis: a study using unilateral ureteral obstruction.Exp Anim. 2005; 54: 53-60Crossref PubMed Scopus (10) Google Scholar, 5.Yang J. Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 19.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 These models focus exclusively on the effects it has on the proximal tubule and the role of EMT in these segments.20.Morrissey J. Guo G. Moridaira K. et al.Transforming growth factor-beta induces renal epithelial jagged-1 expression in fibrotic disease.J Am Soc Nephrol. 2002; 13: 1499-1508Crossref PubMed Scopus (82) Google Scholar, 21.Zeisberg M. Hanai J. Sugimoto H. et al.BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury.Nat Med. 2003; 9: 964-968Crossref PubMed Scopus (1107) Google Scholar In general, the response to injury is different in the fetus, neonate, and adult, with fibrosis being a major component of the latter.22.Colwell A.S. Longaker M.T. Lorenz H.P. Fetal wound healing.Front Biosci. 2003; 8: s1240-s1248Crossref PubMed Google Scholar More specifically, while the fetal kidney's response to obstruction includes the development of reactive peritubular collars and the expansion of the interstitial compartment, which may be harbingers of fibrosis, the effects may be more dramatic and include an interruption in normal nephrogenesis and growth of the kidney. Unfortunately, and perhaps due to the limitations of the models, there has been very little attention focused on the medulla and medullary CD. Given the extensive changes seen in the medulla following in utero urinary tract obstruction in both human and non-human primates, we explored the possibility that CD EMT contributes to medullary interstitial fibrosis. To our knowledge, neither the ability of these cells to undergo EMT nor the contribution of the medullary CD to fibrosis has been previously reported. In this study, the phenotypic conversion of CD epithelial cells in a manner consistent with EMT was shown. The onset of this conversion is highlighted by the degradation of interepithelial E-cadherin and diffuse cytoplasmic immunoreactivity that demonstrates the injury-induced disassociation of adherens junctions.23.Han S.U. Lee H.Y. Lee J.H. et al.Modulation of E-cadherin by hepatocyte growth factor induces aggressiveness of gastric carcinoma.Ann Surg. 2005; 242: 676-683Crossref PubMed Scopus (34) Google Scholar Similarly, this conversion is highlighted by the de novo expression of Vim in the CD epithelium adjacent to developing peritubular collars, the associated disruption of the tubular basement membrane, and evidence of SMA+/CAII+ CD epithelial cell migration. As expected, evidence of cellular migration was infrequently documented given the short window of time during which it occurs and the likelihood that it occurs soon after obstruction. Finally, the phenotypic transition of CD epithelium was also temporally and spatially associated with the formation of peritubular SMA+ collars, lending further support for the probable role of the CD transition in this peritubular pathology. Interestingly, the increase in basolateral Vim expression is seen not only in the migrating SMA+/CAII+ population, but also in the AQP2+ principal cell population. In migrating epithelial and CD-derived peritubular collar cells, this Vim expression may indicate the conversion of these cells to a myofibroblastic phenotype where Vim is critical for growth, motility, and structural stiffness of fibroblasts.24.Eckes B. Dogic D. Colucci-Guyon E. et al.Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts.J Cell Sci. 1998; 111: 1897-1907PubMed Google Scholar, 25.Holwell T.A. Schweitzer S.C. Evans R.M. Tetracycline regulated expression of vimentin in fibroblasts derived from vimentin null mice.J Cell Sci. 1997; 110: 1947-1956PubMed Google Scholar Interestingly, the expression of Vim has also been demonstrated to be of critical importance to the maintenance of endothelial cell structural integrity in response to mechanical stress24.Eckes B. Dogic D. Colucci-Guyon E. et al.Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts.J Cell Sci. 1998; 111: 1897-1907PubMed Google Scholar and to be regulated by changes in physiologic fluid flow.26.Helmke B.P. Thakker D.B. Goldman R.D. et al.Spatiotemporal analysis of flow-induced intermediate filament displacement in living endothelial cells.Biophys J. 2001; 80: 184-194Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar Therefore, the de novo expression of Vim in the non-migrating cells of the injured CD epithelium, along with the formation of muscular collars not unlike those found in arterioles, may represent an equivalent cellular adaptation to the reduced flow and increased luminal pressure resulting from urinary tract obstruction. Unlike the proximal tubule, the CD is composed of two distinct epithelial cell types, the principal and intercalated cells. Embryologically, these CD cell lineages originate from a common ureteric duct-derived progenitor, but have specific and distinct differentiated functions and phenotypes. However, there is in vivo and in vitro evidence that CD cells, and in particular β-intercalated cells, have the capacity to differentiate into various cell types comprising the normal CD epithelium.27.Fejes-Toth G. Naray-Fejes-Toth A. Differentiation of renal beta-intercalated cells to alpha-intercalated and principal cells in culture.Proc Natl Acad Sci USA. 1992; 89: 5487-5491Crossref PubMed Scopus (83) Google Scholar, 28.Fejes-Toth G. Naray-Fejes-Toth A. Differentiation of intercalated cells in culture.Pediatr Nephrol. 1993; 7: 780-784Crossref PubMed Scopus (13) Google Scholar More recent evidence with gene knockout mice suggests the presence of an upstream CD epithelial progenitor. The elimination of a crucial embryologic transcription factor Foxi1 was shown to result in a loss of distinct principal and intercalated cell phenotypes with clonal expansion of a hybrid, putative progenitor population with primitive functional characteristics of both cell types.29.Blomqvist S.R. Vidarsson H. Fitzgerald S. et al.Distal renal tubular acidosis in mice that lack the forkhead transcription factor Foxi1.J Clin Invest. 2004; 113: 1560-1570Crossref PubMed Scopus (135) Google Scholar Putative progenitors have also been identified in the CD and throughout the kidney as 5-bromo-2′-deoxyuridine label-retaining cells in pulse-chase studies,30.Cotsarelis G. Cheng S.Z. Dong G. et al.Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells.Cell. 1989; 57: 201-209Abstract Full Text PDF PubMed Scopus (1096) Google Scholar, 31.Johansson C.B. Momma S. Clarke D.L. et al.Identification of a neural stem cell in the adult mammalian central nervous system.Cell. 1999; 96: 25-34Abstract Full Text Full Text PDF PubMed Scopus (1606) Google Scholar, 32.Lavker R.M. Sun T.T. Epidermal stem cells: properties, markers, and location.Proc Natl Acad Sci USA. 2000; 97: 13473-13475Crossref PubMed Scopus (343) Google Scholar with the majority of slowest cycling label-retaining cells localized to the tubular epithelium and interstitium of the renal papilla.33.Oliver J.A. Maarouf O. Cheema F.H. et al.The renal papilla is a niche for adult kidney stem cells.J Clin Invest. 2004; 114: 795-804Crossref PubMed Scopus (445) Google Scholar, 34.Maeshima A. Yamashita S. Nojima Y. Identification of renal progenitor-like tubular cells that participate in the regeneration processes of the kidney.J Am Soc Nephrol. 2003; 14: 3138-3146Crossref PubMed Scopus (235) Google Scholar These label-retaining cells have been implicated in organ repair following renal ischemia and unilateral urinary tract obstruction.35.Yamashita S. Maeshima A. Nojima Y. Involvement of renal progenitor tubular cells in epithelial-to-mesenchymal transition in fibrotic rat kidneys.J Am Soc Nephrol. 2005; 16: 2044-2051Crossref PubMed Scopus (75) Google Scholar These studies also suggest that while initially the number of label-retaining cells increases, this population is ultimately depleted in the long-term indicating their activation and proliferation in response to injury. In this study, we identified a subpopulation of cells abundant in the inner medulla of the fetal monkey kidney that expresses diffuse SMA and the intercalated cell marker, CAII. Although previous studies with postnatal human, rabbit, and rat kidneys have suggested the complete absence of intercalated cells in inner medullary and papillary CDs,36.Kaissling B. Kriz W. Structural analysis of the rabbit kidney.Adv Anat Embryol Cell Biol. 1979; 56: 1-123Crossref PubMed Google Scholar, 37.LeFurgey A. Tisher C.C. Morphology of rabbit collecting duct.Am J Anat. 1979; 155: 111-124Crossref PubMed Scopus (66) Google Scholar, 38.Myers C.E. Bulger R.E. Tisher C.C. et al.Human ultrastructure. IV. Collecting duct of healthy individuals.Lab Invest. 1966; 15: 1921-1950PubMed Google Scholar our data shows that the normal fetal monkey kidney contains 18% SMA+/CAII+ cells throughout the CDs of the entire inner medulla. As acid–base regulation is not yet an essential function of fetal kidneys, the abundance of these cells suggests that they are less likely to be differentiated intercalated cells, but may represent the CD epithelial progenitors which are destined to become principal cells or intercalated cells as needed postnatally. In addition to retaining the ability to undergo EMT, these putative progenitors are morphologically distinct from their neighboring columnar epithelia and are non-proliferative. Although these cells did not proliferate in response to injury, we did observe an increase in proliferation in the principal cell and myofibroblast populations. These data suggest that the progenitors may commit to epithelial or myofibroblastic differentiation and then proliferate to expand these cell populations. Furthermore, these data also revealed that SMA+/CAII+ cell numbers in the CD of the obstructed kidney decreased from 18 to 8% in severely dilated, fully collared CDs (generally representing two or less cells per duct). This decrease was not associated with an increase in epithelial apoptosis (Figure 7) as shown by TUNEL analysis. Intriguingly, many of the most severely injured ducts exhibited a complete depletion of the SMA+/CAII+ cell population. These results are consistent with the observation of the depletion of renal medullary progenitor cells in a transient renal ischemia model.33.Oliver J.A. Maarouf O. Cheema F.H. et al.The renal papilla is a niche for adult kidney stem cells.J Clin Invest. 2004; 114: 795-804Crossref PubMed Scopus (445) Google Scholar It is possible that this depletion may impair the potential for further CD cell renewal and repair following prolonged injury such as occurs with sustained obstruction, and could result in attenuation of the epithelium, epithelial apoptosis, and denuding of the basement membrane. In conclusion, these findings suggest that these cells may represent a progenitor population within the CD epithelium that responds to tubular dilation and injury in two distinct ways: (1) by clonal expansion of principal cells to maintain epithelial integrity and (2) by committing to a myofibroblastic phenotype through EMT and forming peritubular collars in response to increased luminal pressure (Figure 9). Commitment to these pathways appears to be at the cost of the progenitor population, which is significantly depleted following injury. This depletion may impact the postnatal formation of intercalated cells with not only functional consequences such as defects in bicarbonate and water re-absorption, but also may result in a reduced capacity of the kidney to respond to injury. Loss of repair potential, in combination with the observed decrease in glomerular number observed with this disorder, may contribute to a predisposition to chronic kidney disease in later life. All animal procedures conformed to the requirements of the Animal Welfare Act (USA), and all protocols were approved by the Institutional Animal Care and Use Committee at the University of California, Davis and the Committee on Animal Care at University of British Columbia before implementation. Using an ultrasound-guided technique, unilateral ureteric obstruction was performed in fetal monkeys by the injection of alginate spheres at 70 days gestation.8.Tarantal A.F. Han V.K. Cochrum K.C. et al.Fetal rhesus monkey model of obstructive renal dysplasia.Kidney Int. 2001; 59: 446-456Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Gravid adult rhesus monkeys were selected for this study (N=3). The animals were scheduled for hysterotomy near term (150 days of gestation; term=165±10 days) and complete fetal tissue harvests were performed. Representative sections of the right and left kidneys were processed for immunohistochemistry, placed in OCT, cortex and medulla were separated and snap-frozen for future mRNA studies, and immersed in 10% buffered formalin for histopathology. Comparable studies were conducted in control specimens of similar gestational age (N=3). Total RNA was isolated from cortex or medulla of control, obstructed, and contralateral kidneys using the RNeasy Mini Kit protocol (Qiagen, Valencia, CA, USA) with RNA-free DNase treatment (Ambion, Austin, TX, USA) before treatment with PowerScript reverse transcriptase (BD Biosciences Clontech, Palo Alto, CA, USA). For quantitative PCR analysis, custom-designed primers and 6Fam- or Vic-labeled TaqMan MGB probes (ABI, Foster City, CA, USA; Table 1) were designed for each gene target. All samples were amplified in triplicate on a 7000 Sequence Detection System using TaqMan Universal PCR MasterMix (ABI). Optimization and relative quantification of the assay was determined as described in User's Bulletin #2 (ABI). Briefly, each set of probes and primers was assayed individually to ensure that their amplification efficiencies were <90%. Lastly, each primer and probe set was validated as per User Bulletin #2 to ensure accurate results for relative quantification via the comparative method. Normalization of RNA quantity between samples was accounted for using the expression of the housekeeping gene cyclophilin A.39.de Kok J.B. Roelofs R.W. Giesendorf B.A. et al.Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes.Lab Invest. 2005; 85: 154-159Crossref PubMed Scopus (451) Google Scholar The fold difference of each gene between samples was then determined relative to control kidneys.Table 1Custom designed, Macaca mulatta-specific probe and primer setsGenePrimer/ProbeSequence (Probe reporter dye)E-cadherinForward primer5′-CAGTGACCAACGATGGCATT-3′MGB Probe5′-(6FAM)-AAACAGCAAAGGGC-3′Reverse primer5′-GCTTGGCCTCAAAATCCA-3′VimentinForward primer5′-AACCTGACCGAGGACATCATG-3′Probe5′-(6FAM)-TCCGGGAGAAATT-3′Reverse primer5′-TTCTCGGCTTCCTCTCTCTGAA-3′Pan-TGF-βForward primer5′-GCCCACTGCTCCTGTGACA-3′MGB Probe5′-(VIC)-CAAAGATAACACACTGCAAGT-3′Reverse primer5′-CGGTAGTGAACCCGTTGATGT-3′CyclophilinForward primer5′-GTCGACGGCGAGCCTTT-3′MGB probe5′-(VIC)-CTCCTTCGAGCTGTTTG-3′Reverse primer5′-CTGCTGTCTTTGGAACCTTGTCT-3′TGF-β, transforming growth factor-β.Probes are MGB Probes™ (ABI) and are dual-labeled with a non-fluorescent quencher and either 6FAM or VIC reporter dyes for use with 7000 Sequence Detection System (ABI). Open table in a new tab TGF-β, transforming growth factor-β. Probes are MGB Probes™ (ABI) and are dual-labeled with a non-fluorescent quencher and either 6FAM or VIC reporter dyes for use with 7000 Sequence Detection System (ABI). Formaldehyde-fixed, paraffin-embedded tissue sections were first deparaffinized in xylene and dehydrated by passage through graded ethanols. The sections were then exposed to 40 min of heat-induced antigen retrieval in pre-heated 10 mM citrate buffer (pH 6.0) using a domestic food steamer. After heat-induced antigen retrieval, the sections were cooled for 40 min and incubated at room temperature for 1 h in blocking buffer with 2% of either goat or horse normal serum. Excess blocking buffer was shaken off of the sections and the diluted primary antibodies were added immediately and incubated overnight at 4°C. The primary antibodies used and their dilutions are as follows: mouse anti-Vimentin and anti-fibroblast surface protein (1:100; Sigma, St Louis, MO, USA); rabbit anti-cytokeratin and mouse anti-Ki-67 (1:200 and 1:100, respectively; Dako, Capinteria, CA, USA); anti-E-cadherin (1:30; Neomarkers, Fremont, CA, USA); anti-type IV collagen (1:50; Fitzgerald, Concord, MA, USA); and goat anti-aquaporin 2 and anti-carbonic anhydrase II (1:50 and 1:200, respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Next, sections were incubated for 1 h with fluorescently conjugated secondary antibodies diluted in 0.1 M phosphate-buffered saline containing 0.5% sodium azide. The secondary antibodies used and their dilutions are as follows: goat anti-mouse-IgG (H+L) AlexaFluor-350 and -488 conjugates (1:50 and 1:100, respectively), goat anti-rabbit-IgG (H+L) AlexaFluor-350 and -568 conjugates (1:50 and 1:100, respectively), and donkey anti-goat-IgG (H+L) Alexafluor-568 (1:100; Invitrogen, Carlsbad, CA). For three-color staining, an additional 1 h incubation with mouse anti-αSMA-FITC conjugate (1:50; Sigma) was performed following incubation with the relevant secondary antibodies. In some sections, TUNEL analysis was performed using the ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA) to assess the occurrence of apoptosis. Finally, sections were mounted in the Prolong Gold mounting media with or without the nuclear stain 4′,6-diamidino-2-phenylindole (Invitrogen). Immunofluorescent microscopy was performed on an epifluorescence microscope (Leica, Wetzlar, Germany). Images were captured with a Retiga 1300i camera with RGB filter wheel (QImaging, Surrey, Canada) and were processed and merged using Openlab software (Improvision, Lexinton, MA, USA). The percentage of SMA+/CAII+ cells was determined by manually counting CD cells in several separate fields from control, contralateral, and obstructed kidneys. Obstructed CDs were classified into mild (collar less than 50% enveloped), moderate (collars 50–95% enveloped), and severe (collars 96–100% enveloped) based on the degree of peritubular collar formation. The frequency of Ki-67 immunoreactivity in obstructed medullary segments exhibiting significant dilation was compared against equivalent CDs of control kidneys. For determination of the frequency of Ki-67 interstitial Ki-67 immunoreactivity, only staining of interstitial cells immediately adjacent to CDs was included and expressed as a percentage of the number of CD epithelial cells comprising the associated CD. Separate qPCR reactions were run for each sample. All results were expressed as mean±s.e.m. Statistical analysis was performed using the Student's t-test. A P<0.05 was considered statistically significant. For comparisons of SMA+/CAII+ cell numbers and of Ki-67 immunoreactivity between obstructed and control kidneys, statistical significance was determined by applying a two-tailed unequal variance t-test. Results for each classification of obstructed ducts and for all obstructed ducts when pooled was significant to P<0.05 when compared with controls. This work was supported by grants from the Kidney Foundation of Canada (DGM), the Canadian Institutes of Health Research (DGM), and NIH Grant nos. HL085036 (AFT) and RR00169 (AFT)." @default.
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• W2043343174 title "Collecting duct epithelial–mesenchymal transition in fetal urinary tract obstruction" @default.
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• W2043343174 doi "https://doi.org/10.1038/sj.ki.5002457" @default.
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