Introduction 1998 2001 2004 2005 2007 2005 2007 2007 1997 2005 2007 2007 2007 2006 2003 2004 2007 2007 The aim of our present investigation was to study the morphology of cortical peritubular interstitium, with a focus on the resident fibroblasts, during the first 4 days following ureteral ligature in rats. UUO is a commonly used experimental model for inducing renal fibrosis and is furthermore of outmost clinical relevance. We choose to study the early events after ureteral ligature because in early stages the identification of cell types and their putative phenotypical changes might be better traceable than in advanced stages of fibrosis. We include in our study also the early effects of UUO on tubular epithelia. We performed our investigation by light and electron microscopy, as well as immunofluorescence, and we included morphometric quantification of tissue components and analysis of changes in the proliferation rates of interstitial cells. We demonstrate that resident fibroblasts progressively gain the phenotype of myofibroblasts and massively increase their rate of cell division during the first days after ureteral ligature. Our data suggest that resident peritubular fibroblasts transform to myofibroblsts and participate importantly in the development of renal fibrosis. Methods Experimental design For induction of UUO male 20 Sprague–Dawley rats weighing 160 ± 3 g were anesthetized using isoflurane and the abdominal cavity was opened by a midline incision. The right ureter was tied with 4/0 silk 3–5 mm below the renal hilum. The rats of the control group (four rats) were also laparatomized, the ureter exposed, but no ligature was made. After closure of the abdomen the animals were returned to the cage with free access to standard lab chow and water ad libitum. The experimental procedure conformed to the Institutional Guidelines of Experimental Animal Care and Use and was approved by the Veterinary office of the Kanton Zürich. Tissue fixation 1989 Light and electron microscopy From the unligated left and from the ligated right kidney large tissue blocks extending from the renal capsule to at least the upper third of the inner zone were immersed for at least 24 h in the fixative solution described above to which 1% glutardialdehyde was added. This tissue was then embedded into epoxy resin and used for light and electron microscopy. Semithin (1 μm) sections and ultrathin (80 nm) sections were cut with an ultramicrotome. Semithin sections were stained with 1% methylene blue and 1% azure II and were studied with a Polyvar microscope (Reichert Jung, Vienna, Austria). Ultrathin sections were postfixed with osmium tetroxyde and contrasted with uranyl acetate and studied with a CM100 Philips electron microscope. Immunofluorescence microscopy 1 Table 1 List of primary antibodies Primary antibodies Host Source S100A4 Rabbit Dako, Glostrup, Denmark Ecto-5′nucleotidase (5′NT) Rabbit M. Le Hir Ecto-5′nucleotidase (5′NT) Mouse BD Biosciences, Franklin Lakes, NJ, USA Alpha smooth muscle actin (αSMA) Mouse Dako, Glostrup, Denmark Alpha smooth muscle actin (αSMA) Rabbit Abcam, Cambridge, UK Rat MHC class II Mouse Clone OX 6 Phospho-S6-kinase Rabbit Cell Signaling, Danvers, MA, USA Collagen Type I Mouse MD Biosciences, Zurich, Switzerland Vimentin Mouse Chemicon, Temecula, CA, USA Sections were studied by epifluorescence with a Polyvar microscope (Reichert Jung, Vienna, Austria), and digital images were acquired with a CCD camera. Quantitative evaluation In order to quantify changes in the structural composition of the renal cortex and to assess the mitotic rates of interstitial cells within this area, a morphometrical analysis was undertaken on sections of kidneys from three rats in each of the five experimental groups. 2 The images were overlaid with a 15 × 15 grid to assign the histological structures covered by each of the 225 intersections to one of five categories, namely tubules, capillaries, interstitium, renal corpuscles and large vessels. The fractional areas of these histological structures were defined as the ratio of the number of category-specific intersections to the total number of intersections (225). Furthermore, the total numbers of peritubular interstitial cells in the image were counted and the numbers of mitotic cells in the cortical interstitium were recorded. This allowed calculating the fraction of mitotic cells in relation to all interstitial cells. The identity of each mitotic cell in the cortical interstitium was attributed to one of the two categories “fibroblasts” or “mononuclear cells” (dendritic cells, infiltrating leucocytes) independently by two investigators. Mitotic cells that could not be assigned to one of these two categories or that were classified differently by the two investigators recorded as ‘non-identified’. Statistics n Results Macroscopy The ligature of the right ureter resulted invariably in each rat in a large expansion of the renal pelvis within the renal sinus and of the ureter above the ligature. The enlargement appeared more prominent at the first and second day after the operation than after the third and fourth day. The fluid contained in the expanded renal pelvis was clear up to the second day and appeared trouble and viscous the third and fourth day. In sham operated rats no expansion of the renal pelvis was observed. Microscopy Tubular epithelia 1 2 Fig. 1 a e b d f h a d d h asterisk D P arrow arrowhead Bar a d e h  Fig. 2 a b c b c d c arrowheads Bars a b c d  Starting from day 1 the most striking feature in tubules of kidneys with occluded ureter was the heterogeneity of tubular diameter. Proximal tubules 1 1 1 2 2 1 2 Distal segments and collecting ducts 1 1 3 1 Fig. 3 a b b c b arrowheads Bars a b d  Quantitative estimation of changes of cortical tubular volume and peritubular spaces 4 4 Fig. 4 n  1 4 4 Cortical peritubular interstitium 1989 1990 1994 1996 1994 2005 2006 1994 Phenotypical changes of peritubular fibroblasts 1 1996 1 5 5 6 1994 1996 Fig. 5 a b a arrow b arrows dots arrowheads lower right and upper left corners Bars a b  Fig. 6 a c a arrows b c d arrowheads arrows Bars d  4 1996 1 5 1 5 6 6 6 6 6 6 1989 1994 1994 1997 1998 7 8 7 8 8 14 8 Fig. 7 a b c 5′NT red channel αSMA green channel blue channel a a b c d f Bar a c d f  Fig. 8 5′NT red channel αSMA green channel blue channel a b c Bar a–c  Fig. 9 Renal cortex after green channel Col I red channel blue channel a a c 13 d f Bar  Fig. 10 a e f G G d f asterisks f Bars a c d f  Fig. 11 red channel green channel G arrow  Fig. 12 green channel G insert Bar insert  Fig. 13 a prophase upper left corner b arrowheads 9 c d Bars Bars  Fig. 14 green channel red channel blue channel a Bar  Accumulation of collagen type I in the peritubular interstitium Accumulation of collagen I (Col I) in the interstitial space is a hallmark of renal fibrosis. Collagen type I is produced by (myo)fibroblasts. By immunofluorescence Col I was virtually absent in the cortical peritubular interstitial space of controls. A modest immunofluorescence was apparent along the tubular profiles. 9 9 5 13 Increases in mononuclear cells in the cortical interstitium 1994 1996 dendritic cells 10 1994 1996 10 FSP1/S100A4-positive cells (FSP1/S100A4) FSP1/S100A4 2002 11 12 2005 10 12 Proliferation of interstitial cells 1 13 14 14 2007 15 13 Fig. 15 a b n  13 14 Mitotic figures in tubules and in capillaries were rare in control kidneys. They were easily found in occluded kidneys on day 2, and less so on days 1, 3 and 4. Quantification was not carried out. Discussion The main purpose of the present study was to investigate the early response to ureter obstruction of resident fibroblasts in the renal cortex. Two major alterations were observed, starting from the first day after ligature of the ureter in the rat. Firstly, ultrastructural features and the expression of specific proteins suggested the progressive acquisition by virtually all fibroblasts of characteristic features of myofibroblasts. Because the transformation did not affect all fibroblasts simultaneously a large heterogeneity in the phenotypes of fibroblasts was present. Secondly, the incidence of mitosis of fibroblasts increased dramatically, affecting the various phenotypes of fibroblasts. Transformation of resident fibroblasts to the myofibroblast phenotype 1994 1996 2005 2005 2007 2007 1999 1994 1996 2005 1996 2005 2007 Controversial identity of FSP1/S100A4-expressing cells 2002 2002 2002 2002 2000 2003 2005 Proliferation of interstitial cells 2007 2007 2005 2000 1999 1999 2001 Changes of tubular morphology 2007 2001 2008 2001 2003 2006 2008 2003 2005 2005 2000 2005 2008 2005 2003 2002 2007 2007 In conclusion, during the first days following ureter ligature the resident peritubular fibroblasts differentiate into myofibroblasts and they proliferate strongly. These events are likely essential to fibrosis in UUO.