A Rat Model of Progressive Chronic Renal Failure Produced by Microembolism
1999; Elsevier BV; Volume: 155; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)65239-x
ISSN1525-2191
AutoresMasato Kimura, Takayuki Suzuki, Akira Hishida,
Tópico(s)Metabolism and Genetic Disorders
ResumoWe report a new model of chronic progressive renal failure in rats, produced by a single injection of microspheres (20 to 30 μm in diameter) into the left renal artery after right nephrectomy. Significant proteinuria appeared after 4 weeks, followed by hypoalbuminemia and hypercholesterolemia, in rats that received approximately 5 × 105 microspheres (0.8 mg). Renal function partially recovered by 4 weeks after nephrectomy and injection from postoperative dysfunction, but deteriorated again 12 weeks after operation. In the early stage, histologic examination showed tubules with cuff-like thickening of basement membranes scattered among apparently intact tubules. Many epithelial cells in the atrophic tubuli were immunoreactive for proliferating cell nuclear antigen (PCNA). Dilated tubules became apparent several weeks after development of tubular atrophy, most likely representing distal tubules. Dilated tubuli were mostly negative for the proliferation marker. These results showed similarity to findings in human chronic renal failure and strongly suggested that tubular atrophy and dilation in chronic tubulointerstitial lesions differ in pathogenesis. This new model of renal failure induced by microembolism should be useful for studying the interaction between normal and diseased tissue elements in histologically heterogenous lesions as well as the pathogenesis of interstitial fibrosis in disturbance of microcirculation. We report a new model of chronic progressive renal failure in rats, produced by a single injection of microspheres (20 to 30 μm in diameter) into the left renal artery after right nephrectomy. Significant proteinuria appeared after 4 weeks, followed by hypoalbuminemia and hypercholesterolemia, in rats that received approximately 5 × 105 microspheres (0.8 mg). Renal function partially recovered by 4 weeks after nephrectomy and injection from postoperative dysfunction, but deteriorated again 12 weeks after operation. In the early stage, histologic examination showed tubules with cuff-like thickening of basement membranes scattered among apparently intact tubules. Many epithelial cells in the atrophic tubuli were immunoreactive for proliferating cell nuclear antigen (PCNA). Dilated tubules became apparent several weeks after development of tubular atrophy, most likely representing distal tubules. Dilated tubuli were mostly negative for the proliferation marker. These results showed similarity to findings in human chronic renal failure and strongly suggested that tubular atrophy and dilation in chronic tubulointerstitial lesions differ in pathogenesis. This new model of renal failure induced by microembolism should be useful for studying the interaction between normal and diseased tissue elements in histologically heterogenous lesions as well as the pathogenesis of interstitial fibrosis in disturbance of microcirculation. Renal function is thought to deteriorate spontaneously and progressively after the number of functioning nephrons has decreased below a certain threshold. Thus, a common process appears to underlie functional deterioration in various renal diseases, irrespective of cause. The hyperfiltration theory1Brenner BM Meyer TW Hostetter TH Dietary protein intake and progressive nature of kidney disease: the role of hemodynamically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in aging, renal ablation, and intrinsic renal disease.N Engl J Med. 1982; 307: 652-659Crossref PubMed Scopus (147) Google Scholar proposes nonimmunological mechanisms underlying renal functional deterioration and is supported by many observations in animal models in which nephron numbers are reduced by simple excision of tissue2Chanutin A Ferris EB Experimental renal insufficiency produced by partial nephrectomy.Arch Intern Med. 1932; : 767-787Crossref Scopus (210) Google Scholar, 3Koletsky S Goodsitt AM Natural history and pathogenesis of renal ablation.AMA Arch Pathol. 1960; 69: 654-662Google Scholar, 4Kaufman JM DiMeola HJ Siegel NJ Lytton B Kashgarian M Hayslett J Compensatory adaptation of structure and function following progressive renal ablation.Kidney Int. 1974; 6: 10-17Crossref PubMed Scopus (120) Google Scholar or by ligation of specific branches of the renal artery.5Pearce RM The influence of kidney substance upon nitrogenous metabolism.J Exp Med. 1908; 10: 632-644Crossref PubMed Scopus (4) Google Scholar, 6Carrel A Note on the production of kidney insufficiency by reduction of the arterial circulation of the kidney.Proc Soc Exper Biol Med. 1909; 6: 107-108Crossref Scopus (9) Google Scholar, 7Olson JL Hostetter TH Rennke HG Brenner BM Venkatachalam MA Altered glomerular permselectivity and progressive sclerosis following extreme ablation of renal mass.Kidney Int. 1982; : 112-126Crossref PubMed Scopus (295) Google Scholar In these models, the remaining kidney is histologically normal at the beginning of progressive impairment of renal function. However, in various severe human renal diseases, the decrease in the number of functioning nephrons is associated with marked tubulointerstitial changes, and relatively undamaged nephrons are admixed with those that are extensively damaged until the intact nephrons spontaneously deteriorate. These features are shared by both immunologically and nonimmunologically mediated kidney diseases. Progressive chronic renal failure is characterized histologically by tubulointerstitial and vascular scarring as well as glomerular scarring. Renal dysfunction and outcome correlate better with tubulointerstitial scarring than with glomerular scarring. The extent of tubulointerstitial scarring sometimes exceeds that of glomerular sclerosis in rats with remnant kidneys,8Kleinknecht C Laouari D The influence of dietary components on experimental renal disease.Contemp Issues Nephrol. 1986; 14: 17-35Google Scholar in nephrotoxic serum nephritis,9El Nahas AM Zoop SN Evans DJ Rees AJ Chronic renal failure after nephrotoxic nephritis in rats: Contribution to progression.Kidney Int. 1987; 36: 173-180Crossref Scopus (25) Google Scholar and in adriamycin nephropathy.10Bertani T Cutillo F Zoja C Broggini M Remuzzi G Tubulo-interstitial lesions mediate renal damage in adriamycin glomerulopathy.Kidney Int. 1986; 30: 488-496Crossref PubMed Scopus (182) Google Scholar Tubular cells in damaged kidneys are known to express or secrete various cytokines and growth factors.11Frank J Enbler-Blum G Rodemann HP Müller GA Human renal tubular cells as a cytokine source: PDGF-B, GM-CSF and IL-6 mRNA expression in vitro.Exp Nephrol. 1993; 1: 26-35PubMed Google Scholar, 12Gröne H-J Simon M Gröne EF Expression of vascular endothelial growth factor in renal vascular disease and renal allografts.J Pathol. 1995; 177: 259-267Crossref PubMed Scopus (117) Google Scholar Furthermore, tubular epithelial cells are capable of secreting interstitial collagens,13Haverty TP Kelly CJ Hines WH Amenta PS Watanabe M Harper RA Kefalides NA Neilson EG Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis.J Cell Biol. 1988; 107: 1359-1367Crossref PubMed Scopus (276) Google Scholar proteoglycans, and fibronectin.14Humes HD Nakamura T Cieslinski DA Miller D Emmons RV Border WA Role of proteoglycans and cytoskeleton in the effects of TGF-β1 on renal proximal tubule cells.Kidney Int. 1993; 43: 575-584Crossref PubMed Scopus (58) Google Scholar Strutz et al15Strutz F Caron R Tomaszewski J Fumo P Ziyadeh F Neilson EG Transdifferentiation: a new concept in renal fibrosis (abstr.).J Am Soc Nephrol. 1994; 5: 819Google Scholar have shown in experimental models of renal disease that certain tubular cells expressed FSP1, a specific marker for fibroblasts, which might indicate some degree of transformation of tubular epithelial cells into fibroblasts. In addition, Nadasdy et al16Nadasdy T Laszik Z Blick KE Johnson DL Silva FG Tubular atrophy in the end-stage kidney: A lectin and immunohistochemical study.Hum Pathol. 1994; 25: 22-28Abstract Full Text PDF PubMed Scopus (110) Google Scholar have detected a high proliferation index in the atrophic tubules of human end-stage kidneys with interstitial fibrosis. Thus, the tubular cells in damaged kidneys may play a role in the progression of renal disease. Interactions between damaged and relatively undamaged nephrons has been neglected in studies of progression of end-stage renal disease, partly because of lack of an appropriate animal model. We now present a model of nonimmunological progressive renal failure produced by a single injection of microspheres, in which relatively undamaged nephrons mingle with severely damaged ones beginning in the early stage of renal disease. This lesion distribution could overcome the drawbacks of conventional ablation models discussed above. In addition, the microembolization model should be useful in the study of mechanisms of progression of damage specifically related to disturbances of the renal microcirculation, such as arteriolosclerosis. Renal failure was induced by arterial injection of microspheres into the remaining kidney of nephrectomized rats. Male Wistar rats 12 weeks of age were obtained from SLC (Hamamatsu, Japan) and were allowed free access to standard laboratory chow and water. Under anesthesia with sodium pentobarbital (40 mg/kg body weight, i.p.), the right kidney was removed and microspheres (acryl beads, 20 to 30 μm in diameter; kindly provided by Dr. Takabayashi, Hamamatsu College, University of Shizuoka) suspended in 0.5 ml of physiological saline were injected slowly into the aorta through a 27-gauge needle placed immediately caudal to the ostium of the left renal artery. During microsphere injection, the aorta caudal to the site of needle insertion as well as the anterior mesenteric and celiac arteries were clamped to ensure flow of microspheres into the left renal artery. After injection, the inserted needle was removed and the site of aortic puncture was gently compressed with a ball of cotton for approximately 2 minutes to stop bleeding. Blood flow through the left renal artery was maintained throughout this procedure. Animals were grouped according to number of injected microspheres: group 1 received saline without microspheres (control); group 2 received 0.8 mg of microspheres (approximately 5 × 105 per rat); group 3 received 0.4 mg of microspheres; group 4 received 0.2 mg of microspheres; and group 5 received 0.1 mg of microspheres. To evaluate the obstructed vascular volume resulting from microsphere injection, we measured renal blood flow before and shortly after injection in five rats in group 2 with an electromagnetic flowmeter (MFV-1200; Nihon Koden, Tokyo) connected to a flow probe (FJ-007TS, ϕ 0.7 mm; Nihon Koden) placed upon the left renal artery. Body weight was measured before and 4, 8, and 12 weeks after surgery in all groups. The weight of the left kidney was determined after the rats were killed. Blood pressure was measured in conscious animals by the tail cuff method with a warmed restraining device (PS-300; Riken Kaihatsu, Tokyo) before and 4, 8, and 12 weeks after surgery in groups 1 and 2. Urinary excretion of albumin and serum concentrations of creatinine, albumin, and total cholesterol were determined at the same intervals for all groups. The animals were kept in metabolic cages while 24-hour urine specimens were collected. Urinary albumin was assayed by single radial immunodiffusion method using rabbit antiserum to rat albumin. For planning the histological study, we made a preliminary examination of all groups for 12 weeks with a small number of animals. We found that the histological changes in group 2 were representative ones and in other groups, abnormal histological findings were scarce, especially in the early stage. Therefore, we focused our histological study on group 1 and 2. Five or six rats were killed at 2, 4, 8, and 12 weeks after surgery from each of groups 1 and 2, and at 12 weeks for groups 3, 4, and 5. Before killing, rats were anesthetized with pentobarbital (40 mg/kg body weight, i.p.) and the kidneys were perfused with 10 ml of cold saline through a 23 gauge needle placed in the abdominal aorta and connected to a bottle of saline placed 110 cm higher. The left kidney was removed, fixed in 10% buffeted formalin, embedded in paraffin, sliced into 2-μm-thick sections, and stained with periodic acid-Schiff (PAS) or Masson's trichrome (MT) stain for light microscopic observation. The point-counting method17Weibel ER Principles and methods for the morphometric study of the lung and other organs.Lab Invest. 1963; 12: 131-155PubMed Google Scholar was employed for morphometric evaluation of interstitial fibrosis, tubular basement membrane thickening, dilatation of tubular lumen, and cast formation. Twenty photographs with a final magnification of ×100 were analyzed from each kidney. Interstitial fibrosis was assessed by counting points in the cortical area stained green by the trichrome stain including the tubular basement membrane, except in the area immediately surrounding large vessels. Thickened basement membranes, which were discerned by their acellular homogeneous character and circular shape, were separately counted again. Areas of tubular lumens with and without casts were measured to assess dilation. Areas of casts were measured separately. All areas above were expressed as a percentage of total cortical area. Another portion of the kidney was fixed in 4% paraformaldehyde in phosphate-buffeted saline and sections 4 μm thick were prepared for histochemical staining with a mouse monoclonal antibody reactive to proliferating-cell nuclear antigen (PCNA; Oncogene Science, Cambridge, MA), rabbit polyclonal antibody to cytokeratin (Dako, Japan), and sheep polyclonal antibody to human Tamm-Horsfall glycoprotein (THP; Chemicon International Inc., Temmecula, CA). Biotin-labeled peanut agglutinin (PNA; Biomeda, Foster City, CA) was used for detection of T antigen by the avidin-biotin-horseradish peroxidase method (Histofine SAB-PO kit; Nitirei, Tokyo). Data are expressed as means ± SE. The statistical significance of differences was determined by analysis of variance (ANOVA) and either an unpaired or a paired t-test. A P value of <0.05 was considered statistically significant. After an initial decrease in the first 2 weeks after surgery (not shown), the body weight of rats in all groups gradually increased (Table 1). The body weights of surviving rats in group 2 (receiving 0.8 mg of microspheres) did not increase after 8 weeks, possibly because of the onset of renal failure. The wet weight of the kidney remained virtually identical in groups 1 and 2 through 12 weeks after surgery, indicating no further increase in hypertrophy by microembolism in group 2 (Figure 1).Table 1Body Weight (g) of Animals by Group at Various Times after SurgeryTimeGroup0 weeks4 weeks8 weeks12 weeks1 (control)271 ± 3.6289 ± 5.7311 ± 6.0d: P < 0.01 vs. 0 weeks and group 1, respectively.332 ± 7.0d: P < 0.01 vs. 0 weeks and group 1, respectively.2 (0.8 mg of microspheres)292 ± 3.7e: P < 0.05 vs. 0 weeks, group 1, and group 3, respectively.299 ± 4.9d: P < 0.01 vs. 0 weeks and group 1, respectively.330 ± 6.0d: P < 0.01 vs. 0 weeks and group 1, respectively.324 ± 12.9d: P < 0.01 vs. 0 weeks and group 1, respectively.3 (0.4 mg of microspheres)287 ± 4.1b: P < 0.05 vs. 0 weeks, group 1, and group 3, respectively.313 ± 3.6d,e: P < 0.01 vs. 0 weeks and group 1, respectively.335 ± 10.2a,b,c: P < 0.05 vs. 0 weeks, group 1, and group 3, respectively., d,e: P < 0.01 vs. 0 weeks and group 1, respectively.347 ± 14.8d: P < 0.01 vs. 0 weeks and group 1, respectively.4 (0.2 mg of microspheres)285 ± 4.4308 ± 8.6a,b,c: P < 0.05 vs. 0 weeks, group 1, and group 3, respectively., d,e: P < 0.01 vs. 0 weeks and group 1, respectively.340 ± 13.3a: P < 0.05 vs. 0 weeks, group 1, and group 3, respectively.366 ± 7.6a,b,c: P < 0.05 vs. 0 weeks, group 1, and group 3, respectively., d,e: P < 0.01 vs. 0 weeks and group 1, respectively.5 (0.1 mg of microspheres)282 ± 5.2307 ± 4.9a,b,c: P < 0.05 vs. 0 weeks, group 1, and group 3, respectively., d,e: P < 0.01 vs. 0 weeks and group 1, respectively.345 ± 6.0d,e: P < 0.01 vs. 0 weeks and group 1, respectively.360 ± 3.8a,b,c: P < 0.05 vs. 0 weeks, group 1, and group 3, respectively., d,e: P < 0.01 vs. 0 weeks and group 1, respectively.a,b,c : P < 0.05 vs. 0 weeks, group 1, and group 3, respectively.d,e : P < 0.01 vs. 0 weeks and group 1, respectively. Open table in a new tab Microsphere injection induced a significant increase in the blood pressure of rats in group 2 at 12 weeks (Table 2). No significant change in blood pressure was apparent in control rats. Microsphere injection induced a dose-dependent increase in urinary excretion of albumin that was statistically significant by 4 weeks in group 2, by 8 weeks in group 3, and by 12 weeks in group 4 (Figure 2). No significant change in the extent of albuminuria was apparent in control rats (group 1) over 12 weeks.Table 2Systolic Blood Pressure (mm Hg) of Animals in Groups 1 and 2 at Various Times after SurgeryTimeGroup0 weeks4 weeks8 weeks12 weeks1 (control)117 ± 4.0115 ± 1.5116 ± 2.7114 ± 1.92 (0.8 mg of microspheres)118 ± 2.4127 ± 3.8132 ± 4.4135 ± 3.7*P < 0.01 vs. 0 weeks and group 1.* P < 0.01 vs. 0 weeks and group 1. Open table in a new tab Injection of microspheres induced a biphasic increase in serum creatinine concentration in groups 2 (Figure 3). The first phase was apparent 2 weeks after injection. The second phase was more marked, with the serum creatinine concentration reaching 2.13 ± 0.46 mg/dL by 12 weeks. The serum concentration of creatinine had increased at 2 weeks in groups 3, 4, and 5 but subsequently demonstrated no further significant change over 12 weeks. A significant decrease in serum albumin concentration was apparent in groups 2 and 3; the rate and extent of the decrease was dependent on the number of injected microspheres (Table 3). The injection of microspheres also significantly increased the total serum cholesterol concentration in groups 2 and 3 compared to group 1. The time courses of the changes in serum concentrations of albumin and cholesterol were similar to that of the increase in urinary excretion of albumin (Figure 2 and Table 3).Table 3Serum Concentrations of Albumin (g/dL) and Total Cholesterol (mg/dL) at Various Times after SurgeryTime (weeks)Group04812Albumin14.53 ± 0.064.56 ± 0.054.61 ± 0.034.34 ± 0.06a: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.24.49 ± 0.074.29 ± 0.08a: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.4.00 ± 0.12f,g: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.3.81 ± 0.15a,b,c,d,e: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively., f,g,h,i,j: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.34.48 ± 0.074.40 ± 0.054.31 ± 0.06a,b,c,d,e: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively., f,g,h,i,j: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.4.11 ± 0.02a,b,c,d,e: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively., f,g,h,i,j: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.44.58 ± 0.084.36 ± 0.08ND4.28 ± 0.0754.44 ± 0.054.50 ± 0.03ND4.30 ± 0.10Total Cholesterol161 ± 372 ± 3a: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively. 70 ± 1a: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively. 74 ± 3f: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.263 ± 395 ± 5a,b,c,d,e: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively., f,g,h,i,j: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.150 ± 20f,g: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.174 ± 19a,b,c,d,e: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively., f,g,h,i,j: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.360 ± 387 ± 4a,b,c,d,e: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively., f,g,h,i,j: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively. 90 ± 6a,b,c,d,e: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively., f,g,h,i,j: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.117 ± 12a,b,c,d,e: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively., f,g,h,i,j: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.459 ± 366 ± 3ND 75 ± 3a: P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.564 ± 570 ± 3ND 81 ± 5f: P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.a,b,c,d,e : P < 0.05 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively.f,g,h,i,j : P < 0.01 vs. 0 weeks, group 1, group 3, group 4, and group 5, respectively. Open table in a new tab Injection of 0.8 mg of microspheres in group 2 resulted in a rapid decrease in renal blood flow to approximately 19% of the preinjection value (3.46 ± 0.18 to 0.66 ± 0.15 mL/min); blood flow then gradually increased to 34% of the preinjection value (1.18 ± 0.07 mL/min) by 15 minutes and remained stable thereafter. The most impressive histological abnormalities 2 to 4 weeks after injection of 0.8 mg of microspheres in group 2 were located predominantly in the interstitium (Figure 4A). At this early stage, atrophic tubules and a small number of dilated tubules were evident, both admixed with normal tubules. The principal feature of atrophic tubules was a cuff-like, thickened basement membrane surrounding the tubular epithelial cells (Figure 4A). This change was not apparent in dilated tubules. In the later stage, the kidney showed an increase in interstitial fibrosis and development of large dilated tubules with flattened epithelial cells and proteinaceous casts in their lumens (Figure 4B). Injected microspheres were trapped in small arterioles and in the glomerular capillary lumen near the vascular pole (Figure 4C). Necrotic areas, however, were detected in only a small superficial portion of the cortex. The limited extent of the necrotic lesions in the cortex might have resulted from the disappearance of established ones before 4 weeks. More necrosis may have been evident sooner after the injection. For some glomeruli containing trapped microspheres, slight ischemic change was apparent in neighboring tufts. In the area without necrotic lesions, ischemic changes, such as wrinkling of capillary walls, were detected in a small percentage of glomeruli. By 8 weeks after microsphere injection, the number of dilated tubules with relatively flattened epithelial cells had increased. The thickness of the cuff-like basement membrane of atrophic tubules had increased further, and some of these tubules were surrounded by infiltrating mononuclear cells (Figure 4, D and E). Cytoplasmic vacuoles of glomerular epithelial cells also was conspicuous in some glomeruli. At 12 weeks after injection, an increase in interstitial fibrosis and development of large dilated tubules and proteinaceous casts in their lumens were evident (Figure 4B). However, almost none of the dilated tubules seen at 8 or 12 weeks were associated with thickening of the tubular basement membrane (Figure 4E). The vacuoles in glomeruli had increased in number and extent (Figure 4F), and some glomeruli had developed segmental sclerosis. The segmentally sclerotic glomeruli also showed an increased amount of PAS-positive mesangial matrix, collapsed capillaries, and PAS-positive inclusions in the glomerular epithelial cells (Figure 4G). Totally obsolescent glomeruli were rarely observed. Morphometric studies confirmed the above impression of progressive histological damage (Figure 5). Interstitial fibrosis increased gradually after embolization in group 2. In contrast, no change in extent of fibrous tissue was apparent in control rats (group 1) during the experimental period. Consequently the extent of fibrous tissue in groups 1 and 2 became statistically significant 12 weeks after surgery. The difference in tubular basement membrane (TBM) thickening was more conspicuous than that of total fibrous tissue; as early as 4 weeks, thickening was evident in group 2. The areas of tubular lumens and casts occupying the lumen increased gradually, becoming significant at 8 weeks after microsphere injection. Although the morphometric score of tubular lumens does not necessarily mean the dilatation of each tubule, it seems to reflect the increase in number of large dilated tubules by light microscopic observation. Atrophic tubules with basement membrane thickening did not show PNA or THP staining (Figure 6, A and C). In contrast, the cytoplasm of dilated tubules were reactive with either PNA, antibody to THP, or both (Figure 6, B and D) which suggested that the dilated tubules were distal tubules.16Nadasdy T Laszik Z Blick KE Johnson DL Silva FG Tubular atrophy in the end-stage kidney: A lectin and immunohistochemical study.Hum Pathol. 1994; 25: 22-28Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 18Ivanyi B Olsen TS Immunohistochemical identification of tubular segments in percutaneous renal biopsies.Histochemistry. 1991; 95: 351-356Crossref PubMed Scopus (12) Google Scholar PNA, which strongly stains the distal tubules, also weakly stains proximal tubular brush border. However, it is not difficult to distinguish them by their intracellular distribution and intensity (Figure 6, E and F). Cytokeratin, which is demonstrable in connecting and collecting tubules,19Hemmi A Mori Y Immunohistochemical and scanning electron microscopic study of cytokeratin distribution in the collecting tubule of the rat kidney.Acta Pathol Jpn. 1990; 40: 307-313PubMed Google Scholar was not immunostained in either atrophic or dilated tubules (data not shown). Numbers of PCNA-positive tubular epithelial cells were increased 2 weeks after microsphere injection, especially in the inner cortex and medulla, but had returned to basal values by 4 weeks (data not shown). During the experimental period, PCNA-positive cells were seen frequently in the atrophic tubules with cuff-like, thickened basement membranes, while only a few positive cells were apparent in intact or dilated tubules (Figure 6, G and H). In control rats, numbers of PCNA-positive cells were slightly increased at 2 weeks, but the increase was not statistically significant. We have described a new model of chronic renal failure induced by injection of microspheres of 20 to 30 μm in diameter. These microspheres occluded the preglomerular arterioles and intraglomerular capillaries near the vascular pole to induce a dose-dependent increase in urinary excretion of albumin and in the serum concentration of creatinine and total cholesterol with a reciprocal decrease in serum albumin. The creatinine concentration increased in group 2 (0.8 mg of microspheres) 12 weeks after embolization. Groups other than group 2 generally did not show significant increases in the creatinine concentration during the experimental period, although we found a significant increase in concentration of creatinine in group 3 (0.4 mg of microspheres) after 20 weeks (1.18 ± 0.11 mg/dl at 20 weeks and 1.64 ± 0.24 mg/dl at 24 weeks; unpublished data). Therefore this model, like the renal mass-reduction model, suggested that proteinuria preceded the increase in serum creatinine concentration. Blood pressure was mildly increased in rats injected with large amounts of microspheres. These experiments are not the first attempt to induce chronic renal failure by injection of small particles. However, most reports published so far have focused on hypertension induced by renal infarction and did not analyze details of histological findings on progression. In addition, particles previously used have been larger than our microspheres or had widely varying sizes; as a consequence, the sizes of arteries affected were different than in the present study.20Apfelbach CW Jensen CR Experimental renal insufficiency in dogs with special reference to arterial hypertension.J Clin Invest. 1931; 10: 162Google Scholar, 21Alexander N Heptinstall RH Poickering GW The effects of embolic obstruction of intrarenal arteries in the rabbit.J Pathol Bact. 1961; 81: 225-237Crossref PubMed Scopus (14) Google Scholar, 22Koletsky S Rivera-Velez JM Renin-angiotensin system in microembolic renal hypertension.Arch Pathol. 1968; 85: 1-9PubMed Google Scholar, 23Solez K Richter GW Microembolic renal disease in rats induced with sephadex.Am J Pathol. 1972; 66: 163-188PubMed Google Scholar Apfelbach and Jensen20Apfelbach CW Jensen CR Experimental renal insufficiency in dogs with special reference to arterial hypertension.J Clin Invest. 1931; 10: 162Google Scholar first reported producing renal failure by injection of charcoal particles into the dog renal artery in 1931. In 1961, Alexander et al21Alexander N Heptinstall RH Poickering GW The effects of embolic obstruction of intrarenal arteries in the rabbit.J Pathol Bact. 1961; 81: 225-237Crossref PubMed Scopus (14) Google Scholar performed embolization of intrarenal arteries (arcuate or interlobular size) with several different particles to study the effect of embolism on renal parenchymal histology, blood pressure, and blood urea level. The typical resulting lesions, large wedge-shaped infarcts, differed from the present ones of small foci of atrophic tubules scattered among intact tubules. Koletsky and Rivera22Koletsky S Rivera
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