Focal-Segmental Glomerulosclerosis The Relationship Between Tubular Atrophy and Segmental Sclerosis

Anatomic Pathology / TUBULAR ATROPHY IN FOCAL-SEGMENL GLOMERULOSCLEROSIS Focal-Segmental Glomerulosclerosis The Relationship Between Tubular Atrophy and Segmental Sclerosis Stephen M. Bonsib, M D Key Words: Focal-segmental glomerulosclerosis; Tubular atrophy; Serial sections Abstract Biopsy specimens with focal-segmental glomerulosclerosis (FSGS) show segmental sclerosis (SS) and tubular atrophy (). The relationship between SS and was studied using serial sections. Based on the location of the SS, 3 and 5 biopsy specimens, respectively, were classified as FSGS, glomerular tip lesion (GTL) or FSGS, not otherwise specified (NOS). The proximal tubule was classified as normal or atrophic. The glomerulus was tracked through serial sections in both directions and classified as normal, SS (graded 1+ -3+ ), or ischemic. Segmental sclerosis was identified in 21 of 24 glomeruli in FSGS, GTL and in 46 of 89 glomeruli in FSGS, NOS; was identified in 13 of 16 glomeruli in FSGS, GTL and 6 of 13 glomeruli in FSGS, NOS, which showed 1+ SS, and in 36 of 38 glomeruli in both forms, which showed 2+ to 3+ SS. Basement membrane disruptions at the glomerular-tubular junction and interstitial expansion with inflammation was encountered. Three biopsy specimens with FSGS, NOS had hypertensive changes and showed in several normal and in all ischemic glomeruli. Chronic tubular injury is present in most glomeruli containing segmental lesions, even small lesions, suggesting that the glomerular and tubular injury may have a common cause or that tubular injury may result from constituent(s) in the glomerular filtrate. Downloaded from https://academic.oup.com/ajcp/article-abstract/111/3/343/175821 on 22 April 218 Tubular atrophy and interstitial fibrosis in renal biopsy specimens have prognostic importance in most forms of glomerulonephritis, including focal-segmental glomerulosclerosis (FSGS), because they indicate irreversible nephron damage, the severity of which correlates with the level of renal insufficiency.1-5 Tubular atrophy is detectable in nephrons in which the glomerulus seems uninvolved by segmental lesions or in which the area of sclerosis seems small. This raises questions about early events in the initiation of injury in the glomerular vs the tubular portion of the nephron and their respective causes. The relationship between proximal tubular atrophy and segmental sclerosis (SS) was explored by using serial sections to identify the extent of SS, and the presence of atrophic changes in the proximal tubule was evaluated at its origin from the glomerulus of interest. In 8 biopsy specimens, 113 glomeruli showing FSGS were evaluated with this approach. Materials and Methods Renal biopsy specimens showing FSGS from 8 adults who underwent biopsy shortly after the detection of significant proteinuria were selected. All biopsy specimens were evaluated by light, immunofluorescent, and electron microscopy to exclude other forms of glomerulonephritis in which SS may develop. No biopsy specimens indicated a collapsing form of FSGS, and no patient had diabetes mellitus. The paraffin blocks were serially sectioned at 3 u.m completely through the block or until at least 15 slides (2 sections per slide) were generated. Alternate sections were stained with periodic acid-schiff stain. Glomeruli were located in which the proximal tubule was identified at its origin with the glomerulus, ie, the tubular pole. The tubule at the tubular pole was evaluated for the Am J Clin Pathol 1999; 111:343-348 3 4 3

Bonsib / TUBULAR ATROPHY IN FOCAL-SEGMENL GLOMERULOSCLEROSIS presence or absence of basement membrane thickening and classified as atrophic or normal, respectively. The degree of tubular atrophy was graded as early or advanced. Early tubular atrophy was recognized by a slightly thickened basement membrane compared with that of adjacent normal tubules. Advanced atrophy showed thickening and an irregular contour or multilayered appearance. The glomerulus was then tracked through the serial sections in both directions until it disappeared. Only glomeruli with complete serial sectioning were evaluated. Glomeruli were classified as normal, ischemic, or affected by segmental sclerosis. Ischemic glomeruli showed capillary loop wrinkling with reduction in the capillary loop luminal area. No foam cells, hyaline deposits, podocyte vacuolization, or epithelial cell enlargement characteristic of segmental sclerosing lesions were present. Glomeruli with SS were evaluated for the location of the lesion. Lesions limited to the tubular pole were classified as glomerular tip lesions (GTLs). Segmental lesions involving the vascular pole or the vascular and tubular poles were classified as FSGS, not otherwise specified (NOS). The area of glomerular tuft that was sclerotic compared with the largest glomerular cross-sectional area was semiquantitated from 1+ to 3+ as follows: 1+, less than 25%; 2+, 25% to 5%; 3+, more than 5%. Results Renal biopsy specimens from 8 patients that showed FSGS were serially sectioned to evaluate the relationship between SS and tubular atrophy as recognized by irregularity and thickening of the tubular basement membrane (TBM) at the tubular pole. A total of 113 glomeruli were analyzed in which the proximal tubule ostium was identified and the entire glomerulus was tracked in serial sections in both directions until it disappeared. The 8 biopsy specimens were divided into 2 groups and analyzed separately. Two biopsy specimens had lesions of SS limited to the region adjacent to the tubular pole and were designated FSGS, GTL. Five other biopsy specimens had SS that involved the vascular pole or the vascular and tubular poles and were designated FSGS, not otherwise specified (NOS). FSGS, Glomerular Tip Lesion The 3 patients whose biopsy specimens contained FSGS, GTL were 2 women and 1 man aged 24, 31, and 36 years, respectively. At the time of biopsy, all were normotensive and had nephrotic syndrome with proteinuria ranging from 3.4 to 18.1 g/24 h. The serum creatinine levels ranged from.9 to 1.4 mg/dl (8-124 Limol/L). The 3 biopsy specimens contained a total of 24 glomeruli that were entirely examined by serial sections. Although only 7 (3%) of glomeruli contained SS in the original sections generated at the time of biopsy diagnosis, serial sections revealed that 21 (88%) of 24 glomeruli contained a GTL. In 2 biopsy specimens, 16 (1%) of 16 glomeruli were affected by GTLs, and in 1 biopsy specimen, 5 (62%) of 8 glomeruli were affected by GTLs. Of the 3 glomeruli without GTLs, 1 showed mild TBM thickening. Of 21 glomeruli with GTLs, 16 (76%) glomeruli contained small (1+) lesions affecting less than 25% of the glomerular tuft diameter liable II and 5 glomeruli (24%) had 2+ SS affecting 25% to 5% of the tuft. Of the 16 glomeruli with 1+ SS, 13 (81%) showed early tubular atrophy with mild proximal TBM thickening, and 3 (19%) had normal proximal TBM Hmage II and Hmage 21. All 5 glomeruli (1%) with 2+ SS were associated with a thickened TBM. The thickened TBM could be tracked along several tubule profiles in the vicinity of the urinary pole, but no attempt was made to determine at what point, if any, the TBM assumed a normal appearance. Although most SS lesions were small (1+ SS), all were associated with an adhesion to the Bowman capsule at the ostia of proximal tubule and commonly seemed to occlude or compromise the ostia of the proximal tubule (Image 2). Small breaks in the TBM at its juncture with the Bowman capsule were identified in 1% to 2% of the glomeruli in each biopsy specimen (Images 1 and 2). In glomeruli with 1+ SS and tubular atrophy, mild interstitial expansion with small numbers of mononuclear inflammatory cells was often Table II Relationship Between Segmental Sclerosis and Tubular Atrophy FSGS, GTL Normal glomerulus Segmental sclerosis 1+ 2+ 3+ Ischemic changes FSGS, NOS Early Advanced No Early Advanced No 2 1 24 6 4 3 13 4 1 7 2 6 12 2 8 11 7 FSGS = focal-segmental glomerulosclerosis; GTL = glomerular tip lesion; NOS = not otherwise specified; tubular atrophy. 344 Am J Clin Pathol 1999; 111:343-348 Downloaded from https://academic.oup.com/ajcp/article-abstract/111/3/343/175821 on 22 April 218

Anatomic Pathology / ORIGINAL ARTICLE Image I I A glomerulus with focal-segmental glomerulosclerosis, glomerular tip lesion (1 + segmental sclerosis) showing a thin (normal) tubular basement membrane that has a focal disruption (arrow) (periodic acid-schiff, x45). llmage 21A glomerulus with focal-segmental glomerulosclerosis, glomerular tip lesion (1+ segmental sclerosis) showing early tubular atrophy with a capsular adhesion occluding the proximal tubule ostia. A small tubular basement membrane break (arrow) is also present (periodic acid-schiff, x4). present. In the glomeruli with 2+ SS and advanced tubular atrophy, prominent interstitial expansion with mononuclear inflammatory cells was present. atrophy. Foci of Bowman capsular or TBM disruption were noted in 1% to 25% of the glomeruli in each biopsy specimen with FSGS, NOS. In several glomeruli, the TBM disruptions seemed to be circumferential (Image 4). Three biopsy specimens from patients with clinically documented hypertension contained vascular changes with intimal thickening and sclerosis of arteries and arteriolar medial hyperplasia. Focal global glomerulosclerosis and ischemic glomerular changes recognized by wrinkling of capillary loops with reduction in capillary luminal area also were present llmage 51. Nine ischemically affected glomeruli were serially sectioned. All 9 were from the biopsy specimens with severe vascular disease, and all 9 showed TBM thickening. The tubular atrophy was severe in 7 (Image 5) and llmage 61. No TBM breaks at the glomerulotubular junction were identified. FSGS, Not Otherwise Specified The biopsy specimens of 5 patients showed FSGS, NOS in which the lesions of SS were clearly not limited to the region of the glomerular tip. This group included 2 men and 3 women whose ages were 23, 35, 4, 52, and 6 years, respectively. At the time of biopsy, all had nephrotic syndrome with proteinuria ranging form 3.9 to 8.9 g/24 h. Three had hypertension with diastolic blood pressures ranging from 95 to 11 mm Hg. The creatinine levels ranged from 1.4 to 1.8 mg/dl (124-159 iimol/l) in 4 patients and was 2.8 mg/dl (248 Ltmol/L) in the fifth patient. The 5 biopsy specimens contained a total of 89 glomeruli that were entirely examined by serial sections. Although only 18 (21%) of glomeruli showed SS in the original sections generated at the time of biopsy diagnosis, serial sections revealed that 46 (52%) of 89 glomeruli contained SS. Of 46 glomeruli with SS, 13 (28%) had small areas of SS (1+), and 6 of these showed early tubular atrophy with mild TBM thickening (Table 1). Of 46 glomeruli with SS, 33 (72%) had large areas of SS (2+ or 3+) and TBM thickening was noted in 31 (94%) of 33 glomeruli llmage 31 and llmage 41. Interstitial expansion with inflammation was more prominent compared with FSGS, GTL but seemed proportional to the more extensive global glomerular sclerosis and tubular Downloaded from https://academic.oup.com/ajcp/article-abstract/111/3/343/175821 on 22 April 218 Thirty-four additional glomeruli were serially sectioned and demonstrated no abnormality; specifically neither SS nor ischemic changes were present. However TBM thickening was present in 1 (29%) of 34 glomeruli and was advanced in 4 of those glomeruli (Image 6). All 1 glomeruli were from the 3 biopsy specimens obtained from patients with vascular disease. Discussion Despite a glomerular target for injury in glomerulonephritis, tubulointerstitial alterations correlate most closely Am J Clin Pathol 1999;111:343-348 345

Bonsib / TUBULAR ATROPHY IN FOCAL-SEGMENL GLOMERULOSCLEROSIS Image 31A glomerulus with focal-segmental glomerulosclerosis, not otherwise specified pattern (2+ segmental sclerosis) showing Bowman capsule disruption and adhesion with mild tubular basement membrane thickening (periodic acid-schiff, x4). Image 41A glomerulus with focal-segmental glomerulosclerosis, not otherwise specified pattern (3+ segmental sclerosis) showing an obliteration of the urinary outflow with advanced tubular atrophy and circumferential basement membrane disruption (arrows) at the tubular ostium (periodic acid-schiff, x4). Image 51A glomerulus showing ischemic glomerular basement membrane wrinkling and advanced tubular atrophy (periodic acid-schiff, x4). Image 61A histologically normal glomerulus showing advanced tubular atrophy. A sclerotic interlobular artery also is present (periodic acid-schiff, x4). with renal insufficiency.1-8 Understanding the sequential events in this glomerular-tubulointerstitial axis is crucial to understanding the pathogenesis of nephron damage. Irreversible damage is recognized in renal biopsy specimens by alterations in matrix material and basal lamina. In the glomeruli in the presence of FSGS, this is characterized by capillary loop obliteration, mesangial matrix expansion, and adhesion to the Bowman capsule. In the tubular portion of the nephron, several forms of atrophy have been described.9 The most common form is classic tubular atrophy characterized by initial thickening of the TBM lamina densa, followed by irregularity and multilayering of the TBM. 91 Reciprocal interstitial changes evolve with fibrosis and chronic inflammation. 346 Am J Clin Pathol 1999; 111:343-348 Downloaded from https://academic.oup.com/ajcp/article-abstract/111/3/343/175821 on 22 April 218

Anatomic Pathology / ORIGINAL ARTICLE In the present study of FSGS, the relationship between glomerular sclerosis and the appearance of classic tubular atrophy was explored to determine whether irreversible tubular damage develops in parallel or follows substantial glomerular damage. This study was stimulated by the observation that atrophic tubular changes may be observed in nephrons in which the glomerulus is nonsclerotic or minimally altered in several disorders, such as diabetes, IgA nephropathy, hypertensive nephrosclerosis (personal observations), and in FSGS as described in this report. Only glomeruli in which the entire glomerulus was serially sectioned were evaluated, permitting direct correlation between the extent of glomerular sclerosis and atrophic changes in the proximal tubule. Compared with the original diagnostic sections, the use of serial sections substantially increased the yield of glomeruli affected by segmental lesions, from 18 of 89 (2%) to 46 of 89 (52%) in FSGS, NOS and from 7 of 24 (29%) to 21 of 24 (88%) in FSGS, GTL. A comparable 2- to 3-fold increase in the incidence of SS also has been observed by others in serial section studies. 11-14 It confirms that in the initial phase of the disease, lesions of SS are focal in classic FSGS (FSGS, NOS in the present article) but are often diffuse in FSGS, GTL in which the majority of glomeruli, in some cases possibly every glomerulus, are affected by tubular pole lesions. Although the pathogenesis of FSGS remains unknown, the contrasting incidence and patterns of segmental lesions and the diverse clinical situations in which FSGS develops imply that differences in pathogenesis likely exist within the disease known as FSGS. 15-2 Despite possible pathogenic differences within FSGS, the present study demonstrated that tubular atrophy can be detected in a substantial fraction of glomeruli in which only a modest portion of the glomerular tuft is sclerotic. Severe tubular atrophy also was noted in ischemic glomeruli and in several normal-appearing glomeruli in biopsy specimens from 3 patients with prominent hypertensive changes, suggesting that hypertension may damage not only the vascular and glomerular compartments, but also the tubular compartment. Although segmental glomerular sclerosis and tubular injury may develop in unison, it is unclear whether this derives from a common injury affecting glomerular and tubular cells or whether the tubular injury is secondary to glomerular dysfunction. The common embryologic derivation of glomerular epithelium and tubular epithelium could result in vulnerability to a similar injurious agent as observed in experimental FSGS produced by toxic agents (doxorubicin hydrochloride and puromycin aminonucleoside), in which chronic tubulointerstitial injury can develop in parallel or even precede the glomerular SS.21"23 Conversely, tubular injury could result from glomerular dysfunction since the level of proteinuria has prognostic implications in many Downloaded from https://academic.oup.com/ajcp/article-abstract/111/3/343/175821 on 22 April 218 forms of glomerular disease.6-8 The nonselective proteinuria exposes tubules to an abnormal milieu containing albumin and a variety of potentially toxic molecules such as transferrin, complement components, and lipoproteins that can affect tubular cell function.6-8-24-25 Tubules with mild TBM alterations often were associated with adjacent interstitial expansion and mild inflammation, indicating that interstitial changes develop before advanced tubular injury. The interstitial changes may result directly from tubular cell-derived factors, with an additional contribution from TBM disruptions. Although the term atrophy is used for tubules with TBM thickening, this does not equate with reduced cellular activities. Tubular cells produce new layers of basal lamina, have enhanced proliferative activities, and produce a variety of biologically active molecules, such as chemokines, cytokines, chemoattractants, and matrix metalloproteinases, that affect interstitial cells and recruit inflammatory cells.6"1-24"33 The TBM defects permit the escape of the glomerular filtrate into the interstitium, which could contribute to interstitial inflammation and provide an avenue for interstitial cells to enter the Bowman space with deposition of interstitial collagen.34-35 The present study of biopsy specimens showing FSGS has shown that tubular atrophy is detectable in most nephrons that harbor lesions of SS, and it develops early when the glomerular lesions are very small. This is believed to indicate a glomerular source for the tubular injury related to the same factor(s) that produced glomerular injury or to the constituents of the abnormal glomerular filtrate. From the Department of Pathology, University of Arkansas for Medical Sciences, Little Rock. Address reprint requests to Dr Bonsih: Department of Pathology, Mail Slot 517, University of Arkansas for Medical Sciences, 431 W Markham St, Little Rock, AR 7225-7199. References 1. Risdon RA, Sloper JC, de Wardener HE. Relationship between renal function and histological changesfoundin renal-biopsy specimens from patients with persistent glomerular nephritis. Lancet. 1968;11:363-366. 2. Schnainuck LI, Striker GE, Luther RE, et al. Structutalfunctional correlations in renal disease, II: the correlations. HumPathol. 197;1:631-641. 3. Bohle A, Glomb D, Grund KE, et al. Correlations between relative interstitial volume of the renal cortex and serum creatinine concentration in minimal changes with nephrotic syndrome and infocalsclerosing glomerulonephritis. Virchows Arch A Pathol AnatHiswl. 1977;376:221-232. 4. Wehrmann M, Bohle A, Held H, et al. Long-term prognosis offocalsclerosing glomerulonephritis: an analysis of 25 cases with particular regard to tubulointerstitial changes. Clin Nephrol. 199;33:115-122. Am J Clin Pathol 1999; 111:343-348 347

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