Focal glomerular immune complex deposition: possible role of periglomerular fibrosis/atubular glomeruli.
Abstract: * Context.--Consensus exists among renal pathologists that, in biopsies with immune complex glomerulonephritis, even a single glomerulus with open capillary loops may be sufficient for immunofluorescence and/or electron microscopy evaluation because immune complex deposition is a diffuse phenomenon. However, we have encountered renal biopsies with focal absence of immune complexes in glomeruli on either immunofluorescence or electron microscopy examination despite presence of open glomerular capillary loops.

Objective.--To evaluate renal biopsies with focal immune complex deposition and look for any subtle or unusual morphologic changes in the glomeruli (and in the biopsy in general).

Design.--Native and transplant renal biopsies were reviewed.

All biopsies had been triaged and processed according to our routine protocol for light microscopy, immunofluorescence, and electron microscopy examination.

Results.--Of 2018 renal biopsies from December 2005 to December 2007, we found 10 such biopsies; 5 native and 5 transplant kidney biopsies. We found that the glomeruli with absent immune complex deposits had periglomerular fibrosis with open, albeit, wrinkled appearing capillary loops but no glomerular sclerosis.

Conclusions.--We hypothesize that these histologic features are indicative of nonfunctional glomeruli and may be associated with disconnection between the Bowman capsule and proximal tubule (atubular glomeruli). These glomeruli may not have effective filtration, despite some degree of circulation through the open capillary loops, and therefore are unable to accumulate immune complex deposits.

If biopsies are small and only such glomeruli are available for immunofluorescence or electron microscopy examination, the absence of immune complex deposition in them should be evaluated carefully.

(Arch Pathol Lab Med. 2009;133:283-288)
Article Type: Report
Subject: Glomerulonephritis (Diagnosis)
Immune response (Research)
Immune response (Physiological aspects)
Fibrosis (Research)
Fibrosis (Physiological aspects)
Kidneys (Biopsy)
Kidneys (Usage)
Authors: Satoskar, Anjali A.
Calomeni, Edward
Bott, Cherri
Nadasdy, Gyongyi M.
Nadasdy, Tibor
Pub Date: 02/01/2009
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 College of American Pathologists ISSN: 1543-2165
Issue: Date: Feb, 2009 Source Volume: 133 Source Issue: 2
Topic: Event Code: 310 Science & research
Accession Number: 230151963
Full Text: There is a consensus among renal pathologists that immune complex deposition is a diffuse phenomenon, meaning that if immune complex glomerulonephritis occurs, all glomeruli with open capillaries will show immune complex deposits, even if the light microscopic lesions are focal (in many glomerulonephritides not all glomeruli show light microscopic abnormalities). Therefore, it appears that even a single glomerulus with open capillary loops in the tissue for immunofluorescence (IF) and/ or electron microscopy (EM) is sufficient to evaluate for immune complex deposition. In most biopsies with immune complex glomerular diseases, there is correlation between the IF and the EM findings: if IF reveals glomerular immune complexes, EM will show discrete electron-dense immune-type deposits in the glomeruli. However, there have been few reports with discrepant findings between IF and EM regarding immune complex deposition. (1-3) We have encountered few renal biopsies in which the immune complex deposition showed a focal distribution pattern on IF or EM. This is not problematic if there are numerous glomeruli available for examination and only a few glo meruli show absence of immune complex deposits. However, it is a potential diagnostic pitfall, if the biopsy is small with only one or a few glomeruli available for examination, because in such biopsies immune complex deposition may remain undetected. We studied cases with focal immune complex deposition to elucidate the probable cause for this apparent focal pattern of glomerular immune complex deposition.

MATERIALS AND METHODS

From December 2005 to December 2007, we examined a total of 2018 renal biopsies, including native kidney as well as renal allograft biopsies. Native kidney biopsies and renal allograft biopsies performed 6 or more months after transplantation were triaged for light microscopy, full panel IF (see later), and EM as per our routine protocol. Most renal allograft biopsies performed within the first 6 months of transplant received only light microscopic examination and IF examination for C4d. Among the biopsies that received full panel IF and EM, we found 10 biopsies showing focal immune complex deposition on either IF or EM examination.

Light Microscopy

Tissue for light microscopy was fixed in 10% formalin, embedded in paraffin, sectioned (2-3 [micro]m), and stained with hematoxylineosin, periodic acid-Schiff, Masson trichrome, and methenamine silver. Five serial sections were routinely cut at 3 consecutive levels in the block. Serial sectioning of the blocks was not performed because these were all recent diagnostic renal biopsies. Selected resin blocks for EM (see later) with glomeruli were serially sectioned to examine the glomerulotubular junction. Only resin blocks from cases with multiple blocks containing multiple glomeruli were cut to avoid depleting diagnostic EM tissue.

Direct IF

Tissue was embedded in optimal cutting temperature compound and snap frozen in liquid nitrogen. Fluorescein isothiocyanateconjugated primary polyclonal rabbit anti-human antibodies to immunoglobulin (Ig) G, IgA, IgM, C1q, C3, fibrinogen, and [kappa] and [lambda] light chains (all from Dako Cytomation, Carpinteria, Calif) were used. The intensity of staining was graded on a scale from 0 to 3+ (1+, mild staining; 2+, moderate staining; 3+, strong staining; [+ or -], trace staining).

Electron Microscopy

Tissue was placed in vials containing 3% glutaraldehyde (pH 7.4) and postfixed in osmium tetroxide. Following routine processing and embedding into Spurr resin, thin sections were cut and contrasted with uranyl acetate and lead citrate. Ultrastructural examination was performed using a Carl Zeiss EM 900 transmission electron microscope (Peabody, Mass).

RESULTS

The 10 biopsies showing focal immune complex deposition either on IF or EM examination included 5 native and 5 transplant kidney biopsies (Table). In all of these biopsies, the few glomeruli with absent immune complex deposits (other than those with global sclerosis) (Figure 1, A; Figure 2, A; Figure 3, A) did have open capillary loops, but they were somewhat wrinkled and ischemic appearing (Figure 1, B; Figure 2, B; Figure 3, B). These glomeruli also had periglomerular fibrosis in and around the Bowman capsule but no evidence of segmental sclerosis of the glomerular capillary tufts. Overall, these glomeruli did appear smaller than the glomeruli with immune complex deposits, but no morphometric measurements were done in this study. The detailed description of the glomeruli on light microscopy as well as the tissue for IF and EM are shown in the Table. These glomeruli did not specifically show glomerular basement membrane thickening or podocyte foot process effacement. In selected cases, serial sectioning of the resin block for EM was performed. At least some, but not all, of these glomeruli appear to be atubular (disconnection between the Bowman capsule and proximal tubule). Periglomerular fibrosis is not always associated with complete occlusion of the glomerulotubular junction, as seen on serial sections. Sometimes only narrowing of the junction was seen, associated with prominent atrophy of the adjoining tubule. The remaining glomeruli in the biopsies, having immune complex deposits (Figure 1, C; Figure 2, C; Figure 3, C), showed open well-distended capillary loops (Figure 1, D; Figure 2, D; Figure 3, D), were relatively larger in size, and did not have lamellar fibrosis around the Bowman capsule. However, it should be noted that periglomerular fibrosis is not always associated with absence of immune complex deposition. Three of the 10 biopsies (cases 4, 7, and 10 in the Table) did reveal moderate arterial fibrointimal thickening; 1 biopsy showed mild arterial fibrointimal thickening (case 3, Table). One biopsy (from renal allograft) had features of collapsing glomerulopathy (case 6, Table), but no arteries were sampled in the biopsy specimen. In 2 of the patients, no arteries were seen in the biopsy tissue (but 1 had severe arteriolar hyaline) and in 3 patients arterial thickening and narrowing were not present.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

COMMENT

Involvement of glomeruli in immune complex glomerulonephritides is conventionally considered to be a diffuse phenomenon. Although on light microscopic examination, histologic features such as proliferative glomerular lesions and necrotizing or crescentic lesions may be focal (focal glomerulonephritis) with some glomeruli being normal, on IF and EM examination, usually immune complex deposition is diffuse and involves even the light microscopically normal-appearing glomeruli. Finding focal absence of immune complex deposition in glomeruli with open capillary loops, by IF and/or EM examination in occasional kidney biopsies with immune complex glomerulonephritis, prompted us to look more critically at these biopsies. On closer examination, the nonsclerotic glomeruli with absent immune complexes did have subtle but characteristic histologic features, which differed from the remaining glomeruli in the same biopsy, containing distinct immune complex deposits. The most distinctive finding was the presence of periglomerular fibrosis. The somewhat wrinkled ischemic appearance of the glomerular capillaries in these glomeruli was suggestive of poor glomerular perfusion. This along with arterial narrowing, seen in some of the biopsies, raises the possibility that obliterative arterial/arteriolar lesions within the kidney may contribute to poorly perfused glomeruli with little or no filtration. The apparent random distribution of the glomeruli lacking immune complex deposits in many of the sections does not appear to support this vascular pathogenesis, but a 2-dimensional image in a small kidney biopsy specimen does not prove whether these glomeruli are random or in a zonal distribution. In fact, poor glomerular perfusion and ischemia (obliterative preglomerular vascular changes) may play an important role in the pathogenesis of the described phenomenon. Rare cases of unilateral glomerulonephritis in patients with renal artery stenosis have been reported in the older literature.4 The absence of proliferative glomerular changes in kidneys with renal artery stenosis clearly indicates the role of poor renal/glomerular blood perfusion in preventing glomerular proliferative lesions. Unfortunately, the presence or absence of glomerular immune complex deposits was not addressed in these old publications.

[FIGURE 3 OMITTED]

Vascular etiology alone is probably not sufficient to explain the lack of immune complex deposition in glomeruli with periglomerular fibrosis. Some of these glomeruli with periglomerular fibrosis appeared to have short segments of patent proximal tubule with subsequent narrowing or closure, indicating an underlying disconnection of the Bowman space from the proximal tubule. Such glomeruli are described as "atubular" glomeruli and have reduced or absent glomerular filtration. Obviously, if there is no outflow from the Bowman capsule, glomerular capillary filtration will cease. It is likely that absent glomerular capillary filtration can inhibit glomerular immune complex deposition.

Atubular glomeruli were first described by Oliver (5) in his experiments with microdissection of the kidney in proliferative glomerulonephritis (Bright disease) and scanning EM of the glomerular tufts. Atubular glomeruli are glomeruli that are disconnected from their proximal tubule at the glomerulotubular junction, or those that have just a short length of proximal tubule, with narrowing or obstruction along the length of the tubule because of tubular injury/interstitial fibrosis. Further work by Marcussen and others (6-9) has shown that atubular glomeruli constitute a significant portion of the glomerular population in many chronic kidney diseases in humans such as chronic pyelonephritis, chronic allograft nephropathy, diabetic nephropathy, and chronic ischemia secondary to renal artery sclerosis, as well as in animal models of renal disease. In kidney biopsies with chronic pyelonephritis, a significant negative correlation was found between the percentage of glomeruli without connection to a normal proximal tubule and the total volume fraction of proximal tubules and a significant positive correlation with the volume fraction of the interstitial tissue. (10) These morphometric findings indicate that the larger the number of atubular glomeruli, the greater the degree of tubular atrophy and interstitial volume (commonly due to interstitial fibrosis), which all correlate with worsening renal dysfunction. This has been further emphasized in a recent review. (11) These studies indicate that atubular glomeruli are common in chronic renal diseases due to primary vascular or tubulointerstitial etiologies. According to Marcussen (10,12) these atubular glomeruli tend to be smaller in volume than glomeruli with intact tubular connection, but they have open capillary loops and only minor ultrastructural changes are seen, including a slight increase in mesangium and thickened basement membranes. These reported findings are consistent with our description of glomeruli lacking immune complex deposits. Many of the glomeruli we are describing could very well have glomerulotubular disconnection as a result of periglomerular fibrosis.

Atubular glomeruli and glomeruli with atrophic tubules do not contribute to glomerular filtration but function as a pathway for blood that is delivered without prior filtration to the peritubular capillaries. (10) Therefore, we can hypothesize that these nonfiltering glomeruli, despite some degree of blood flow through them, are unable to accumulate immune complex deposits because of the absence of filtration. As discussed previously, periglomerular fibrosis is common in a variety of chronic renal diseases, including chronic ischemia. Poorly perfused glomeruli have low filtration pressure, which may inhibit glomerular immune complex deposition. How exactly atubular glomeruli develop is unclear. It is quite possible that chronic ischemia is one of the causes. Fibrosis in the periglomerular interstitium (a common finding in chronic ischemia) has been suggested to be at least a contributory factor in the development of atubular glomeruli. (12) There may be other preceding transient intraglomerular events that may also play a role, such as herniation of glomerular tuft into the origin of the tubule, formation of glomerular tip lesions with associated sclerosis of nearby capillary loops, and damage to the beginning of the proximal tubule. (13) Despite these events, the remaining glomerular tuft may show only minor morphologic abnormalities and this can be deceiving because it is difficult to appreciate the underlying loss of function in these nephrons.

In summary, absence of immune complex deposition in glomeruli with periglomerular fibrosis is a potential diagnostic pitfall, if the number of glomeruli for examination in the renal biopsy is limited. One has to be aware of this phenomenon and make a thorough search for glomeruli without periglomerular fibrosis in the available specimen with repeat IF (or immunoperoxidase) and EM studies, if possible.

Accepted for publication July 16, 2008.

References

(1.) Laasonen A, Rantala I, Mustonen J, Pasternack A. Immunoelectron microscopic localization of immune deposits in IgA glomerulonephritis. Acta Pathol Microbiol Immunol Scand [A]. 1984;92(4):249-256.

(2.) Lowance DC, Mullins JD, McPhaul JJ. Immunoglobulin A (IgA) associated glomerulonephritis. Kidney Int. 1973;3:167-176.

(3.) Skjorten FJ, Brorson SH, Roald B, Strom EH, Lund B. The use of post-embedding immunoelectron microscopy in the diagnosis of glomerular diseases: comparison of immunoelectron microscopic and immunofluorescence studies. Acta Pathol Microbiol Immunol Scand. 1992;100:1001-1007.

(4.) Dikman SH, Strauss L, Berman LJ, Taylor NS, Churg J. Unilateral glomerulonephritis. Arch Pathol Lab Med. 1976;100:280-483.

(5.) Oliver J. The aglomerular nephrons of terminal Bright's disease. In: Architecture of the Kidney in Chronic Bright's Disease. New York, NY: Paul B Hoeber; 1937:43-57.

(6.) Marcussen N. Atubular glomeruli in chronic renal disease. Curr Top Pathol. 1995;88:145-174.

(7.) Marcussen N, Olsen TS. Atubular glomeruli in patients with chronic pyelonephritis. Lab Invest. 1990;62:467-473.

(8.) Marcussen N, Ottosen PD, Christensen S, Olsen TS. Atubular glomeruli in lithium-induced chronic nephropathy in rats. Lab Invest. 1989;61:295-302.

(9.) Tanner GA, Tielker MA, Connors BA, Phillips CL, Tanner JA, Evan AP. Atubular glomeruli in a rat model of polycystic kidney disease. Kidney Int. 2002;62: 1947-1957.

(10.) Marcussen N. Atubular glomeruli and the structural basis for chronic renal failure. Lab Invest. 1992;66:265-284.

(11.) Chevalier RL, Forbes MS. Generation and evolution of atubular glomeruli in the progression of renal disorders. J Am Soc Nephrol. 2008;19:197-206.

(12.) Marcussen N. Tubulointerstitial damage leads to atubular glomeruli: significance and possible role in progression. Nephrol Dial Transplant. 2000; 15(suppl 6):74-75.

(13.) Najafian B, Crosson JT, Kim Y, Mauer M. Glomerulotubular junction abnormalities are associated with proteinuria in type 1 diabetes. J Am Soc Nephrol. 2006;17:S53-S60.

Anjali A. Satoskar, MD; Edward Calomeni, BS; Cherri Bott, BS; Gyongyi M. Nadasdy, MD; Tibor Nadasdy, MD, PhD

From the Department of Pathology, The Ohio State University Medical Center, Columbus.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Tibor Nadasdy, MD, PhD, The Ohio State University Medical Center, Renal Pathology, 320 W 10th Ave, M018 Starling Loving Hall, Columbus, OH 43210 (e-mail: tibor.nadasdy@osumc.edu).
Biopsy Diagnosis and Glomerular Findings in the 10 Cases With Focal
Immune Complex Deposition on Immunofluorescence or Electron
Microscopy *

Case   Kidney
No.    Biop     Diagnosis       Light Microscopy    Immunofluorescence

1      Native   Membranous      8 Glomeruli,        14 Glomeruli, 5
                GN              1 sclerotic         sclerotic, 6
                                                    glomeruli absent
                                                    staining, 3 bright
                                                    IgC, [kappa] and
                                                    [lambda] staining
2      Native   Lupus           Diffuse             7 Glomeruli, 3
                nephritis       proliferative GN    remaining have
                                extensive intra-    hyaline
                                capillary hya-      thrombi; thrombi
                                line thrombi, 9     and GBM positive
                                glomeruli, 3        for IgG, IgM, IgA,
                                sclerotic, 2        [kappa] and
                                wrinkled loops      [lambda]; no
                                                    extraglomerular
                                                    deposits
3      Native   Crescentic      7 Glomeruli, 3      3 Glomeruli, 2
                IgA             with crescents,     show crescents;
                nephropathy     remaining           strong diffuse
                                glomeruli show      mesangial IgA
                                moderate            [kappa] and
                                mesangial           [lambda]
                                hypercellularity
4      Tx       Membranous      17 Glomeruli, 4     7 Glomeruli, 4
                GN chronic      sclerotic, re-      sclerotic, 2 have
                allograft       maining diffuse     periglomerular
                nephropathy     capillary loop      fibrosis, no
                                thickening          staining, 1
                                                    glomerulus with
                                                    strong capillary
                                                    wall IgG
5      Tx       Mild ATN        3 unremarkable      3 Glomeruli, mild
                with            glomeruli           mesangial IgG,
                mesangial                           IgM, C3 [kappa]
                immune                              and [lambda]
                complexes
6      Tx       Late acute      Plasma cell-rich    6 Glomeruli, 3
                rejection,      interstitial        glomeruli show
                recurrent       inflammatory        diffuse mesangial
                lupus           cell infiltrate     and glomerular
                nephritis       7 glomeruli         capillary wall IgG,
                                with features       [kappa], [lambda]
                                of collapsing       3 glomeruli show
                                glomerulopathy      no staining
7      Tx       Polyoma virus   Diffuse             6 Glomeruli, 4
                nephropathy     interstitial        glomeruli show
                with            inflammation        bright mesangial
                mesangial IgG   with tubular        IgG, C3 and
                [kappa]         epithelial          and [kappa] and
                deposits        viral inclusions,   trace [lambda]
                                27 glomeruli        light chain, 2
                                with mild focal     glomeruli absent
                                glomerulitis        straining
8      Tx       Recurrent IgA   Unremarkable        9 Glomeruli with
                phropathy,      glomeruli           moderate diffuse
                late acute                          granular mesangial
                rejection                           IgA
9      Native   Lupus           27 Glomeruli        6 Glomeruli, 2
                nephritis       with focal          sclerotic, 1 with
                                proliferative       periglomerular
                                GN, segmental       fibrosis and no
                                necrotizing         staining, diffuse,
                                lesions and         IgG, and IgA, IgM,
                                cellular            C1q, [kappa] and
                                crescents, 4        [lambda] in
                                glomeruli           remaining glomeruli
                                sclerotic
                                and 2 with
                                periglomerular
                                fibrosis
10     Native   Cryoglobulin    8 Glomeruli,        3 Glomeruli, mild
                deposits,       mild focal          capillary wall and
                lymphoprolif-   proliferative       small
                erative         lesions             intracapillary IgG
                disorder                            deposits

Case   Kidney
No.    Biop     Electron

1      Native   1 Glomerulus with subepithelial
                ICs
2      Native   2 Glomeruli, one with wrinkled
                loops and no ICs, 1 glomerulus
                has dense ICs with focal
                microtubular substructure
3      Native   3 Glomeruli, 1 sclerotic, 1 with
                wrinkled loops, periglomerular
                fibrosis and rare ICs, 1 glomerulus
                with mesangial ICS
4      Tx       2 Glomeruli with abundant
                subepithelial ICs
5      Tx       3 Glomeruli, 1 sclerotic, 1 with
                wrinkled capillaries and periglomerular
                fibrosis with no ICs, 1 glomerulus
                with mesangial ICs
6      Tx       5 Glomeruli, 4 glomeruli scarred, remaining
                glomerulus shows, intramembranous and
                paramesangial ICs
7      Tx       3 Glomeruli, 1 shows wringkling of capillary
                loops, periglomerular fibrosis, no ICs,
                other glomeruli show mesangial and subendothelial
                ICs
8      Tx       8 Glomeruli, 4 glomeruli sclerotic, 2 glomeruli with
                wrinkled capillary loops with no ICs, remaining show
                mesangial ICs
9      Native   3 Glomeruli with open capillary loops, mainly
                mesangial, few capillary loop ICs, extensive
                arteriolar wall ICs
10     Native   3 Glomeruli, 2 with periglomerular fibrosis with
                no ICs, 1 glomerulus has ICs with microtubular
                substructure

* Boldface indicates those glomeruli in the biopsy tissue that showed
open capillary loops but absent immune complex deposits, which we
hypothesize are atubular nonfiltering glomeruli. GN indicates
glomerulonephritis; Ig, immunoglobulin; ICs, immune complexes; GBM,
glomerular basement membrane; Tx, transplant; and ATN, acute tubular
necrosis.
Gale Copyright: Copyright 2009 Gale, Cengage Learning. All rights reserved.