Glomerular staining
- 1. G L O M E R U L A R S T A I N I N G T E C H N I Q U E S A N D A D V A N C E S N T I S S U E D I A G N O S I S O F G L O M E R U L A R D I S E A S E S B Y D R M O N I K A P G D M N E P H R O L O G Y S A V E E T H A M E D I C A L C O L L E G E A N D H O S P I T A L
- 2. STEPWISE APPROACH • diagnosis of glomerular disease in renal biopsy specimens often has at least 5 steps • 1) preliminary review of available clinical data prior to specimen examination • 2) light microscopic examination • 3) immunohistologic examination, • 4) electron microscopic examination • 5) integration of all pathologic and clinical data into a final interpretation and diagnosis
- 3. ADEQUACY OF TISSUE SAMPLING • to confidently exclude a segmental glomerular disease that is affecting about 5% of the glomeruli a biopsy specimen containing 20 glomeruli is needed • comparing the amount of glomerular involvement in two different biopsy specimens- To detect differences of 25% to 40% glomerular involvement the minimum specimen size is 20 to 25 glomeruli. • for lupus GN - a biopsy specimen should contain a minimum of 10 glomeruli. • In transplant biopsies a group in Banff has suggested that the requirements for biopsy specimen adequacy are 10 or more glomeruli and at least two arteries.
- 5. TISSUE EXAMINATION AND INTERPRETATION • first a low-power screening examination of the specimen – give an idea of area of defect – help in localizing that the defect is in glomerulus, tubule, and interstitium, and/or blood vessels • site of lesions, the distribution of lesion • categorize whether the lesion is active or chronic type
- 10. SECTIONING AND FIXATION • LM – • hematoxylin and eosin (H&E),periodic acid–Schiff (PAS), Masson’s trichrome, Jones methenamine silver (JMS),and Congo red—are routinely used • Tissue sections should be thinner 2–3 microns thick • Bouin’s or Zenker’s fixative provide better preservation of certain morphologic details. • Bouin’s fixative contains picric acid, which is explosive when dry. • Methacarn, a modified Carnoy’s fixative - good fixation for LM and EM , may allow the IHC detection of Ag that are not detected in formalin-fixed tissue. • 4% Paraformaldehyde • The standard method of processing tissue for light microscopy is by dehydrating in graded alcohols, transferring to a clearing agent such as xylene, and embedding in paraffin wax • Multiple slides can then be stained with each stain, with some intervening unstained sections kept either for potential IHC examination or for staining with other special stains as
- 14. STAINING AND LIGHT MICROSCOPY
- 20. COMMON TERMINOLOGY AND DEFINITION
- 31. ATN
- 46. Globally sclerosed glomerulus with ischemic origin of the injury. Remaining material of the capillary tuft is seen more intensively stained with PAS (left) and methenamine -silver stains (right) because sclerosed glomeruli contain abundant type IV collagen. The fibrous tissue that surrounds the sclerosed capillary tuft proliferates from the capsule and is negative, or weakly positive, with PAS and silver stains because contains a different collagen, predominantly type I. (PAS, and methenamine -silver, X300).
- 47. Glomerulus with segmental sclerosis (left superior corner). There are segments with preserved architecture. Notice small hyaline segments (positive with the PAS stain, arrow); they are proteins and other components. (Pas, x400).
- 48. Adhesion and sclerosis at the “tip domain” of the tuft, in other words, at the tubular lumen or neck in the bowman’s capsule (arrows) (although the origin of tubule is not perfectly identified here). This lesion has been associated with a less aggressive course in some works, but we do not found this “good prognosis” in our hispanic patients. (Masson’s trichrome, X400).
- 49. Almost global collapse of glomerular tuft. Notice the loss of capillary lumens and notorious proliferation of visceral epithelial cells (podocytes). Sometimes the proliferation is so intense that it can fill the bowman’s space and be confused with an epithelial crescent. The collapsing lesion can be global or segmental. (Methenamine - silver, X400).
- 50. Global cellular proliferation with increase of the glomerular size; there is loss of capillary lumens and nuclei with variable aspect in form and size, suggesting that there are several proliferating cell types. In some cases the mesangial proliferation contributes to accentuate the lobules of the glomeruli: “lobulation”. (H&e, x400).
- 51. Severe cellular proliferation that obstructs the capillary lumen (endocapillary). Abundant polymorphonuclear leukocytes in the tuft: exudative form. This aspect suggests post-infectious GN. (H&e, x600).
- 52. Endocapillary proliferative GN with a segment of necrosis (among the arrows) that is eosinophilic (pink); it is accompanied by nucleus fragments (karyorrhexis) and polymorphous. In these lesions there is capillary wall rupture and they are frequently associated to crescents (H&E, X600).
- 53. Extracapillary proliferation. Notice the collapsed tuft in the center of the microphotography. There is a clear space between tuft and the crescent. The green arrows point the bowman’s capsule and the blue arrows point the internal edge of the crescent. This proliferation is formed by epithelial cells and mononuclear inflammatory cells. (H&e, x400).
- 54. Compressed tuft, in the center of the glomerulus, by a dense circumferential cellular proliferation that occupies the entire bowman’s space. The arrows indicate rupture of capillary walls, a frequently associated phenomenon to the crescent. (Methenamine -silver, X300).
- 55. In this case the crescent has evolved with collagen proliferation, which is demonstrated by the fibres stained with silver stain: green arrows (they are also easily demonstrated with trichrome stain). The lesions in this stage are called fibroepithelial crescents. When the entire crescent has been replaced by fibrous tissue and it has not epithelial cells, is called fibrous crescent and it is a chronic, irreversible lesion. In the microphotography also there is a zone with bowman’s capsule destruction: red arrow. (Methenamine -silver, X300).
- 56. In this glomerulus we can see cellular proliferation in diverse mesangial areas. We consider proliferation when there are more than 3 nuclei in a mesangial area. The arrows point areas with increase of cellularity, in this case: mild proliferation. ( H&e, x.400).
- 57. Just as in the previous photo, we can see, in this image, cellular proliferation limited to the mesangium, with well preserved peripheral capillary walls and without obstruction of its lumens, although these appear diminished. Unlike to the previous one, in this photo we see a severe increase of the cellularity, which give a lobulated aspect to the tuft. See the relative homogeneity of nuclei and compare with the endocapillary proliferation in figures 5 and 6. (H&e, x400).
- 58. With stains that highlight the mesangial matrix (silver and PAS) is notorious the increase of the extracellular components of the mesangium that usually accompany mesangial hypercellularity. Pure increase of the matrix (without cellular increase) can be seen in some types of glomerular injury. (Pas, x400).
- 59. GBM reaction to the subepithelial deposits is seen as perpendicular projections to the GBM in its external part: “spikes”, demonstrated here with silver stain (arrows). The immune deposits (no visible here) are between spikes. (Silver, X1000).
- 60. This case corresponds to type I membranoproliferative GN. There is cellular proliferation and images in double contour (sometimes termed: tram-tracking, splitting, or duplication of the GBM) (arrows). (Methenamine-silver, X400).
- 61. Many and extensive subendothelial deposits. The immune deposits are positive with the fuchsin used in trichrome stain. The double contour forms due to synthesis of basement - membrane-like material between the endothelial cells and the deposits. (Masson’s trichrome, X400).
- 62. Glomerulus from a kidney allograft with chronic transplant glomerulopathy. The double contours found in this alteration (arrows) are not caused by subendothelial immune deposits. Cellular proliferation is not seen in these cases. (Methenamine -silver, X400)
- 63. Acellular deposits in interstitium. Congo-red demonstrate positivity for amyloid. On the left we see the reddish colour that gives the staining, but the true positivity is marked by apple green birefringence when polarized lenses are used (right). (Congo -red stain, X200; on the left with normal lenses; on the right with polarized lenses).
- 64. IMMUNOHISTOCHEMICAL TESTING • Immunohistochemical testing is performed either on cryostat sections of a piece of snap-frozen tissue (IF)or on paraffin sections(IP) • Immunofluorescence - Antigen detection on frozen sections is usually performed using an antibody labeled with a fluorochrome, and this preparation is then viewed using a fluorescence microscope • technically straightforward and very sensitive since the antigens have not been altered by fixation. • Drawbacks – – Require separate piece of tissue at the time of biopsy. – Morphology of frozen sections is never as good as that of paraffin sections, – more difficult to define the site of the antigen within the glomerulus. – IF sections fade over time, but if they are appropriately mounted and refrigerated in the dark they will retain the staining for weeks to months
- 65. IMMUNOFLUORESCENCE • best performed on unfixed, frozen sections • Tissue can be transported to the laboratory fresh on saline-soaked gauze or in Michel's fixative • Serial sections are cut at 2-4 μm in a cryostat • Fluorescein-labeled antibodies used for the antigens that should be routinely examined include immunoglobulins (primarily IgG, IgM, and IgA), complement components (primarily C3, C1q, and C4), fibrin, and kappa and lambda light chains • Additional antibodies may be required in specific circumstances – amyloid typing – collagen IV alpha chains in hereditary nephritis – IgG subclasses – virus identification – lymphocyte phenotyping in allografts in suspected cases of PTLD, and C4d in renal allograft biopsies
- 66. • IMMUNOPEROXIDASE – A. antigen retrieval B. Ag detection C. deposition of a colored reaction product Antigen retrieval - If paraffin sections are used, then some form of antigen retrieval is essential for most Ag, because they become masked during fixation and processing. • some form of protease digestion - For the detection of Igs and complement • length of time required for protease digestion is critically dependent on a number of factors such as the length of time the biopsy specimen has been in fixative and the particular processing schedule used. • Some of these factors may be difficult to control. • This variability of the antigen retrieval process is the major drawback of immunohistochemical analysis of paraffin sections • Results are highly dependent on the skills of the technician performing the staining. • Ag detection- • using a primary Ab, f/b a detection system that leads to the deposition of a colored reaction product that is visible by LM. • Commonly this product is developed by a reaction that utilizes the enzyme horseradish peroxidase
- 67. • major advantage - – not necessary to take a separate piece of tissue – possible to specifically localize antigens and compare these sections with adjacent sections examined by LM • Drawback – – detection of the linear capillary wall staining of anti-GBM antibodies is more difficult in paraffin sections. – more difficult to detect very early deposits in membranous GN in paraffin sections than in frozen tissue
- 77. Linear positivity for igg demonstrated by immunofluorescence. The capillary walls are seen well demarcated by a continuous, well -defined line. This pattern is observed in anti -GBM disease and in DM(IgG and albumin), being in this last case an unspecific immunostaining. In this image we see a crescent compressing the glomerular tuft (immunofluorescence for IgG, anti-human-IgG antibodies marked with fluorescein, fluorescence microscopy, X400).
- 78. Linear positivity for IgG in DN: "pseudolinear" (immunofluorescence for IgG, anti -human- IgG antibodies marked with fluorescein, fluorescence microscopy, X400).
- 79. Granular subendothelial deposits. This pattern is seen in some glomerular diseases with immune complexes deposits. The photo corresponds to a case of lupus nephritis (immunofluorescence for C3, anti -humn-c3 antibodies marked with fluorescein, fluorescence microscopy, X400).
- 80. Granular positivity for C3 in a case of post -infectious GN. This pattern isdescribed as "bumps and humps" or "lumpy-dumpy", traditionally associated with postinfectious GN (immunofluorescence for C3, anti -humn-c3 antibodies marked with fluorescein, fluorescence microscopy, X400).
- 81. Granular positivity for igg demonstrated by immunofluorescence. There are many “grains” of diverse size in the capillary walls (parietals). In some capilaries a "reticular" appearence can be seen. The photo corresponds to a case of membranous GN, with the more typical appearence of subepithelial deposits (immunofluorescence for IGG, anti -human- IGG antibodies marked with fluorescein, fluorescence microscopy, X400).
- 82. IgA positivity in mesangial areas in a case of IgAN. See that capillary walls are not marked by the immunostaining. Only there is a positivity that draws irregular areas that correspond to the mesangium. (Immunofluorescence for IgA, anti-human- IgA antibodies marked with fluorescein, fluorescence microscopy, X400).
- 83. Tubulointerstitial positivity for Igs or complement can be see in some autoimmune disease, mainly lupus, and exceptionally in other diseases. (Immunofluorescence for IgG, anti -human- IgG antibodies marked with fluorescein, fluorescence microscopy, X400).
- 84. ELECTRON MICROSCOPY o Usefull for assessing structural changes in the glomerulus and for identifying immune complexes, which are seen as areas of electron density o an invaluable technique for the examination of glomeruli in biopsy specimens from in native kidneys , but for determination of causes of dysfunction in transplanted kidneys o separate fixative, then exposed to osmium tetroxide and processed into resin blocks. o To select the areas to be studied, “semithin” 0.5 μm sections are first screened by LM
- 85. • EM Assesment – – GBM and its thickness – endothelium, with note of any thickening or loss of fenestrations – the capillary lumen, and particularly any narrowing by cells or other material – the podocytes, with particular attention to the preservation of the foot processes and any vacuolation or microvillous change in the cell bodies. – The presence of any electron-dense deposits—most commonly due to immune complex deposition, together with their distribution—mesangial, subendothelial, or subepithelial. – EM may also demonstrate a number of other structures, such as fibrils in amyloidosis or fibrillary glomerulonephritis, tubules in immunotactoid glomerulopathy, or the characteristic inclusion bodies of various storage diseases
- 87. • EM is most helpful in the following clinical situations: – Hematuria, especially microscopic, with or without proteinuria – When there is a family history of renal disease. – When there is a symptomatic proteinuria, with normal renal excretory function
- 89. ALGORITHM F OR I NTEGRATING I M AND E M F INDINGS W HEN D IAGNOSING SOM E O F T H E PATHOLOGIC E XPRESSIONS O F GLOMERUL AR D ISEASE
- 93. Scanning electron micrograph of a cast of a glomerulus with its many capillary loops (CL) and adjacent renal vessels. The afferent arteriole (A) takes its origin from an interlobular artery (lower left). The efferent arteriole (E) branches to form the peritubular capillary plexus (upper left). (×300.)
- 94. Electron micrograph of a portion of a glomerulus from normal human kidney in which segments of three capillary loops (CL) are evident. The relationship among mesangial cells (M), endothelial cells (E), and visceral epithelial cells (V) is demonstrated. Several electron-dense erythrocytes lie in the capillary lumens. (×6700.) BS, bowman’s space
- 95. Electron-dense subepithelial deposits in membranous GN (arrows). Notice also the projections of basement membrane -like material surrounding deposits. EM, original magnification, X6.000.
- 96. Subendothelial deposits in membranoproliferative GN (red arrows). Notice also a capillary segment with double contour. EM original magnification, X6.000.
- 97. Mesangial electron -dense deposits in a case of IgA nephropathy (arrows). EM, original magnification, X6.000.
- 98. Complete loss of podocyte processes (effacement or "fusion"). The blue arrow indicates an area of detachment (loss) of the podocyte. EM, original magnification, X8.000.
- 99. Diffuse thinning of glomerular basement membranes. The homogeneous thin appearance suggests the alteration, however, it is necessary to measure its thickness and, of course, correlate with clinical features: this finding does not necessarily mean "disease". EM, original magnification, X4.000.
- 100. Glomerular basement membrane very irregular, with areas of thinning and other very thickened in a case of Alport disease. EM, original magnification, X10.000.
- 101. Diffuse thickening of the glomerular basement membrane (note the result of measuring its thickness) in a case of diabetes mellitus; this change, though unspecific, can help in the early diagnosis of diabetic nephropathy. ME, original magnification, X10.000.
- 107. COMMON ABNORMALITIES IN REL ATION TO GLOMERUL AR CAPSULE
- 108. COMMON ABNORMALITIES IN GLOMERUL AR BASEMENT MEMBRANE
- 109. ABNORMALITIES IN COMMON GLOMERUL AR LESIONS
- 110. COMMON VASCUL AR LESIONS IN RENAL PATHOLOGY
- 111. COMMON TUBUL AR ABNORMALITIES
- 114. INTERSTITIAL LESIONS • edema, inflammation, and fibrosis • Both edema and fibrosis are associated with separation of normally closely apposed tubules. • With interstitial edema only, the basement membranes of tubules are of normal thickness and contour. • In contrast, with fibrosis the tubules are invariably atrophied with thickened and irregularly contoured basement membranes • distinction between an acute and a chronic interstitial process is made based on the presence of edema (acute) or fibrosis (chronic), regardless of the character of any infiltrating leukocytes • interstitial inflammation depend(s) on the nature of the inflammatory process. • Polymorphonuclear leukocytes, as expected, are present in early phases of bacterial infections; however, they are usually replaced by lymphocytes, plasma cells, and monocytes approximately 7-10 days following the onset of infection • On the contrary, other infectious agents may elicit only a “round cell” response. • Cell-mediated forms of acute inflammation, even in very early stages, are characterized by lymphocytic infiltrate, with or without plasma cells, monocytes, and granulomata • interstitium may contain abnormal extracellular material such as amyloid, immunoglobulin light chains, immune complex deposits
- 115. OTHER STUDIES PERFORMED ON THE RENAL BIOPSY SPECIMEN • suspected infection part of the biopsy specimen sent for culture or for PCR testing for infective organisms. • In biopsy specimens with lymphoid infiltrates Ig gene rearrangement studies may allow the confirmation of clonality. • The chemical composition of material in the biopsy specimen—for example, crystalline material—may be determined by energy-dispersive x-ray spectroscopy. • extracting mRNA from biopsy specimens to study differences in gene expression in various pathologic conditions, and to study the range of proteins in the biopsy specimen—the proteome • A results from a group in Edmonton have suggested that transcript analysis of transplant kidney biopsy specimens could play a role in diagnosis of acute antibody-mediated rejection
- 116. BIOPSY SPECIMENS FROM TRANSPLANTED KIDNEYS • To assess the cause of kidney dysfunction in 1st few mths postTx - IHC analysis with a full panel of Ab to Ig and complement, or to perform EM not necessary, unless there is a clinical suspicion of glomerular disease. • IHC assay for C4d - to assess Ab binding and complement activation on peritubular capillary endothelium. • In later biopsy specimens EM is very useful in the diagnosis of chronic allograft glomerulopathy and its differentiation from recurrence of de novo glomerulonephritis. • chronic rejection - involving peritubular capillaries, a/w multilayering of the peritubular capillary BM
- 117. IN-SITU HYBRIDIZATION • uses labeled cDNA or RNA probes • localizes specific DNA/RNA sequence in tissue section which is then quantitated using autoradiography or fluorescence microscopy • commonly used ones are – BK virus. – EB virus probes in the diagnosis of PTLD. – Pathogenic cytokines such as platelet-derived growth factor, epithelial growth factor,
- 118. ADVANCED MICROSCOPY FOR ADVANCED STUDIES • Advanced forms of transmission & scanning electron microscopy have revealed the detailed spatial relationships of GBM, podocytes, and endothelial cells • super resolution microscopy (SRM) – • Diffraction limit - Abbé and Rayleigh independently showed that the microscopes of the time could not image anything smaller than approximately half the wavelength of the illumination source and for the shortest wavelength of visible light (violet, 400 nm), it is 200 nm • combines advantages of IF and EM – offers high resolution identification of specific molecules – images large, physiologically relevant volumes of the glomerulus – Rapidity – ease of use and low cost with some types of SRM – visualizes structures below the classical diffraction limit of conventional light microscopy by adding a time variable to either the illumination of the specimen, or to the fluorescence signal emitted by it
- 119. • Structured Illumination Microscopy (SIM) • Stimulation Emission Depletion Microscopy (STED) • Stochastic Optical Reconstruction Microscopy (STORM) • SIM, STED, and STORM have visualized normal and nephrotic disease podocytes, and have confirmed their appearances to be similar to those seen by EM but with added new information on cell configuration and protein localization. • STORM has also localized podocyte cytoskeleton and adhesion proteins, and GBM proteins at a resolution never before possible • Photoactivated Localization Microscopy (PALM) • PALM & STORM a/k/a “single molecular resolution techniques” or “pointillist” techniques • expansion microscopy and genetic labeling • Block face scanning microscopy (BF-SEM) • detailed and complex three dimensional structure of podocytes relative to each other and to the GBM • focused ion beam milling scanning microscopy (FIB-SEM) • Electron Tomography (goniometer tilt stage) TEM • large scale, high resolution information on the organization of glycocalyces of podocytes and endothelial cells. • demonstrated the intercellular relationships of podocytes to their nearest neighbors, to the subpodocyte space and to the GBM
- 120. • Transmission EM (TEM) • commonly used in diagnosis and research • focuses high energy (100 kV and higher) electrons with lenses to form images in a way similar to conventional light microscopy. • It is most often used with thin sections of kidney tissue • Scanning EM (SEM) • used almost exclusively in research • forms magnified images by scanning lower energy (10–50 kV) electrons focused into a narrow beam over the surface of tissues, and builds an image, point by point, analogous to confocal microscopy, from electrons that are scattered off a metal coating added to the tissue. • This gives a three dimensional contour image in a volume and at a resolution between those of TEM and LM. • particularly useful in defining the complex structure of glomerular podocytes on the glomerular capillary basement membrane
- 121. Transmission electron micrographs(TEM) of glomerular filtration barrier in normal rats perfused with native anionic ferritin (A) or cationic ferritin (C) and in rats treated with heparitinase before perfusion with anionic (B) or cationic (D) ferritin. In normal animals, anionic ferritin is present in the capillary (cap) but does not enter the glomerular basement membrane (GBM), as shown in A. In contrast, cationic ferritin binds to the negatively charged sites in the lamina rara interna (LRI) and lamina rara externa (LRE) of the GBM (see C). After treatment with heparitinase, both anionic (B) and cationic (D) ferritin penetrates into the GBM, but there is no labeling of negatively charged sites by cationic ferritin. En, endothelial fenestrae; FP, foot processes; LD, lamina densa; US, urinary space. (×80,000.)
- 122. Transmission electron micrograph (TEM) of the juxtaglomerular apparatus of rabbit kidney, illustrating macula densa (MD), extraglomerular mesangium (EM), and a portion of an arteriole (on the right) containing numerous electron -dense granules. (×3700.)
- 123. TRANSMISSION ELECTRON MICROGRAPH (TEM) of the s1 segment of rat proximal tubule. the cells are characterized by a tall brush border, a prominent endocytic -lysosomal apparatus, and extensive invaginations of the basolateral plasma membrane. ( ×10,600.)
- 124. TEM of the S2 segment of rat proximal tubule. The brush border is less prominent than in the S1 segment. Note numerous small lateral processes at the base of the cell. ( ×10,600.)
- 125. TEM of the S3 segment of rat proximal tubule. The brush border is tall, but the endocytic - lysosomal apparatus is less prominent than in the S1 and S2 segments. Basolateral invaginations are sparse, and mitochondria are scattered randomly throughout the cytoplasm. (×10,600.)
- 126. Transmission electron micrograph of the apical region of a human proximal tubule illustrating the endocytic apparatus, including coated pits, coated vesicles, apical dense tubules, and endosomes. (×18,500.)
- 127. • Limitations of TEM and SEM • neither TEM nor SEM can easily identify specific proteins • cumbersome and limited by the requirement that antibodies bind specifically to antigens under the harsh specimen processing conditions necessary for EM • illuminating electron beam used with SEM and TEM damages and destroys biological macromolecules, thereby distorting or destroying image detail. • TEM can only image a very thin specimen • time consuming, difficult, • require specialized equipment added to the instrument
- 128. • Expansion microscopy (EXM) • physically rather than optically enlarges whole tissues by permeabilizing and then isotropically expanding them with a polymerizing gel so that structures smaller than the diffraction limit of light microscopy (200 nm) become larger than the diffraction limit and hence visible by IFM
- 132. DIABETIC NEPHROPATHY IN T1DM WITH PROTEINURIA
- 133. • Afferent and efferent glomerular arteriolar hyalinosis can also be detected within 3 to 5 years after onset of diabetes or following transplantation of a normal kidney into the diabetic patient. • This can eventuate in the total replacement of the smooth muscle cells of these small vessels by waxy, homogeneous, translucent-appearing material that is positive for the periodic acid–Schiff reaction and consists of immunoglobulins, complement, fibrinogen, albumin, and other plasma proteins. • Arteriolar hyalinosis, glomerular capillary subendothelial hyaline (hyaline caps), and capsular drops along the parietal surface of Bowman’s capsule make up the so-called exudative lesions of diabetic nephropathy • Progressive increases in the fraction of glomerular afferent and efferent arterioles occupied by ECM and in medial thickness have also been reported in young patients with type 1 diabetes mellitus. • Increases in the fraction of the volume of the glomerulus occupied by the mesangium, or Vv(Mes/glom), can be documented as early as 4 to 5 years after the onset of type 1 diabetes. • In many cases it may take 15 or more years to manifest. • This may be because the relationship of mesangial expansion to diabetes duration is nonlinear, with slow development earlier and more rapid development later in the disease • volume fraction of cortex that is interstitium, or Vv(Int/cortex), is a decrease in this parameter, perhaps due to the expansion of the tubular compartment of the cortex • Approximately 50% of type 1 diabetic patients with proteinuria have at least a few glomeruli with nodular lesions
- 135. FSGS • Light micrographs and diagrams depicting patterns of focal segmental glomerulosclerosis. One pattern has a predilection for sclerosis in the perihilar regions of the glomeruli (A and D). The glomerular tip lesion variant has segmental consolidation confined to the segment adjacent to the origin of the proximal tubule (B and E). The collapsing glomerulopathy variant has segmental collapse of capillaries with hypertrophy and hyperplasia of overlying epithelial cells (C and F). (Jones’ methenamine silver stain, ×100.)
- 136. • Light Microscopy • focal and segmental glomerular sclerosis • sclerosis may begin as segmental consolidation (caused by insudation of plasma proteins causing hyalinosis), by accumulation of foam cells, by swelling of epithelial cells and by collapse of capillaries resulting in obliteration of capillary lumens • increase in extracellular matrix material that ultimately accounts for the sclerosis component of the lesion. • relatively well-circumscribed focal tubular atrophy and interstitial fibrosis with slight chronic inflammation, even when there are no light microscopic glomerular lesions, no immune deposits, and no ultrastructural changes other than foot process effacement
- 137. • five pathologic variants are collapsing FSGS, tip lesion FSGS, cellular FSGS, perihilar FSGS, and FSGS NOS • characteristic feature of the collapsing variant - focal segmental or global collapse of glomerular capillaries with obliteration of capillary lumens. Podocytes overlying collapsed segments are usually enlarged and contain conspicuous resorption droplets. Hyperplasia of podocytes raises the possibility of crescentic glomerulonephritis. Relative to the extent of glomerular sclerosis, tubulointerstitial injury is more severe in collapsing glomerulopathy than in typical FSGS. Tubular epithelial cells have larger resorption droplets, extensive proteinaceous casts, and marked focal dilation of lumens (microcystic change).
- 138. • The tip lesion variant of FSGS is characterized by consolidation of segments contiguous with the proximal tubule. These lesions may be sclerotic or cellular. However, the increased cellularity is predominantly within the tuft, unlike the extracapillary hypercellularity of collapsing FSGS. Foam cells often contribute to this endocapillary hypercellularity • characterized by consolidation of the glomerular segment that is adjacent to the origin of the proximal tubule and thus opposite the hilum • initial consolidation usually has obliteration of capillary lumens by foam cells, swollen endothelial cells, and an increase in collagenous matrix material (sclerosis). Hyalinosis is seen less often than with typical FSGS. Podocytes adjacent to the consolidated segment are enlarged and contain clear vacuoles and hyaline droplets. These altered podocytes often are contiguous to, if not attached to, adjacent parietal epithelial cells and tubular epithelial cells at the origin of the proximal tubule, which also have irregular enlargement and vacuolation. The tip lesion may project into the lumen of the proximal tubule
- 139. • cellular variant of FSGS - has lesions that resemble the cellular lesion for the tip variant, but they are distributed throughout the glomerular tuft. • Perihilar FSGS is characterized by the perihilar predilection of lesions and the presence of hyalinosis. • The FSGS NOS category is a nonspecific category that is used when the lesions do not have the distinctive features of any of the other four specific variants.
- 140. MEMBRANOUS GLOMERULOPATHY • Light Microscopy • diffuse global capillary wall thickening in the absence of significant glomerular hypercellularity. • vary with the stage of the disease and with the degree of secondary chronic sclerosing glomerular and tubulointerstitial injury. • Mild stage I lesions may not be discernible by light microscopy, especially when only a hematoxylin and eosin stain is used. • Stage II, III and IV lesions usually have readily discernible thickening of the capillary walls. • Masson trichrome stains may demonstrate the subepithelial immune complex deposits as tiny fuchsinophilic (red) grains along the outer aspect of the GBM. • However, this is not a sensitive, specific, or technically reliable method for detecting glomerular immune complex deposits. • Special stains that accentuate basement membrane material, such as Jones’ silver methenamine stain, may reveal the basement membrane changes that are induced by the subepithelial immune deposits. • Spikes along the outer aspect of the GBM usually are seen in stage II lesions • Stage III and IV lesions have irregularly thickened and trabeculated basement membranes, which resemble changes that occur with membranoproliferative glomerulonephritis and chronic thrombotic microangiopathy. • Overt mesangial hypercellularity is uncommon in idiopathic MN, although it is more frequent in secondary MN • Crescent formation is rare unless there is concurrent anti-GBM disease or ANCA disease • With disease progression, chronic sclerosing glomerular and tubulointerstitial lesions develop. Glomeruli become segmentally and globally sclerotic, and develop adhesions to Bowman’s capsule. Worsening tubular atrophy, interstitial fibrosis, and interstitial infiltration by mononuclear leukocytes parallels progressive loss of renal function
- 141. • Light micrograph of a glomerulus with features of stage II membranous glomerulopathy demonstrating spikes along the outer aspects of the glomerular basement membrane (see Figure 31-2). These correspond to the projections of basement membrane material between the immune deposits. (Jones’ methenamine silver stain, ×300.)
- 142. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS TYPE I • Light Microscopy • diffuse global capillary wall thickening, increased mesangial matrix, and endocapillary hypercellularity • Infiltrating mononuclear leukocytes and neutrophils also contribute to the glomerular hypercellularity • hypersegmentation or lobulation (an earlier name for this phenotype of glomerular injury was lobular glomerulonephritis) • Markedly expanded mesangial regions may develop a nodular appearance with a central zone of sclerosis that may resemble that of diabetic glomerulosclerosis or monoclonal immunoglobulin deposition disease • A distinctive but not completely specific feature of type I MPGN is a doubling or more complex replication of GBMs that can be seen with stains that highlight basement membranes, such as Jones’ silver methenamine stain or periodic acid–Schiff stain • The presence of “hyaline thrombi” within capillary lumens should raise the possibility of cryoglobulinemia or lupus as the cause for the MPGN. • Hyaline thrombi are not true thrombi but rather are aggregates of immune complexes filling capillary lumens. • A minority of patients with type I MPGN have crescents, but these rarely involve more than 50% of glomeruli. • As with other types of glomerulonephritis, substantial crescent formation correlates with a more rapid progression of disease.
- 143. • Light micrograph of a glomerular segment from a patient with type I membranoproliferative glomerulonephritis (MPGN) demonstrating doubling (arrows) and more complex replication of glomerular basement membranes. (Periodic acid–Schiff stain, ×1000.)
- 144. Acute poststreptococcal glomerulonephritis • Light Microscopy • acute histologic change is influx of neutrophils, which results in diffuse global hypercellularity • Endocapillary proliferation of mesangial cells and endothelial cells also contributes to the hypercellularity. • The hypercellularity often is very marked and results in enlarged consolidated glomeruli. • The description acute diffuse proliferative glomerulonephritis often is used as a pathologic designation for this stage of acute PSGN. • A minority of patients have crescent formation, which usually affects only a small proportion of glomeruli. • Extensive crescent formation is rare. • Special stains that have differential reactions with immune deposits may demonstrate subepithelial deposits. • For example, the subepithelial deposits may stain red (fuchsinophilic) with Masson trichrome stain. • Interstitial edema and interstitial infiltration of predominantly mononuclear leukocytes • Focal tubular epithelial cell simplification (flattening) also may accompany severe disease. • Arteries and arterioles typically have no acute changes, although preexisting sclerotic changes may be present in older patients
- 145. • Diagram depicting the continuum of structural changes that can be caused by glomerular inflammation (top), the usual clinical syndromes that are caused by each expression of glomerular injury (middle), and the portion of the continuum that most often corresponds to several specific categories of glomerular disease (bottom). ANCA, Antineutrophil cytoplasmic antibody; ESRD, end-stage renal disease; GBM, glomerular basement membrane; IgA, immunoglobulin A.
- 146. • Light micrograph of a glomerulus with features of acute poststreptococcal glomerulonephritis demonstrating marked influx of neutrophils (arrows). (Masson trichrome stain, ×700.
- 147. IgA NEPHROPATHY • Light Microscopy • any of the light microscopic phenotypes of proliferative glomerulonephritis or may cause no discernible histologic changes. • usually manifests as a focal or diffuse mesangioproliferative or proliferative glomerulonephritis, although specimens from a few patients will have no lesion by light microscopy, those from a few will show aggressive disease with crescents, and occasional specimens will already demonstrate chronic sclerosing disease. • Different criteria for performing renal biopsy result in different frequencies of the various phenotypes of IgA nephropathy among distinct populations of patients. Of 668 consecutive native kidney IgA nephropathy specimens diagnosed in the UNC Nephropathology Laboratory, 4% showed no lesion by light microscopy, 13% had exclusively mesangioproliferative glomerulonephritis, 37% had focal proliferative glomerulonephritis (25% of these had <50% crescents), 28% had diffuse proliferative glomerulonephritis (45% of these had <50% crescents), 4% had crescentic glomerulonephritis (50% or more crescents), 6% had focal sclerosing glomerulonephritis without residual proliferative activity, 6% had diffuse chronic sclerosing glomerulonephritis, and 2% had lesions that did not fall into any of these categories.
- 149. Thank you