Glomerular diseases

1. GLOMERULAR DISEASE AND ROLE OFIMMUNOFLUORESENCE IN DIAGONOSIS

2. RENAL BIOPSY In order to evaluate a kidney biopsy, the pathologist should correlate complete clinical and laboratory information with light microscope, immunofluorescence and ultrastructural findings.Biopsy adequacy:  1-2 glomeruli Electron Microscopy  3-5 glomeruli Immunofluoresence  5-10 glomeruli light Microscopy

3. RENAL BIOPSY1- Fixation (Immediate): 10% NB Formalin /Zenker’s/Bouin’s (paraffin sections) 4%Gluteraldehyde (EM) No fixation (Immunofluorescence)2- Paraffin sections cut at 3μ thickness3- Stains: PAS is the most useful, easiest to perform. Hematoxylin and eosin. Masson trichrome Silver preprations4- Immunohistochemistry (IG, C, other antigens)

4. NEEDLE BIOPSY OPEN BIOPSY Most of renal biopsy are done by ether thepercutaneous route using cutting needle or by direct exposure of kidney (open Biopsy).

5. Ideally, two biopsy cores should be obtained when a needle biopsy is performed.

6. STAINS FOR RENAL BIOPSY1. H&E General2. PAS Basement M. & Mesangial matrix3. Trichrome Fibrosis4. Silver Basement M. & Mesangial matrix5. Congo red Amyloid

7. A BRIEF INTRODUCTION ABOUT IMMUNOFLORESCENCE TECHNIQUE Immunofluorescence is a technique for light microscopy with a fluorescence microscope and is used primarily on biological samples.  This technique makes it possible to visualize antigens in the tissue section , cultured cell lines, or individual cells, and may be used to analyze the distribution of proteins, glycans, and small biological and non-biological molecules.  A fluorochrome is a dye that absorbs light and then emits its own light at a longer wavelength. This phenomenon of absorption and emission of light is called fluorescence. When the fluorochrome is attached or conjugated to antibody, the sites of reaction between antigen and labeled antibody can be visualized easily.

8. A BRIEF INTRODUCTION ABOUT IMMUNOFLUORESCENCE TECHNIQUE  The most commonly used fluorochromes in immunofluorescence technique are fluorescein isothiocyanate (FITC) and rhodamine. Both of these dyes absorb light that is not visible (UV)to the human eye and emit light that is visible.  Virtually any antigen can be detected by immunofluorescence. In most routine histopathology laboratories, kidney and skin biopsy specimens are examined with immunofluorescence technique.

9. A BRIEF INTRODUCTION ABOUT IMMUNOFLORESCENCE TECHNIQUE• Immunofluorescence microscopy provided insight not only into the pathogenesis of glomerular diseases but also is very useful in diagnosing primary renal diseases, assessing the nature and severity of renal involvement in various systemic disorders and in addition, yields important correlations and prognostic features.• Correct diagnosis of glomerulonephritis requires renal biopsy and histopathological examination by light, immunofluorescence and electron microscopic examination, and correlation with clinical features and biochemical parameters.• Facilities for electron microscopic study is not readily available in many institutions. In most cases light microscopy (LM) and direct immunofluorescence (DIF) study are more than enough for definitive diagnosis of glomerulonephritis.

10. OVERVIEW OF A NORMAL GLOMERULUS The glomerulus is a vascular structure composed of a tuft of specialized capillaries that arises from the afferent arteriole to form lobules that rejoin the vascular pole to drain into efferent arteriole. Normally, the lobules are poorly defined, but they are highlighted in some disease processes. Each lobule is supported by branching framework, the mesengium. This tuft of capillaries lies within the lumen of the expanded proximal end of the nephron, which is lined by the epithelial cells overlying a thick basement membrane.

11. NORMAL GLOMERULUS The normal glomerulus of the kidney at high powerhas thin, delicate capillary loops and the mesangium is not prominent.

12. NORMAL GLOMERULUS NORMAL GLOMERULUS Light micrograph of a normal glomerulus. There are only 1 or 2 cells percapillary tuft, the capillary lumens are open, the thickness of the glomerular capillary wall (long arrow) is similar to that of the tubular basementmembranes (short arrow), and the mesangial cells and mesangial matrix are located in the central or stalk regions of the tuft (arrows).

13. NORMAL GLOMERULUS Each glomerulus measures approximately 200 micrometers. The cellularity of the glomerulus varies in different diseases, and an accurate assessment requires histological preparations 2 to 4 microns thick. The presence of more than three cells in an individual glomerular mesangial region away from the vascular pole is considered hypercellularity. The glomerular basement membrane is a trilaminar structure composed of central lamina densa, bordered by lamina rara interna and externa. In adults GBM measures 310 to 380 nanometers and its thickness is altered in various glomerular diseases. Visceral epithelial cells have foot processes are involved in basement membrane synthesis and plays a role in glomerular permeability.

14. GLOMERULAR DISEASES  Glomerular diseases constitute some of the major problems in nephrology. Glomerulonephritis is an inflammation of the glomerulus, while glomerulopathy is a term for disorder affecting this structure.  Glomeruli may be injured by variety of factors and in course of several systemic diseases.  Most of the glomerular diseases are immunologically mediated, whereas tubular and interstitial disorders are frequently caused by toxic or infectious agents.

15. CLASSIFICATION OF GLOMERULAR DISEASE BY DISTRIBUTION A)Classification of disease distribution when many glomeruli are considered.FOCAL:- Disease affecting only some of glomeruli.DIFFUSE:- Disease affecting most or all glomeruli. B)Classification of disease distribution when single glomeruli are considered.SEGMENTAL:- a lesion involving only a part of the glomerulusGLOBAL:- a lesion involving the entire glomerulus.

16. CLINICOPATHOLOGICAL CLASSIFICATION OF GLOMERULAR DISEASE. Primary glomerular disease :- in this the glomeruli are the predominant site of involvement. Secondary glomerular disease:- includes certain systemic and hereditary diseases which secondarily involve the glomeruli.

17. THE GLOMERULAR SYNDROMES SYNDROME MANIFESTATIONSNephritic syndrome Hematuria, azotemia, variable proteinuria, oliguria,edema and hypertensionRapidly progressive Acute nephritis, proteinuria and acute renal failureglomerulonephritisNephrotic syndrome >3.5 gm/day proteinuria, hypoalbuminemia, hyperlipidemia,lipiduria.Chronic renal failure Azotemia progressing to Uremia over months to years. Isolated urinary Glomerular hematuria and/or subnephrotic proteinuria. abnormalities

18. A schema of the average patient ages associated with various common forms of nephrotic syndrome

19. HISTOLOGICAL ALTERATIONS IN GLOMERULOPATHIES Characterized by one or more of four basic tissue reactions :- Hypercellularity characterized by Increase in the number of cells inthe glomerular tufts. This hypercellularity is comprised of one or morecombination of mesangeal or endothelial cell proliferation, leukocyteinfiltration or formation of crescents. Basement Membrane Thickening on light microscopy appears asthickening of the capillary walls and is best seen by PAS staining. Onelectron microscopy it can have one of the two forms:- a) Deposition of amorphous electron dense material on endothelial orepithelial side of GBM or within the GBM itself. b) Thickening of the basement membrane due to increased synthesisof its protein components as occurs in diabetic glomerulosclerosis.

20. HISTOLOGICAL ALTERATIONS IN GLOMERULOPATHIES contt…Hyalinosis denotes the accumulation of material that ishomogenous and eosinophilic by light microscopy. By electronmicroscopy the hyaline is extracellular ,amorphous made up ofleaked plasma proteins from circulation into glomerularstructures. Hyalinosis is a consequence of endothelial orcapillary wall injury.Sclerosis is characterized by accumulation of extracellularcollagenous matrix either confined to mesengeal areas orinvolving the capillary loops or both.

21. PATHOGENESIS OF GLOMERULAR INJURY Immune mechanisms underlie most forms of primary glomerulopathies and many of secondary glomerular disorders. ANTIBODY MEDIATED IN SITU IMMUNE COMPLEX DEPOSITION CIRCULATING IMMUNE COMPLEX MEDIATED CYTOTOXIC ANTIBODIES CELL MEDIATED IMMUNE INJURY ACTIVATION OF ALTERNATIVE COMPLEMENT PATHWAY

22. Pathogenesis of Glomerular DiseaseImmunedisorder Kidney involvement Injury by inflammation and other mediators Glomerular dysfunction

23. IMMUNE MECHANISM OF CELLULAR INJURY IN GLOMERULUSANTIBODY MEDIATED IMMUNE COMPLEX MEDIATEDFixed Antigens Planted Antigens Endogenous Antigens Exogenous Antigens-NC 1 domain -Exogenous - DNA - Infectious products(anti GBM Nephritis) infectious agents - Tumor antigens-Heymann antigen Drugs-Mesangial antigens -Endogenous DNA, nuclear proteins, immunoglobulins.

24. NON IMMUNE MECHANISM OF CELLULAR INJURY IN GLOMERULUS METABOLIC GLOMERULAR INJURY Diabetic nephropathy, Fabry’s disease HAEMODYNAMIC GLOMERULAR INJURY Systemic hypertension DEPOSITION DISEASES Amyloidosis, cryoglobulinaemia. INFECTIOUS DISEASES HBV, HCV, HIV, E.coli-derived nephrotoxin INHERITED GLOMERULAR DISEASES Alports syndrome

25. ACUTE PROLIFERATIVEGLOMERULONEPHRITIS

26. ACUTE PROLIFERATIVE GLOMERULONEPHRITIS This condition is characterized histologically by diffuse proliferation of glomerular cells, associated with influx of leucocytes. These lesions are typically caused by immune complexes. The inciting antigen may be exogenous or endogenous. The prototype exogenous antigen-induced disease pattern is postinfectious glomerulonephritis.

27. ACUTE PROLIFERATIVE GLOMERULONEPHRITIS APGN has mainly two types- POST STREPTOCOCCAL GLOMERULONEPHRITIS. NON - STREPTOCOCCAL GLOMERULONEPHRITIS.

28. ACUTE PROLIFERATIVE GLOMERULONEPHRITISPOST STREPTOCOCCAL GLOMERULONEPHRITIS.Produces the nephritic syndrome (hematuria, red cell casts, moderate proteinuria and edema) in children two weeks following a respiratory or skin infection with a "nephritogenic strain" of group A, beta-hemolytic streptococci.Clinically, this disease is manifested by a rather abrupt onset of gross hematuria, edema, proteinuria, hypertension and impaired renal function.The serum levels of hemolytic complement activity and C3 protein are abnormally reduced.

29. ACUTE PROLIFERATIVE GLOMERULONEPHRITISPOST STREPTOCOCCAL GLOMERULONEPHRITIS.Latent period between infection and onset of nephritis is compatible with the time required for the production of antibodies and formation of immune complexes.There is deposition of circulating immune complexes which fix complement and attract PMNs.This chokes off their blood supply, making the glomeruli hypercellular and bloodless.This explains the oliguria, edema, and hypertension2% to 5% patient die during acute episode.

30. ACUTE PROLIFERATIVE GLOMERULONEPHRITISLIGHT MICROSCOPY  Enlarged , hypercellular glomeruli.  Hypercellularity is caused by infiltration of both leucocytes and monocytes, proliferation of endothelial and mesenchymal cells and in severe cases there crescent formation.  Interstitial edema  Tubules often contain red cell casts

31. ACUTE PROLIFERATIVE GLOMERULONEPHRITISIMMUNOFLUORESCENCE  Granular deposits of IgG,IgM and C3 in the mesengium and along the GBM.  Immune complex deposits are almost universally present and can even be focal or sparse.

32. ACUTE PROLIFERATIVE GLOMERULONEPHRITISELECTRON MICROSCOPY The findings are discrete, amorphous, electron dense deposits on the epithelial side of basement membrane, often having appearance of humps

33. ACUTE PROLIFERATIVE GLOMERULONEPHRITIS

34. ACUTE PROLIFERATIVE GLOMERULONEPHRITIS NON – STREPTOCOCCAL GLOMERULONEPHRITIS This similar form of glomerulonephritis occurs sporadically in association with other infections, including Bacterial:- staphylococcal endocarditis, pneumococcal pneumonia and meningococcemia. Viral:-hepatitis B, hepatitis C, mumps, HIV, varicella. Parasitic:-malaria, toxoplasmosis. In these settings, granular immunofluorescent deposits and subepithelial humps characteristic of immune complex nephritis are present.

35. RAPIDLY PROGRESSIVEGLOMERULONEPHRITIS

36. RAPIDLY PROGRESSIVE GLOMERULONEPHRITIS Severe form of glomerulonephritis in which majority of the glomeruli are involved by epithelial crescents. RPGN is characterized by rapid and progressive loss of renal function accompanied by hematuria, variable proteinuria and severe oliguria. Most common histological picture is the presence of crescents in most of glomeruli.

37. RAPIDLY PROGRESSIVE GLOMERULONEPHRITISCLASSIFICATION BASED ON IMMUNOLOGICAL FINDINGS TYPE I (ANTI-GBM ANTIBODY) Renal limited Goodpasture syndrome TYPE II (IMMUNE COMPLEX) Idiopathic The common Post infectious glomerulonephritis denominator in all Lupus Nephritis types of RPGN is Henoch-Schonlein purpura severe glomerular Others TYPE III (PAUCI- IMMUNE) injury ANCA-associated Idiopathic Wegener granulomatosis Microscopic Polyangitis

38. RAPIDLY PROGRESSIVE GLOMERULONEPHRITISTYPE I It is anti-GBM antibody induced disease, characterized byRPGN linear deposits of IgG and C3 in the GBM. In some individuals, the anti- GBM antibodies cross react with pulmonary aleveolar basement membraneand produces pulmonary hemorrhage associated with renal failure (Goodpastures Syndrome) The Goodpasture antigen is a peptide within the noncollagenous portion of the α3 chain of collagen type IV.TYPE II It is a result of immune complex deposition. RPGN It can be a complication of any of the immune complex nephritides. In all its types I/F study reveals granular pattern of staining characterized of immune complex deposition. Also called pauci-immune type.TYPE III Defined by lack of anti-GBM antibodies or immune RPGN complexes by immunofluorescence and electron microscopy. Most patients with this type of RPGN have circulating antineutrophil cytoplasmic antibody (ANCAs), hence there is a component of systemic vasculitis seen.

39. RAPIDLY PROGRESSIVE GLOMERULONEPHRITISGROSS Enlarged ,pale kidney with petechial hemorrhage on cortical surface

40. RAPIDLY PROGRESSIVE GLOMERULONEPHRITIS Light microscopy can provide useful information to help distinguish which subclass (RPGN I, II, III) of crescentic glomulonephritis is present. RPGN type II have more segmental hypercellularity than types I and III. Necrotizing changes of glomerular tufts are more common in RPGN types I and III. In particular, comparing RPGN Type I to RPGN type III, the pauci-immune disease is more often associated with necrotizing arteritis

41. RAPIDLY PROGRESSIVE GLOMERULONEPHRITIS LIGHTMICROSCOPY Glomeruli may show focalnecrosis, diffuse or focal endothelialproliferation and mesengial proliferation.The histological picture is dominated by distinctive crescents. Crescentric glomerulonephritis. Collapsed glomerular tuft and crescent-shaped mass of proliferating parietal epithelial cells.

42. RAPIDLY PROGRESSIVE GLOMERULONEPHRITIS WHAT ARECRESCENTS ?  Crescents are formed by proliferation of partial cells and by migration of monocytes and macrophages into urinary space.  The crescents eventually obliterate bowman space and compress the glomerular tuft.  Fibrin strands are frequently prominent between the cellular layers in the crescents.  The escape of fibrinogen into bowman space and its conversion to fibrin are an important contributor to crescent formation.

43. RAPIDLY PROGRESSIVE GLOMERULONEPHRITISIMMUNOFLUORESCENCE Type I RPGN•Anti GBMdisease•Good pasturesyndrome Direct immunofluorescence shows smooth, linear staining of the glomerular capillary basement membranes for IgG. Similar staining pattern would also be seen for C3.

44. RAPIDLY PROGRESSIVE GLOMERULONEPHRITISIMMUNOFLUORESCENCE Type II RPGN Immunofluorescence reveals the granular pattern of staining characteristic of immune complex deposition

45. RAPIDLY PROGRESSIVE GLOMERULONEPHRITISIMMUNOFLUORESCENCE Type III RPGN It has weak or no demonstrable immunoglobulin / complement deposition,

46. RAPIDLY PROGRESSIVE GLOMERULONEPHRITIS ELECTRONMICROSCOPY Crescentric glomerulonephritis showing characteristic wrinkling of GBM with focal disruptions (arrows)

47. MEMBRANOUS NEPHROPATHY

48. MEMBRANOUS NEPHROPATHY Common cause of nephrotic syndrome in adults. Characterized by diffuse accumulation of electron dense, Ig-containing deposits along the subepithelial side of basement membrane. In about 85% patients no associated condition can be uncovered and are considered idiopathic. In about 15% patients there is other systemic condition associated with it and is referred to as secondary membranous glomerulopathy. Formation of membrane attack protein {C5b-C9} causes the capillary damage and hence leakage of proteins.

49. MEMBRANOUS NEPHROPATHY contt…SECONDARY MEMBRANOUS GLOMERULOPATHY It is a form of chronic immune complex-mediated disease. The inciting antigens can sometimes be identified in the immune complexes. For eg exogenous antigen (hepatitis B or Treponema), Endogenous nonrenal antigens (thyroglobin), Endogenous renal antigens (membrane protein antigen) The most notable associations are seen with Drugs— penicillamine, captopril,gold NSAIDs Malignant tumors --- Ca lung, Ca colon , Melanoma SLE--- 10%-15% of glomerulonephritis in SLE is of membranous type Infections--- Hepatitis B, Hepatitios C, Syphilis, malaria. Auto immune disorders--- Thyroiditis.

50. MEMBRANOUS NEPHROPATHY contt… LIGHT MICROSCOPY Uniform, diffuse thickening of the glomerular capillary wall. Basement membrane material is laid down between these deposits, appearing as irregular spikes protruding from the GBM. These spikes are best seen by silver stains, which color the basement membrane black.Marked diffuse thickening of the capillary walls without an increase in the number of cells. There are prominent spikes projecting from basement membrane.

51. MEMBRANOUS NEPHROPATHY contt…

52. MEMBRANOUS NEPHROPATHY contt… IMMUNOFLUORESCENCE Immunofluorescence microscopy: granular deposits contain both immunoglobulins and various amounts of complement . The course of the disease is variable but generally indolent. Although proteinuria persists in more than 60% of patients, only about 10% die or progress to renal failure within 10 years, and no more than 40% eventually develop renal insufficiency.

53. MEMBRANOUS NEPHROPATHY contt…ELECTRON MICROSCOPY Electron micrograph showing electron dense deposits along the epithelial side of the basement membrane. There is effacement of foot processes overlying deposits.

54. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS

55. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS Derived from the characteristic histological changes by light microscopy Accounts for 10% to 20% of cases of nephrotic syndrome. MPGN is characterized by alteration of glomerular cells and leukocyte infiltration. Proliferation is predominantly in the mesengium and involves capillary loops also , hence a synonym mesengiocapillary glomerulonephritis is used. Persistent and slowly progressive.

56. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS CLASSIFICATION Primary MPGN :- when the cause is idiopathic. on the basis of distinct ultra structural, immunofluorescence and pathological findings it is divided into:- A) Type I MPGN B) Type II MPGN (dense deposit disease) C) Type III MPGN (very rare, it is characterized by a mixture of subepithelial deposits and the typical pathological findings of Type I disease) Secondary MPGN :- when associated with other systemic disorders.

57. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS TYPE I MPGNAccount for approx 5% of the cases of end stage renal disease. Children and young adults more frequently involved.Approx one third of patients present with nephritic syndrome.Approx two thirds of the patients develophypocomplementemia with predominant depletion in C3 levels.Evidence of immune complexes in the glomerulus andactivation of both classical and alternative complement pathways.Antigens involved are unknown, sometimes believed to beprotein derived from infectious agents like hepatitis C and Bviruses.(planted antigens)

58. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS TYPE II MPGN (Dense Deposit Disease)Shows unique morphological appearance of basementmembrane best seen by electron microscopy.Much more rare than type 1.Type II MPGN tends to present with nephritis while MPGNtype I presents more often with nephrotic features.There is activation of alternative complement pathway.Serum levels of C3 remains low for a longer period than type Idisease.More than 70% of patients have a circulating antibody termedC3 nephiritic factor ( C3NeF ).

59. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS LIGHT MICROSCOPY Light microscopy of both types of MPGN are similar , but the cellularproliferation, and especially the circumferential mesengeal interposition isless prominent in type 2 MPGN.Glomeruli are large and hypercellular.Hypercellularity is produced both by proliferation of cells in themesengium and endocapillary also.Glomeruli have lobular appearance due to proliferating mesengial cells andincreased mesengeal matrix.The GBM is thickened, often segementally.The glomerular capillary wall often show a double contour or tram-trackappearance especially evident in silver or PAS stains. This is caused bysplitting of GBM.

60. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS LIGHT MICROSCOPY Thickening of capillary walls, usually global and diffuse. There is also hypercellularity. Much of this hypercellularity is mesangial proliferation, and some of the capillary wall thickening is caused by mesangial interposition into the subendothelial zone of the capillary loops.

61. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITISIMMUNOFLUORESCENCE Type I MPGNThere are C3, IgG and IgM deposits, being those of C3 more frequentand constant. These deposits are granular in the capillary walls.Often they are elongated and smooth in their external edge becausethey are subendothelial and they are molded to the GBM.

62. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITISIMMUNOFLUORESCENCE Type II MPGNThe bright deposits scattered along capillary walls and in themesangium by immunofluorescence microscopy with antibody tocomplement component C3 are typical for membranoproliferativeglomerulonephritis, type II.

63. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITISELECTRON MICROSCOPY TYPE I (MPGN) Marked thickening of glomerular capillary wall by immune deposits and by Normal glomerular capillary loop showing interposition of mesengeal cell processes.normal endothelial cells, GBM and epithelial There are two layers of GBM surrounding cells.GBM is thin and no electron dense the mesengeal interposition that account deposits are present. for double contour appearance on light microscopy.

64. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS ELECTRON MICROSCOPY TYPE II (MPGN)Normal glomerular capillary loop Dense ribbon like appearance of sub-showing normal endothelial cells, GBM endothelial and intramembranousand epithelial cells.GBM is thin and no material and narrowing of theelectron dense deposits are present. capillary lumen due to proliferation of cells.

65. MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS SECONDARY MPGNMore common in adults and arises in the following conditions Infections HepatitisB and C, endocarditis, Visceral abscesses, malaria, schistisomiasis, mycoplasma, HIV and EB virus infectionImmunological SLE, Scleroderma, sjogren’s syndrome, rheumatoid disorders arthritis, sarcoidosis. Neoplastic Carcinoma, CLL, NHL, melanoma. diseases Hereditary α1 antitrypsin deficiency, complement deficiency, diseases hereditary angioedemaMiscellaneous Drug abuse, kartagener’s syndrome, turner’s syndrome, down’s syndrome

66. IgA NEPHROPATHY

67. IgA NEPHROPATHYIt is the commonest form of glomerulonephritis resulting inESRD throughout the world. Male predominance.Also known as IgA nephritis, Bergers disease, Synpharyngitic glomerulonephritisFrequent cause of gross and microscopic hematuriaCharacterized by the presence of prominent IgA deposits in themesengeal region.Suspected by light microscopy, but diagnosis is made only byimmunochemical method.

68. IgA NEPHROPATHYPATHOGENESIS Abnormalities of immune regulation leads to increased IgA synthesis in response to respiratory or gastrointestinal exposure to environmental agents. IgA1 (nephritogenic form) and IgA1-containing immune complexes are then trapped in the mesengium, where they activate the alternative complement pathway and initiate glomerular injury. IgA nephropathy occurs with increased frequency in individuals with gluten enteropathy and in liver diseases (defective hepatobiliary clearence of IgA complexes)

69. IgA NEPHROPATHYLIGHT MICROSCOPY Glomeruli may be normal or may show mesengial widening and endocapillaryproliferation.The mesengial widening is due to cell proliferation, accumulation of matrix and immune deposits.

70. IgA NEPHROPATHYIMMUNOFLUORESCENCE The IF pattern parallels the distribution of deposits seem by EM. Itshows deposits of IgA often with C3 and properdin and lesser amounts of IgG or IgM. Early complement components are usually absent.

71. IgA NEPHROPATHY ELECTRON MICROSCOPYPortion of a glomerulus from a patient with IgA nephropathy showing electron-dense mesengial deposits

72. MINIMAL CHANGE DISEASE

73. MINIMAL CHANGE DISEASEAccounts for 80% of all cases of the idiopathic nephrotic syndrome in children.Majority of cases seen in 3 to 4 year age groups.Male predominance of 2.5:1 in children but no difference seen in adults.80-90% idiopathic.Associated with infectious disease, recent immunization, ingestion of heavymetals.In adults related to use of NSAIDS.A full blown nephrotic syndrome with heavy proteinuria often of selective type isthe most common presentation.On light microscopy changes are seen in the convoluted tubules where largeamounts of lipid and protein droplets accumulate in the cell cytoplasm.(lipoidnephrosis)In contrast all the glomeruli appear normal.

74. MINIMAL CHANGE DISEASELIGHT MICROSCOPY The glomerulus is normocellular, the capillary loops are patent, and the basement membrane is normal in thickness

75. MINIMAL CHANGE DISEASEIMMUNOFLUORESCENCENo deposits of complement or immunoglobulins are recognized in IF. (nil deposit disease)

76. MINIMAL CHANGE DISEASE ELECTRON MICROSCOPY Portion of a glomerulus from a patient with minimal change glomerulopathy showing obliteration of foot processes. The epithelialcell cytoplasm is hyperactive and shows microvillus and cyst formation.

77. FOCAL SEGMENTALGLOMERULOSCLEROSIS

78. FOCAL SEGMENTAL GLOMERULOSCLEROSISFocal segmental glomerulosclerosis (FSGS) defines acharacteristic pathologic pattern of glomerular injuryand is not necessarily a distinct disease.The hallmark of kidney biopsy is an increaseddegree of scarring seen on light microscopy of somebut not all of the glomeruli present (focal) thatinvolves some but not all portions of the affectedglomeruli (segmental).Characterized by proteinuria commonly innephrotic range.

79. FOCAL SEGMENTAL GLOMERULOSCLEROSIS CLASSIFICATIONFSGS may be primary (idiopathic) or secondary to various etiologies. PRIMARY SECONDARY Typical FSGS Unilateral renal agenesis Collapsing HIV infection glomerulopathy Heroin addiction Glomerular tip lesion Morbid obesity Sickle cell disease Congenital heart disease Glycogen storage disease Hypertensive nephropathy

80. FOCAL SEGMENTAL GLOMERULOSCLEROSISPrimary FSGS makes up approx 10% to 15% of nephroticsyndrome in children and 20% to 30% in adults.It is the predominant cause of idiopathic nephrotic syndromein adults.Mainly sporadic type.Involves children under 5 years of age and adults in 3rd and4th decades.Onset is insidious.40% to 60% of patients progress to end stage renal diseasewithin 10 to 20 years.

81. FOCAL SEGMENTAL GLOMERULOSCLEROSIS LIGHT MICROSCOPYFocal and segmental lesions may involve only a minority of the glomeruli andmay be missed if the biopsy specimen contains an insufficient number ofglomeruli.The lesion initially tends to involve the juxtamedullary glomeruli andsubsequently becomes generalized.In the sclerotic segments there is collapse of capillary loops, increase inmatrix and segmental deposition of plasma proteins along the capillary wall(hyalinosis).The hyalinosis may become so pronounced as to occlude the capillary lumen.Lipid and foam cells are often present.Glomeruli that do not show segmental lesion usually appear normal.

82. FOCAL SEGMENTAL GLOMERULOSCLEROSISLIGHT MICROSCOPYBiopsy from apatient withFSGS.One of theglomeruli showssegmentalsclerosis whileothers appearunremarkable.Tubular atrophyis also seen.

83. FOCAL SEGMENTAL GLOMERULOSCLEROSIS COLLAPSING GLOMERULOPATHY It is clinically andpathologically distinct variantof FSGS Characterized bywidespread collapse ofglomerular capillary loops. Poor prognosis with rapidloss of renal function and noresponse to therapy. It can occur as an idiopathicdisease or as secondaryprocess associated with i/vdrug abuse or HIV infection. Collapsing glomerulopathy. Visible retraction of the glomerular tuft, narrowing of capillary lumens, proliferation and swelling of visceral epithelial cells, and prominent accumulation of intracellular protein absorption droplets in the visceral epithelial cells.

84. FOCAL SEGMENTAL GLOMERULOSCLEROSISGLOMERULAR TIP LESION Variant of FSGS. Characterized by aconsolidation of the glomerularsegment adjacent to the origin ofproximal tubule. The capillary Lumina ofsclerotic loops may appearobliterated by swelling ofendothelial cells and presence offoamy cells. The epithelial cells adjacent tothe involved segment areenlarged, vacuolated and oftencontain hyaline droplets.In this glomerulus we can see the location of the lesions characterizedby a "tip" (from the glomerular tip). There are adhesions and sclerosis, hyaline deposits and endocapillary hypercellularity.

85. FOCAL SEGMENTAL GLOMERULOSCLEROSISIMMUNOFLUORESCENCE Immunofluorescence microscopy demonstrating segmental deposition of IgM in a biopsy from a patient with focal and segmental glomerulosclerosis.

86. FOCAL SEGMENTAL GLOMERULOSCLEROSIS ELECTRON MICROSCOPYEarly focal and segmental glomerulosclerosis. There is mild segmental prominence of the mesangium (upper third) and vacuolization of the epithelial cell cytoplasm. A lipid-laden intracapillary cell with foamy cytoplasm is also present (arrows).

87. DIABETIC NEPHROPATHY

88. DIABETIC NEPHROPATHYDiabetic nephropathy is the leading cause of chronic renal failure in the industrialised world.It is also one of the most significant long-term complications in terms of morbidity and mortality for individual patients with diabetes.Diabetes is responsible for 30-40% of all end-stage renal disease (ESRD) cases.Most of the patients who develop diabetic nephropathy have had diabetes for at least 10 years, so it is more frequent in type I DM than in type II DM. By far the most common lesions involve the glomeruli and are associated clinically with three glomerular syndromes: non- nephrotic proteinuria, nephrotic syndrome and chronic renal failure.

89. DIABETIC NEPHROPATHYThree lesions that are encountered in diabetic nephropathy are :- GLOMERULAR LESIONS. CAPILLARY BASEMENT MEMBRANE THICKENING DIFFUSE GLOMERULOSCLEROSIS NODULAR GLOMERULOSCLEROSIS INSUDATIVE LESIONS RENAL VASCULAR LESIONS, PRINCIPALLY ARTERIOLOSCLEROSIS. PYELONEPHRITIS, INCLUDING NECROTIZING PAPILLITIS.

90. DIABETIC NEPHROPATHY CAPILLARY BASEMENT MEMBRANE THICKENING There is wide spread thickening ofthe GBM and occurs virtually in allcases of diabetic nephropathy andforms the part and parcel of diabeticmicroangiopathy. Pure capillary basementmembrane thickening can bedetected only by electronmicroscopy. Thickening begins as early as 2years after the onset of type I DMand by5 years amounts to about a 30% increase. RENAL GLOMERULUS SHOWING MARKEDLY THICKNED GLOMERULAR BASEMENT MEMBRANE

91. DIABETIC NEPHROPATHY DIFFUSE GLOMERULOSCLEROSIS  Most common lesion in diabetic nephropathy.  Diffuse increase in mesangial matrix and thickening of capillary wall.  Mesangial increase is typicallyassociated with overall thickening of GBM. Matrix deposition is PAS-positive.  As the disease progresses, the expansion of mesangial areas can extend to nodular configuration. Early diffuse diabetic glomerulosclerosis showing a mild increase inmesangial matrix and thickened capillary walls. The arteriole shows the typical hyaline appearance of an insudative lesion.

92. DIABETIC NEPHROPATHY NODULAR GLOMERULOSCLEROSIS Also known as Intercapillaryglomerulosclerosis or Kimmelstiel-wilson disease. Consists of largely acellular nodules that are located in the intercapillary regions. Nodules vary in size and often have laminated appearance.They are ecsinophilic, argyrophilic, PAS- positive and stain green with masson’s trichrome stain and blue with mallory’s stain.Ultrastructurally they are composed ofmasses of extra cellular mesangial matrix which is the result of both increasedsynthesis and decreased degradation of mesangial matrix. NODULE OF DIABETIC GLOMERULOSCLEROSIS

93. DIABETIC NEPHROPATHY INSUDATIVE LESIONS The nodular lesion are frequently accompanied by prominent accumulations of hyalinematerial in capillary loops (fibrin caps) or adherent to bowman’s capsule (capsular drops).  Histological and immunofluorescence studies indicate that this insudative material represents infiltration by constituents of the plasma, including protein, lipids and mucopolysaccharides. FIBRIN CAP CAPSULAR DROP

94. DIABETIC NEPHROPATHY IMMUNOFLUORESCENCEDiffuse linear localization of IgG along glomerular and tubular basement membranes andbowman’s capsule is the most common immunofluorescence finding in diabetic nephropathy. Diabetic glomerulosclerosis with linear staining for IgG along the glomerular basement membrane.

95. LUPUS NEPHRITIS

96. LUPUS NEPHRITIS Immune mediated nephritis is the common complication of SLE. The pathogenesis of this lesion is likely to be related to the inflammation response resulting from the presence of immune aggregates at the site of injury. The most constant feature, which is found in nearly all patients with clinical lupus nerhritis is proteinuria. A kidney biopsy is essential in the renal assessment of patients with SLE. The pathological findings of lupus nephritis are extremely diverse and may occur in any or all four renal compartments: glomeruli, tubules, interstitsium and blood vessels. This diversity may be the result of differences in the immune response in different patients or in same individual over a period of time.

97. WHO LUPUS WITH PATHOLOGICAL CORRELATIONCLASSIFICATION OF NEPHRITISClass I Light microscopy findings NormalMinimal mesangial lupus IF/EM Findings Mesangial immune depositsnephritisClass II Light microscopy findings Purely mesangial hypercellularity or mesangial matrixMesangial proliferative expansion with mesangial immune depositslupus nephritis IF/EM Findings Mesangial immune deposits; few immune deposits in subepithelial or subendothelial deposits possibleClass III Light microscopy findings Active or inactive focal, segmental, or globalFocal lupus nephritis glomerulonephritis involving <50% of all glomeruli IF/EM Findings Subendothelial and mesangial immune depositsClass IV Active or inactive diffuse, segmental or globalDiffuse lupus nephritis glomerulonephritis involving >50% of all glomeruli; Light microscopy findings subdivided into diffuse segmental (class IV-S)and diffuse global (class IV-G) IF/EM Findings Subendothelial immune depositsClass V Light microscopy findings Diffuse thickening of glomerular basement membraneMembranous lupus without inflammatory infiltrate .nephritis IF/EM Findings Subepithelial and intramembranous immune depositsClass VI Light microscopy findings Advanced glomerular sclerosis involving >90% ofAdvanced sclerosis lupus glomeruli, interstitial fibrosis, and tubular atrophy.nephritis IF/EM Findings Few , if any immune deposits.

98. LUPUS NEPHRITIS CLASS II : There is mild diffuse mesangial hypercellularity and an increase in matrix. These mesangial deposits can be identified by immunofluorescence.CLASS III : There is focal andsegmental glomerulonephritischaracterized by segmentalnecrosis, adhesions to bowman’scapsule, and leucocyticinfiltration.

99. LUPUS NEPHRITIS CLASS IV : lupus nephritis showing a well circumscribed area of necrosis containing small hematoxylin bodies.CLASS IV: glomerulus showingseveral wire-loop lesions

100. LUPUS NEPHRITIS CLASS IV: Glomerulus with an epithelial crescent showing two hyaline thrombi.CLASS IV: IF prepration for IgGshowing large amounts ofimmunocomplex deposits not onlyin the mesengium and along theglomerular capillary loops, but alsoaround the tubular basementmembrane, interstitium.

101. LUPUS NEPHRITIS CLASS V : The capillary walls are thickened and the mesangial matrix increased.Advanced glomerularsclerosis in lupus nephritisclass VI

102. RENALAMYLOIDOSIS

103. RENAL AMYLOIDOSIS It designates a group of conditions characterized by extracellular deposition of fibrillar proteins that have a β-pleated sheet configuration on X-ray diffraction analysis. Nonselective proteinuria, with or without nephrotic syndrome is the most common manifestation of renal involvement by amyloidosis. Most significant deposition of amyloid in the kidneys is in the glomeruli, but it also takes place around tubules, within interstitium and the walls of blood vessels. The glomerular deposits first appear as subtle thickenings of the mesangial matrix, accompanied by uneven widening of the basement membranes. Eventually the amyloid deposits obliterate the glomerulus completely. The morphological features of the deposits do not differ in AL or AA amyloidosis.

104. RENAL AMYLOIDOSIS H & E STAINING Panoramic image of a renal biopsy showing glomeruli with massivedistension of the mesangial areas by amyloid. The wall of an arteriole (right side of image) is also laden with amyloid (H&E stain)..

105. RENAL AMYLOIDOSIS CONGO RED STAININGThe production of an apple green colorby polarized light in congo red stainedsections is probably the most reliablelight microscopic method for diagnosingamyloidosis.Staining procedure must be performedon sections that are at least 8 μm thick.The fibrils of primary AL and secondaryAA amyloidosis can be distinguished bypretreating tissue sections withpotassium permanganate before congored staining. Under these conditions,AAamyloid fibrils lose their affinity forcongo red stain and the birefringence islost, where as AL amyloid is not affected. Glomerulus from renal biopsy stained with congo red and examined by polarization microscopy. The characteristic "apple-green" birefringence of amyloid is apparent (Congo red stain).

106. RENAL AMYLOIDOSIS FLUORESCENT STAINING Fluorescent stains like thioflavin-S or T bind to amyloid and fluoresceyellow under ultraviolet light (i.e amyloid emits secondary fluorescence)

107. RENAL AMYLOIDOSISELECTRON MICROSCOPYElectron micrograph of a glomerulus showing the characteristic non- branching fibrils of amyloid in a random "felt-like" pattern of distribution.

108. RENAL AMYLOIDOSIS Immunofluorescence may reveal the accumulation of immunoglobulins in a non specific pattern.  By using antibodies specific for amyloid AA and light chains, it is possible to differentiate between amyloids AA and AL. KAPPA LAMBDAAnti-immunoglobulin light chains (κ or λ) are useful for amyloid AL diagnosis

109. FIBRILLARY GLOMERULONEPHRITIS ANDIMMUNOTACTOID GLOMERULOPATHY

110. Fibrillary glomerulonephritis and immunotactoid glomerulopathy FIBRILLARY GLOMERULONEPHRITIS It is a morphological variant of glomerulonephritis associated with characteristicfibrillar deposits in the mesangium and glomerular capillary walls. These fibrillar deposits resemble amyloid fibrils superficially but differultrastructurally and do not stain with congo red. The fibrils most often are 18 t0 24 nm in diameter and hence are larger than 10 to 12 nmfibrils characteristic of amyloid. The glomerular lesions usually show MPGN like pattern under light microscopy, andby immunofluorescence microscopy, there is selective deposition of polyclonal IgG, C3and light chains. Clinically the patient develops nephrotic syndrome. IMMUNOTACTOID GLOMERULOPATHY It is much rare condition. The deposits are microtubular in structure and are 30 to 50 nm in width

111. Fibrillary glomerulonephritis and immunotactoid glomerulopathy Fibrillary glomerulonephritis. Immunotactoid glomerulopathyRandomly arranged microfibrillary showing intramembranous fibrils withdeposits (diameter: 18–23 nm). MES, a diameter of around 45nm. (× 20,000) mesangium.

112. Renal involvement in Plasma cell dyscrasias

113. Renal involvement in Plasma cell dyscrasias Patients with plasma cell dyscrasias (myeloma) may exhibit a variety of renal manifestations as a result of damage from circulating light- and heavy-chain immunoglobulin components produced by the neoplastic plasma cells. It is associated with:- [1] Amylodosis, in which the fibrils are usually composed of monoclonal λ light chains. [2] Deposition of monoclonal immunoglobins or light chains in GBM. [3] Distinctive nodular glomerular lesions resulting from the deposition of nonfibrillar light chains.

114. Renal involvement in Plasma cell dyscrasias LIGHT MICROSCOPYAffected glomeruli are enlarged and the deposition of the markedly PAS- positive materialproduces capillary wall thickening and nodular expansion of mesangium.The extend of glomerular involvement can vary in a biopsy from mild mesangial expansion to afully developed nodular glomerulosclerosis that resembles diabetic glomerulosclerosis. LIGHT CHAIN DISEASE HEAVY CHAIN DISEASE Light and heavy chain deposition disease. There are similar ﬁndings in light and heavy chaindeposition disease with mesangial nodularity. Prominent hypercellularity is associated with heavy chain deposition disease compared with light chain deposition disease

115. Renal involvement in Plasma cell dyscrasias IMMUNOFLUORESCENCEImmunofluorescence microscopy demonstrates staining of the abnormal lightchain along the glomerular and tubular basement membranes, as well as inthe mesengium, vessel walls, and interstitium. A B Light chain deposition disease. Note linear staining along peripheralcapillary walls in glomeruli and tubular basement membranes in A and predominantly granular mesangial staining for light chains in B.

116. Henoch-SchӦnlein purpura

117. Henoch-SchӦnlein purpura Henoch-SchӦnlein purpura (HSP) is a systemic vasculitis characterized by purpuric skin lesions unrelated to any underlying coagulopathy. The classic clinical triad of HSP is palpable purpura, joint symptoms, and abdominal pain. However, renal involvement is the most serious complication. A small number of patients develop a rapidly progressive form of glomerulonephritis with many crescents. IgA is deposited in the glomerular mesangium in a distribution similar to that of IgA nephropathy

118. Henoch-SchӦnlein purpura Light microscopic finding varies from mild focal mesangial proliferation todiffuse mesangial proliferation and/or crescentric glomerulonephritis. Immunofluorescence microscopy shows deposition of IgA, sometimes withIgG and C3, in the mesangial regionModerate to marked mesangial Glomerular deposits of IgA show amatrix expansion can be seen in global homogenous and granular this glomerulus mesangial pattern by

119. MIXEDCRYOGLOBULINEMIA

120. MIXED CRYOGLOBULINEMIA  Cryoglobulins are complexes of one or more different classes of immunoglobulins that precipitate at lower temperature (4 degree) and become soluble again when temperatures are elevated. Mixed cryoglobulinemia ia a systemic condition in which deposits of cryoglobulins composed principally of IgG-IgM complexes induce vasculitis, synovitis and a proliferative glomerulonephritis typically MPGN.

121. MIXED CRYOGLOBULINEMIA LIGHT MICROSCOPYThe most common finding in renal biopsies from patients with mixed cryoglobulinemia is a diffuse proliferative glomerulonephritis often with membranoproliferative pattern. In more acute cases, the depositsproduce the appearance of thrombior wire loops comparable to what is seen in lupus glomerulonephritis.IMMUNOFLUORESCENCE  Immunofluorescence usually demonstrates positivity for the immunoglobins present in the cryoglobulins in the glomeruli and vessels. C3 is often found in these locations.

122. Goodpasture syndrome,Microscopic polyangitis,Wegener granulomatosis

123. Goodpasture syndrome, Microscopic polyangitis, Wegener granulomatosis They are all associated with glomerular lesions. Glomerular lesions in these three conditions can be histologically similar and are principally characterized by foci of glomerular necrosis and crescent formation. In early or mild form of involvement, there is focal and segmental, sometimes necrotizing, glomerulonephritis. In more severe cases associated with RPGN , there is more extensive necrosis, fibrin deposition, and extensive formation of epithelial crescents and leads to global scarring.

124. ALPORT’S SYNDROME

125. ALPORT’S SYNDROME Alports Syndrome is a primary basement membrane disorder manifested by progressive nephritis(hematuria and proteinuria), deafness and ocular abnormalities. Approx 80%-85% of patients have X linked form of syndrome resulting from mutation of COL4A5. Gross or microscopic hematuria is the most common and earliest manifestation.

126. ALPORT’S SYNDROME LIGHT MICROSCOPY Light microscopy findings are nonspecific.  There is focal and segmental glomerular hypercellularity of the mesangial and endothelial cells.  Renal interstitial foam cells can be found and represent lipid-laden macrophages which can be seen in many renal diseases.

127. ALPORT’S SYNDROME IMMUNOFLUORESCENCE Monoclonal antibodies directed against α3(IV), α4(IV), and α5(IV) chains of type IV collagen can be used to evaluate the GBM for the presence or absence of these chains. The absence of these chains from the GBM is diagnostic of AS and has not been described in any other condition.Normal diffuse linear staining for α5(IV). Staining for α5(IV) completely negative

128. ALPORT’S SYNDROMEELECTRON MICROSCOPY The most significant morphological finding ofalports syndrome can only be seen by electron microscopy. The typical lesion is thickening of glomerular basement membrane with transformation of lamina densa into multipleinterwoven lamellae which enclose electron-lucent areas containing roundgranules of variable density. Glomerular capillary loop showing diffuse, irregular thickening of GBM . The lamina densa is split into multiple interwoven lamellae

129. THIN BASEMENT MEMBRANE LESION

130. THIN BASEMENT MEMBRANE LESION It is a common hereditary entity manifested by familial asymptomatic hematuria. There is thinning of GBM to between 150- 250 nm. The anomaly is due to mutation in genes encoding α3 or α4 chains of type IV collagen. This disorder is to be distinguished from IgA nephropathy (other cause of hematuria) and Alports syndrome.

131. THIN BASEMENT MEMBRANE LESIONELECTRON MICROSCOPY Ultrastructural nature of the glomerular basement membrane in thin basementmembrane nephropathy (A) Normal adult male kidney (B) In TBMN, the GBM does not revel any structural abnormalities, but it is characteristically thinned, sometimes having only approximately half of the thickness in a normal kidney

132. FABRY’S DISEASE

133. FABRY’S DISEASE Also called Angiokerotoma corporis diffusum universale. Fabry’s disease is an X-linked recessive lysosomal storage disease that is caused by deficient activity of the lysosomal enzyme α-galactosidase A (α-Gal A). This deficiency results in accumulation of globotriaosylceramide (Gb3). Gb3 accumulates in many cells, particularly in renal epithelial cells, endothelial cells, pericytes, vascular smooth muscle cells, cardiomyocytes, and neurons of the autonomic nervous system .

134. FABRY’S DISEASE LIGHT MICROSCOPY Glomeruli on light microscopy show hypertrophic glomerular visceral epithelial cells(podocytes) distended with foamy appearing vacuoles, mesangial widening, andvarying degrees of glomerular obsolescence .Within the glomerulus, the largest amount of lipid material is seen in podocytes,followed by the parietal epithelial, mesangial, and glomerular endothelial cells. Glomerulus showing extensive inclusion Plastic embedded tissue showing in-site deposition bodies of glycolipid in podocytes and mild of glycolipid in glomerular podocytes (toluidine mesangial widening (PAS stain) blue stain)

135. FABRY’S DISEASE ELECTRON MICROSCOPYVast majority oflaminatedinclusion bodiesare present in thecytoplasm ofaffected cells.They are eitherround with aconcentric myelin-like structure, orovoid with parallellayers called Zebrabodies.Portion of a glomerulus from a patient with Fabry’s disease demonstrating numerous laminated inclusions in the epithelial cell cytoplasm.

136. NAIL-PATELLA SYNDROME

137. Nail-Patella syndromeAlso called Onycho-osteodysplasia or Fog’s syndrome.Is an autosomal dominant disorder.Occurs due to point mutation in chromosome no 9.Renal involvement occurs in approx 30% to 55% of patients.Light microscopy shows non specific changes.By electron microscopy, the GBM appears irregularly thickened and oftenexhibits electron-lucent areas giving it “moth eaten” appearance.These areas are occupied by collagen like fibers. Collagen-like fibers in the GBM in nail– patella syndrome