Section B -

Vasculopathies Volker Nickeleit, M.D. J. Charles Jennette, M.D.

The following section briefly highlights key aspects characterizing different vasculopathies commonly encountered by the pathologist. It is far beyond the scope of this handout to provide detailed information on the pathophysiology of each vasculopathy. We focus primarily on morphological aspects. For in depth reading, the interested reader is referred to selected references listed in the "reference section". I) Amyloidosis and monoclonal immunoglobulin deposition disease Amyloidosis and monoclonal immunoglobulin deposition diseases are very heterogeneous groups of disorders (see table 1). They can be associated with underlying hematopoetic neoplasms, in particular plasma cell dyscrasias/plasmocytomas. Amyloid and monoclonal immunoglobulins are typically deposited along vessel walls and basement membranes resulting in severe functional abnormalities, such as stenosis, leakage, rupture, changes in the filtration barrier, or a loss of adaptive functions (vascular dilatation and contraction). Normally, there is only minimal overlap; amyloidosis is typically not seen in cases of monoclonal immunoglobulin deposition disease and vice versa. 1) Monoclonal Immunoglobulin Deposition Disease (Light – And/or Heavy Chain Deposition Disease) Definition : Deposition of monoclonal immunoglobulins along basement membranes, primarily affecting renal tubules, glomerular capillaries and arteries. Clinical Findings : The kidneys are primarily affected. Proteinuria (in 90% of cases) and the nephrotic syndrome (in 40% of cases) are typical symptoms at time of presentation leading to a renal biopsy. Approximately 50% to 60% of patients suffer from plasmocytoma; many patients present with kappa or lambda (4:1) monoclonal light chains in the serum and or urine. The accumulation of monoclonal heavy chains (mostly gamma) is a rare event. Caveat: The detection of monoclonal light or heavy chains in the serum or urine increases the probability that a patient suffers from immunoglobulin deposition disease, however, does not accurately predict disease. Histology : Most characteristic is a nodular expansion of glomerular mesangial regions, similar to "Kimmelstiel-Wilson" nodules, seen in 60% of cases. Often, glomeruli are diffusely and globally affected and the mesangial nodules are of similar sizes. In rare cases (10% to 20%) focal extracapillary crescents can be detected. Tubular basement membranes are typically thickened, in particular along non-atrophic tubules. In addition, arterioles and small arteries can show wall hypertrophy and hyaline deposits. The disease process can spare glomeruli and can be limited to the tubulo-interstitial compartment, reported in one series in 50% of cases of immunoglobulin deposition disease (E.H. Strom et al.). The light microscopic changes are caused by the deposition of monoclonal immunoglobulins that can easily be demonstrated by immunohistochemistry or immunofluorescence microscopy. Electron microscopy shows typical, finely granular, densely packed electron dense particles along the outer aspect of tubular basement membranes, along the inner aspect (i.e. lamina rara interna) of glomerular basement membranes, in glomerular mesangial regions, around medial smooth muscle cells and along the lamina elastica of arteries. Intrarenal and extrarenal arteries are typically affected (without significant clinical symptoms). The vascular monoclonal immunoglobulin accumulations can be on occasion massive, leading to individual smooth muscle cell necrosis and mimicking vascular "amyloid deposits" by standard light microscopical examination. By light microscopy, monoclonal immunoglobulin deposition disease may be misinterpreted as 'diabetic nephropathy'. Helpful diagnostic clues during the histologic work-up include: 1) in monoclonal immunoglobulin deposition disease, mesangial nodules usually have the same size and demonstrate a global and diffuse distribution pattern. Kimmelstiel-Wilson nodules often show a segmental accentuation and vary considerably in size. In diabetes, glomerular basement membranes are typically thickened; this feature is not seen in immunoglobulin deposition disease. Of course, the detection of hyalinosis in efferent arterioles is highly characteristic for diabetes. In addition, nodular amyloid deposits or membranoproliferative glomerulonephritides may enter into the differential diagnosis. Diagnostic confirmation can easily be achieved by immunofluorescence microscopy and electron microscopy. (Caveat: diabetes often shows linear accentuation of glomerular and tubular basement membranes with antisera specific for IgG and kappa light chains.) Since some rare cases of immunoglobulin deposition disease may not give positive staining signals by immunofluorescence microscopy or vice versa may not demonstrate typical ultrastructural deposist, both IF and EM are required for proper evaluation (also see tables 2 and 3). Table 1: Renal Diseases Induced by Monoclonal Immunoglobulin Molecules (modified from D'Agati V, Jennette JC, Silva FG: Non-Neoplastic Kidney Diseases Fascicle Atlas of nontumor pathology, American Registry of Pathology, Washington , D.C. , 2005) Glomerular/Vascular Diseases (tubules and interstitium may be involved) AL Amyloidosis AH Amyloidosis Light Chain Deposition Disease (LCDD) Heavy Chain Deposition Disease (HCDD) Light & Heavy Chain Deposition Disease (LHCDD) Cryoglobulinemia (Types I & II) Monoclonal Immunotactoid Glomerulopathy Tubulointerstitial Disease Light Chain (Myeloma) Cast Nephropathy Light Chain Tubulopathy (e.g. with Fanconi Syndrome) Acute Tubular Necrosis Table 2 : Differential pathologic glomerular features of diseases with glomerular monoclonal immunoglobulin deposits and their mimics. (modified from D'Agati V, Jennette JC, Silva FG: Non-Neoplastic Kidney Diseases Fascicle Atlas of nontumor pathology, American Registry of Pathology, Washington, D.C. , 2005) AA amyloid AL amyloid AH amyloid LCDD HCDD LHCDD Diabetic GS Fibrillary GN Immunotactoid GN Cryoglobulinemia MPGN USUAL LM PATTERN: fluffy acidophilic deposits fluffy acidophilic deposits fluffy acidophilic deposits nodular sclerosis nodular sclerosis nodular sclerosis nodular sclerosis & thick GBM thick capillaries and expanded mesangium thick capillaries and expanded mesangium thick capillaries and expanded mesangium thick capillaries and expanded mesangium DEPOSIT STAINING: PAS weak/- weak/- weak/- + + + + mottled mottled double GBM double GBM Silver weak/- weak/- weak/- -/weak/+ -/weak/+ -/weak/+ + mottled mottled double GBM double GBM Congo red + + + - - - - - - - - IM STAINING: Ig - one LC, usually one HC, usually one LC, usually one HC, usually one LC & one HC polyclonal HC & LC oligoclonal HC & LC monoclonal > polyclonal polyclonal > monoclonal polyclonal HC & LC C3 - - weak/- weak/- + + - strong + strong + strong + strong + EM PATTERN: fibrils, 8-12 nm fibrils, 8-12 nm fibrils, 8-12 nm dense granules dense granules dense granules thickened GBM and mesangium fibrils,15-30 nm microtubules, 20-50 nm deposits, often 25-35 nm microtubules deposits Abbreviations: DD=deposition disease, EM=electron microscopy, GBM=glomerular basement membrane, GN=glomerulonephritis, GS=glomerulosclerosis, HC=heavy chain, Ig=immunoglobulin, IM=immunofluorescence microscopy, LC=light chain, LM=light microscopy, MP=membranoproliferative, PAS=periodic acid Schiff Table 3: Characteristics of AL amyloidosis, monoclonal immunoglobulin deposition disease, and fibrillary glomerulonephritis identified in renal biopsy specimens evaluated in the University of North Carolina Nephropathology Laboratory. (modified from D'Agati V, Jennette JC, Silva FG: Non-Neoplastic Kidney Diseases Fascicle Atlas of nontumor pathology, American Registry of Pathology, Washington, D.C. , 2005)   AL amyloidosis Light or heavy chain deposition disease Fibrillary glomerulonephritis   n=80 n=25 n=74 Frequency in kidney biopsy specimens 15/1000 4/1000 8/1000 Mean age (range) 63 (38-82) 60 (35-79) 52 (21-75) Sex (male: female) 1:1.1 1:0.6 1:1.2 Race (black: white)* 1:3.5 1:4.6 1:13.0 Mean Proteinuria 7.2 3.7 6.0 Mean creatinine 1.9 5.1 3.8 Light chain staining 23% kappa 75% lambda 76% kappa 20% lambda polyclonal, mostly kappa and lambda *expected black:white ratio in the renal biopsy population is approximately 1:3 2) Amyloidosis Definition : Deposition of amorphous, "waxy" proteinaceous material along arterial walls, basement membranes and in the interstitial compartment. Amyloid has been known for over 150 years; it was first described and the name was coined by Rudolf Virchow. Amyloidosis is a spectrum of diseases with different etiologies that share the deposition of extracellular protein polymers. These polymers have a specific tertiary structure (beta pleated sheets). Amyloid polymers are characteristically Congo Red positive and have an apple-green tinctorial property under polarized light. Ultrastructurally, typical non-branching fibrils are found (9nm – 12 nm in diameter). Different proteins can accumulate as "amyloid" with different organ involvement and various clinical presentations (at present more than 20 different "amyloid" proteins are known; see tables 4 and 5). Thus, "amyloidosis" is more of a descriptive term. However, regardless of the specifics, amyloid deposits can cause severe organ failure (e.g. of the heart or kidneys) that clinically presents as the leading problem (obscuring the diagnosis of amyloidosis). Generalized amyloidoses involving the kidneys and/or heart are associated with marked morbidity and mortality (survival in cases of generalized AA or AL amyloidosis: 2-4 years). Amyloid generally accumulates along basement membranes (including glomerular and tubular basement membranes), in vascular walls and also in the interstitial compartment. Since treatment strategies to "dissolve" amyloid deposits are undefined, much emphasis has to be placed on an early diagnosis in order to treat possible underlying diseases and thereby prevent the further accumulation of amyloid fibrils. In this regard the pathologist plays a pivotal role. Currently, the prevalence of secondary AA type amyloidosis is low due to improved therapeutic protocols to treat infections (such as tuberculosis or osteomyelitis). However, AA type amyloidosis should be suspected in all forms of "chronic inflammatory conditions" (rheumatoid arthritis, psoriatric arthritis, ankylosing spondylitis, Behcet's syndrome, inflammatory bowel disease, sarcoidosis, Hodgkin's disease etc). Generalized AA deposits are typically seen years after the onset of disease (3% to 23% of patients suffering from rheumatoid arthritis show AA type amyloid deposits). The accumulation of AA type amyloid is associated with elevated serum titers of the "acute phase" precursor proteins SAA (serum amyloid A). At present, primary AL types of amyloidosis are clinically most relevant. The gold standard to establish a diagnosis of an amyloidosis is a biopsy. Kidneys, the liver, the GI tract and the subcutaneous tissue are commonly involved in all forms of generalized amyloidoses; these sites are best suited for diagnostic work-up. The sensitivity of a kidney or liver biopsy is >90%, of a rectal biopsy 70% to 85% and of a subcutaneous abdominal skin biopsy 50% to 85%. Rectal and subcutaneous biopsies are most often performed since bleeding complications secondary to massive amyloid deposits in vascular walls of internal organs can be avoided. The interpretation of biopsy specimens is associated with certain caveats. A rectal biopsy has to be deep enough to include submucosal tissue and vessels containing potential amyloid deposits. For the same reason, a subcutaneous fat aspirate has to include fibrous septae. Large amounts of amyloid may be misinterpreted as "elastosis" (in the skin) or "collagenous colitis" (in the intestinal tract). Small amounts of amyloid cannot be readily detected by standard light microscopic examination. Special stains such as Congo Red incubations are required for proper histologic work-up. Of note: Congo Red stains only give a positive staining result if tissue sections are approximately 8 to 10 micrometers thick. Sections have to be analyzed under polarized light to search for diagnostic "apple green" amyloid deposits. The pathologist should make considerable efforts to subclassify the amyloid deposits since amyloidosis can only be indirectly treated by "curing" the underlying disease, i.e. an inflammatory condition in cases of AA amyloidosis or a lymphoproliferative disorder in cases of AL amyloidosis. Most often the diagnostic work-up will reveal AL type amyloid deposits, followed by the accumulation of AA amyloid (see tables 4 and 5); all other forms of amyloid are rare in the western world. Special antibodies are available to subtype the amyloid deposits in formalin fixed and paraffin embedded tissue sections. Alternatively, amyloid may also be extracted from tissue blocks and analyzed by amino acid sequencing. Macroscopic findings : In advanced cases, the organs are enlarged, glassy appearing with a waxy consistency. Small amounts of amyloid deposits do not result in gross abnormalities. Histology : We will use renal amyloidosis as an example. All forms of amyloid basically can give rise to identical histological changes. In the kidneys the earliest amyloid deposits are detected in a focal and segmental distribution pattern in mesangial regions. Amyloid is found as acellular, homogenous, eosinophilic material, only weakly positive in PAS, trichrome and silver stains (also see table 2). These early amyloid deposits are generally not associated with renal dysfunction. In more advanced cases amyloid is diffusely and globally deposited in mesangial regions (sometimes in a nodular fashion, caveat: diabetic nephropathy or monoclonal immunoglobulin deposition disease) and additionally along glomerular basement membranes (resulting in proteinuria). Amyloid is also seen in arterioles, arteries, along tubular basement membranes and in most advanced cases also in the interstitium (encasing preexisting structures). Arterial amyloid deposits are initially detected in the media but they can ultimately replace the entire vascular walls resulting in loss of contractile function, rupture, hemorrhage or stenosis. Thrombosusformation is typically absent. Thus, internal organs carrying a high load of amyloid are at increased risk of severe hemorrhage following biopsy. Of note: Limited cerebral amyloid deposits (A beta type) in arteries of the brain are a very common cause of hemorrhage and stroke. Table 4: Classification of the most relevant types of amyloid Amyloid Precursor in serum Clinical Disease AA apo SAA Secondary amyloidosis Mediterranean fever AL lambda light chains, kappa light chains (3:1) Primary amyloidosis (e.g. multiple myeloma) AH IgG1 Heavy chain amyloid ATTR transthyretin Hereditary amyloid polyneuropathy or cardiomyopathy AGel gelsolin Finnish familial amyloidosis AApoA1 apoA1 Familial amyloid polyneuropathy A(beta2)M beta2-microglobulin Amyloid found with dialysis A (beta) A beta protein precursor Cerebral amyloid (Alzheimer's disease, "aging" cereb. arterial walls) Table 5: Amyloidosis (primary and secondary)   Primary Secondary Location of deposits (frequent) GI tract, muscle, nerves lymph nodes, tongue, carpal tunnel, heart, liver, kidneys kidneys, spleen, liver, adrenals, GI tract Location of deposits (infrequent) adrenals, brain heart, peripheral nerves, tongue, brain Renal symptoms with marked proteinuria 37% 100% Underlying disease plasma cell dyscrasia/multiple myeloma (monoclonal immunoglobulins in serum or urine in 90%) "chronic inflammatory conditions", e.g. tuberculosis, polyarthritis, osteomyelitis, bronchiectasis, tumors Amyloid type AL AA Congo red stain 3+ positive 3+ positive Congo red with KMNO4 pre-treatment (light microscopy) 1+ -2+ positive 0 - +/- Congo red with KMNO4 pre-treatment (polarized light, "apple green") 1+ - 2+ positive 0 Thioflavine T positive* positive* Immunohistochemistry (paraffin; specific antibodies) positive positive Electron Microscopy fibrils 9-12 nm (non-branching) fibrils 9-12 nm (non-branching) * The Thioflavine T stain has a high sensitivity but low specificity II) Diabetic Vasculopathy Seventy percent of all deaths in diabetic patients are caused by vascular disease. In diabetics, (regardless of the type) vascular changes occur at a younger age and progress faster than in non-diabetic patients. Diabetes mellitus is associated with changes in large vessels (macroangiopathy) and small vessels (microangiopathies). Macroangiopathies affect arteries, often in the lower limbs and the heart, and present with typical atherosclerosis and calcifications of the media. These changes are not characteristic for diabetes mellitus. They are promoted by hypertension and hyperlipidemia. Microangiopathies, which affect arterioles, capillaries, venules and lymphatics are often found in the skin, heart, retina, muscle, nerves, brain, and kidneys. They are – at least in part – directly linked to hyperglycemia and significantly contribute to morbidity and mortality. The exact pathophysiologic chain of events leading to diabetic microangiopathies is incompletely understood and vasculopathies are only found in a sub-group of diabetics, such as diabetic nephropathy in only 40% of patients. The development of microangiopathies in insulin dependent diabetics takes years. For example, more than 10 years are required for the onset of typical diabetic nephropathy. Histology (microangiopathies): Hyalinosis develops under the endothelial layer and between atrophic smooth muscle cells and pericytes resulting in vascular wall thickening, vascular twisting and the development of microaneurysms. The basement membranes are abnormally thickened due to increased synthesis and deposition of fibronectin, laminin, type IV collagen, and heparan sulfates. Basement membrane alterations are associated with structural weakening, and changes in the filtration barrier including leakage. In the retina, the most typical cases of diabetic microangiopathies show microaneurysms, hemorrhage, infarcts and angiogenesis with neo-vascularization (proliferative stage of diabetic retinopathy). Diabetic retinopathy is often, but not always associated with microangiopathies in the kidneys (in some series 30% of patients with retinopathy are free of kidney disease). Diabetic nephropathy can serve as a prime example to study diabetic microangiopathies. Diabetic nephropathy involves glomeruli, capillaries and afferent and efferent arterioles. The earliest glomerular change, which is hardly ever biopsied, is enlargement (glomerulomegaly). It corresponds to the early clinical phase of an elevated glomerular filtration rate and "enlarged kidneys". By the time proteinuria is detectable there is typically generalized thickening of glomerular basement membranes and an increase of the mesangial matrix. Initially, morphometry is required to detect these changes but eventually, the GBM thickening and mesangial expansion are so pronounced that they can readily be discerned by routine light microscopy (e.g., PAS, Jones silver, Masson trichrome stains). Electron microscopy shows thickened and layered glomerular basement membranes (GBM) with an irregular lamina rara externa showing minute "crater type lesions". Frequently, GBM thickening is segmentally accentuated and detected adjacent to GBM thinning (in areas of capillary microaneurysms). Occasionally, the GBM can even be normal on ultrastructural examination (likely reflecting a sampling problem if only less affected glomeruli are studied). Basement membrane thickening may also be observed along tubules. Mild to moderate mesangial hypercellularity occasionally accompanies the early phases of mesangial matrix accumulation (caveat: mesangioproliferative glomerulonephritis). Overt glomerular matrix expansion (glomerulosclerosis) manifests as two basic patterns: diffuse glomerulosclerosis and nodular glomerulosclerosis. These two patterns often coexist in a given biopsy specimen, although the number of glomeruli available for examination will influence the likelihood of seeing both patterns. Conceptually, diffuse glomerulosclerosis evolves into nodular glomerulosclerosis as the mesangial matrix expands. In our opinion, the designations "diffuse" versus "nodular" are primarily of descriptive value; the distinction does not carry great clinical significance. The nodular lesions of diabetic glomerulosclerosis were first described by Kimmelstiel and Wilson. The nodules start to develop from the center of mesangial areas, often in a focal and segmental pattern. As the nodules grow, mesangial cells are arranged along the outer edges and the mesangial matrix may appear laminated. Glomerular capillaries are dilated and form microaneurysms, typical for diabetic microangiopathies. In rare instances (in our experience 0.5% of biopsies) these microaneurysms can rupture, fibrin can spill into Bowman's space and small, focal and segmental extracapillary crescents can form (caveat: glomerulonephritis). Hyalinosis is common in diabetic glomerulosclerosis. Hyaline (hyaline = glassy) material is formed secondary to insudation of plasma proteins from leaky vessels. The hyaline deposits may occur anywhere in the tuft; they are characteristically found as hyaline caps or capsular drops. Advanced forms of diabetic glomerulosclerosis often show segmental glomerular scarring (FSGS). Diabetic renal microangiopathies characteristically affect afferent and efferent arterioles with subendothelial and medial 'nodular' hyalinosis. Diabetic arteriolopathy is very similar to toxic changes induced by calcineurin inhibitors or hypertension induced areteriolosclerosis, although, these latter changes are only found in afferent vessels. Immunohistochemistry/-fluorescence microscopy: The characteristic observation is linear staining of glomerular and tubular basement membranes with antisera specific for IgG and kappa light chains as well as other plasma proteins, such as albumin, although the staining for IgG is usually brightest. Immunofluorescence microscopy is useful for excluding other glomerular diseases that can mimic diabetic glomerulosclerosis by light microscopy (e.g. light chain deposition disease, heavy chain deposition disease, membranoproliferative glomerulonephritis, fibrillary glomerulonephritis, amyloidosis) or a second disease process, such as an IgA glomerulonephritis (concurrent in our experience in 1.2% of biopsies with diabetic nephropathy). Immunohistochemistry is particularly helpful in biopsies with extracapillary crescent formation. Differential Diagnosis: Light chain deposition disease (LCDD) and heavy chain deposition disease (HCDD) cause nodular glomerulosclerosis that is very similar to diabetic glomerulosclerosis by light microscopy. This suggests that the IgG localization in basement membranes in diabetic glomerulosclerosis may be the cause for the nodular sclerosis and not merely an epiphenomenon. Some examples of membranoproliferative glomerulonephritis can have conspicuous mesangial nodules that resemble diabetic glomerulosclerosis, but the overall morphology including immunohistochemistry and electron microscopy usually is discriminatory. Fibrillary glomerulonephritis, immunotactoid glomerulopathy, amyloidosis, fibronectin glomerulopathy, collagenofibrotic glomerulopathy and a number of other diseases cause capillary wall thickening and increase in extracellular material in the mesangium that can suggest diabetic glomerulosclerosis, but careful evaluation generally allows proper differentiation. Rare specimens will have pathologic features that are identical to those of diabetic glomerulosclerosis yet the patient will have no clinical evidence of diabetes mellitus. This finding is described as "idiopathic nodular glomerulosclerosis" (a diagnosis of exclusion). When evaluating cases with diabetic nephropathy, it seems important to adequately identify other concomitant, "treatable" disease processes, such as diabetic glomerulosclerosis and ANCA disease or membranous glomerulonephritis. On the other hand, rare cases of diabetic glomerulosclerosis with crescent formation should not be overdiagnosed as evidence of glomerulonephritis. III) Antiphospholipid Syndromes (APS) Definition : Hypercoagulable state with arterial and/or venous thrombus formation secondary to high titers of circulating autoantibodies against phospholipid/ phospholipid- binding-protein complexes. Antiphospholipid syndromes are classified as secondary disease if they are accompanied by other autoimmune disorders, i.e. lupus erythematosus or other connective tissue diseases, or as primary if they only present with a hypercoagulable state. Clinical Findings : The clinical presentation varies greatly and may sometimes mimick a vasculitis. Recurrent thrombosis is the leading symptom. Venous thrombosis is frequently detected in deep leg veins (55%; caveat: pulmonary emboli and hypertension in half of the patients with deep vein thrombosis), as well as in renal, hepatic and retinal veins. Arterial thrombosis is typically seen in cerebro-vascular (50%), coronary (25%), ocular, mesenteric, deep leg and renal arteries (caveat: stroke, myocardial or bowel infarction, ischemia of the lower extremities with skin ulcerations, renal hypertension). The microvasculature of the kidney can additionally show changes resembling a thrombotic microangiopathy (caveat: thrombocytopenia, shistocytes, renal failure). The clinical spectrum also includes livedo reticularis (differential diagnosis: vasculitides including polyarteritis nodosa), repeated miscarriages and cardiac valvular vegetations (Libman-Sacks endocarditis in 4% of patients). In rare instances (less than 1% of cases) multiple organ sites are affected by thrombosis simultaneously with dramatic clinical consequences and a mortality rate of up to 50% (termed: "catastrophic antiphospholipid syndrome). By ELISA high titers of IgG or sometimes IgM antibodies are detected. These antibodies bind to various auto antigens (e.g. beta 2 glycoprotein 1/phospholipid complexes on platelets and endothelial cells) and promote the activation of the coagulation cascade, although the precise pathogenic mechanisms are still undetermined. Autoantibodies are also directed against prothrombin, protein C, protein S and annexin V. Antiphospholipid antibodies (often low titers) can be detected in 5% to 15% of healthy individuals. Histology : The histolgic examination shows fibrin thrombi of different ages, sometimes organized with signs of re-canalization. Conspicuous inflammation and vascular wall necrosis are lacking. Remote thrombus formation in arteries and veins should always raise the suspicion of an underlying APS. In the kidneys not only large caliber vessels (i.e. interlobar and arcuate types) but also arterioles and glomerular capillaries may be affected with evidence of thrombosis and intimal remodeling resembling a thrombotic microangiopathy (see section IV). IV) thrombotic microangiopathies Thrombotic microangiopathy (TMA) – a descriptive term – characterizes stenosing and/or thrombotic changes in small vessels, i.e., capillaries, arterioles and pre-arterioles. Larger arteries are characteristically spared. The brain, kidneys, gastrointestinal tract, pancreas, spleen and adrenal glands are commonly affected whereas the liver and lungs are typically spared. The initial common event in the pathogenesis of all forms of TMA is severe endothelial cell injury caused by a wide variety of different agents or events (see table 6). Endothelial cell injury results in intimal remodeling. Generally the pathologist cannot reliably identify the underlying causative agent or event and can only render a descriptive diagnosis of "TMA". It is the primary responsibility of the managing clinician to identify the underlying cause of the histologically observed "TMA" and to initiate proper treatment. Clinical and Laboratory Findings : Typical clinical signs of TMA are: thrombocytopenia, hemolytic anemia, fragmented red blood cells (i.e. shistocytes) in peripheral smears, and organ dysfunction including seizures or renal failure. Depending on the organ primarily involved (kidney or brain) and the extent of vascular changes, clinical symptoms may vary considerably from mild to severe. A TMA occurring in adults often primarily affects the brain and is referred to as "thrombotic thrombocytopenic purpura" (TTP), whereas children commonly suffer from severe renal failure and hemolysis, i.e. "hemolytic uremic syndrome" (HUS). The clinical distinction, however, is not always clear-cut and the same patient may be described as having HUS by the nephrologist and TTP by hematologists. Because the pathological features seen in biopsies and the initial treatment for HUS and TTP are similar, they are often clinically referred to as (TTP-HUS). Of note: Some cases of TMA do not easily fit into the clinical spectrum of TTP or HUS, such as calcineurin inhibitor or DOTATOC induced TMAs limited to the kidneys. These cases can lack clinical signs of hemolysis or thrombocytopenia. Classical HUS : It is a disease of young children primarily associated with an enteric infection by E. coli 0157:H7 or Shigella dysenteriae (i.e. diarrhea associated HUS). Renal involvement is prominent and up to one third of patients can be anuric. Children most often present approximately one week after onset of diarrhea with pallor, oliguric renal failure, lethargy, and occasionally purpura/petechiae. New onset hypertension and low platelet counts are present in the majority of patients, whereas CNS dysfunction is noted in only 20%. The disease is caused by verotoxins 1 and 2 (i.e. shiga like exotoxins) that bind to specific plamamembrane receptors on endothelial cells (i.e. globotriosyl ceramides, Gb3) and promote endothelial cell necrosis. The density of the receptors appears to be highest in renal vessels explaining the predominance of "acute kidney failure" in the setting of classical HUS. The pathophysiologic chain of events in so-called nondiarrhea forms of HUS is multifactorial and only poorly understood. Nondiarrhea HUS is more frequently seen in adolescents and adults. It shares many clinical symptoms with TTP, however, ADAMTS13 activity (i.e. von Willebrand factor-cleaving protease) seems unchanged. Classical TTP : It was first described by Moschowitz in 1924 as a disorder of unknown cause that predominantly affects adult women and rarely children. It is characterized by the pentad of microangiopathic anemia, profound thrombocytopenia, fever, CNS manifestations including seizures, confusion and coma, and (mild) renal failure. TTP may arise de novo(idiopathic or classical TTP) or may be familial (including rare forms of chronic relapsing TTP that occur at regular intervals of about three weeks). TTP is rarely preceded by diarrhea (unlike classical HUS) but often follows a 'flulike' prodrome with fever, fatigue, nausea, vomiting, and abdominal pain. Increasing evidence suggests that TTP is caused by defects of a von Willebrand cleavage enzyme (i.e. ADAMTS13, which is produced predominately by hepatocytes and secreted into the plasma). The lack of enzymatic activity results in unusually large von Willebrand multimers, platelet aggregation and thrombus formation. The sporadic variants of TTP seem to be frequently caused by autoantibodies directed against ADAMTS13 (which inhibit enzyme function), whereas the familial recurrent cases of TTP demonstrate mutations or structural defects of the enzyme itself. Typically, the activity of ADAMTS13 is very low (<5%, normal plasma activity: 67%-177%). Caveat: Mildly reduced levels of ADAMTS13 activity can also be seen in a variety of clinical settings (e.g. liver cirrhosis, inflammation, preganancy). However, such "mildly" reduced enzyme activity is typically not associated with TTP. Histology : Changes observed in the kidneys will serve as examples to illustrate the morphology of a TMA (see table 7). A TMA affects small vessels (in the kidney: interlobular arteries including branches of arcuate arteries, pre-arterioles, arterioles and glomerular capillaries; interlobar and arcuate caliber arteries are generally spared). Some investigators suggest that a TTP is more frequently characterized by platelet thrombi whereas a HUS more often shows fibrin thrombi. In the kidney the histological changes can be divided into three main patterns that correlate with long term renal function: those predominating in the glomerular compartment (young children, good prognosis); those dominating in the arterial compartment (adults, poor prognosis); and a mixed pattern with both glomerular and vascular involvement (older children and adults, poor prognosis). Early (potentially reversible) "exudative and infiltrative" lesions have to be distinguished from late (irreversible) "sclerosing" changes. Glomerular changes: 1) endothelial cell swelling and separation of endothelial cells from underlying basement membranes with production of new basement membrane material (double contouration of the GBM); 2) fragmented red blood cells and fibrin thrombi in glomerular capillary lumens; 3) mesangiolysis with associated capillary dilatation. Double contouration of peripheral glomerular basement membranes is already detected during the early time course; it can persist for many months to years. Typically, the double contouration of the GBM is not associated with cell interposition (caveat: membranoproliferative glomerulonephritis). Immunohistochemistry and electron microscopy do not reveal diagnostically significant immune complex type deposits (normally only fibrin or non-specific IgM/complement factor C3 deposits are found). Electron microscopy shows characteristic subendothelial new basement membrane formation and deposition of "flocculent" material along the widened lamina rara interna. Glomeruli can also demonstrate non-specific ischemic changes with wrinkling of peripheral basement membranes secondary to severe TMA involving the feeding afferent arterioles. Early changes limited to the glomeruli are fully reversible. Disease progression and vascular involvement results in scarring and nephron loss. Vascular changes: Most biopsies demonstrate severe changes that can be seen individually or in combination: 1) severe endothelial cell swelling and "mucoid" intimal widening with narrowing and occlusion of arteriolar lumens; 2) intraluminal fibrin thrombi and/or fragmented red blood cells in the intima and media. 3) necrosis of individual endothelial or medial smooth muscle cells. 4) PAS positive nodular proteinaceous deposits replacing arteriolar smooth muscle cells. As the disease progresses, the widened intimal zones show increasing numbers of myofibroblasts and marked collagen deposits, ultimately leading to "onion-type" intimal scarring and stenosis. Ischemic damage and renal hypertension are severe clinical consequences of a TMA in arteries causing considerable morbidity and mortality. Of note: alterations of a TMA are typically detected at glomerular vascular poles ("hot-spots" for TMAs) and extend downstream into glomerular capillaries and/or upstream into the arteriolar tree. Often glomerular vascular poles are dilated and show fibrin thrombi that are subsequently organized by ingrowth of myofibroblasts. Table 6: Thrombotic Microangiopathies and Coagulopathies (modified from D'Agati V, Jennette JC, Silva FG: Non-Neoplastic Kidney Diseases Fascicle Atlas of nontumor pathology, American Registry of Pathology, Washington, D.C. , 2005) Hemolytic Uremic Syndrome (HUS) predominant Shiga-like toxin-induced HUS (e.g. E. coli, Shigella dysenteriae) Neuraminadase-induced HUS (e.g., Streptococcus pneumoniae) Other bacterial or viral infections (e.g. typhoid fever, HIV, influenza, enterovirus) Iatrogenic HUS Drug-induced HUS (e.g. cyclosporine, tacrolimus, mitomycin-C, oral contraceptives, quinine) Bone marrow transplantation-induced HUS Radiation-induced HUS, DOTATOC-therapy Systemic sclerosis renal crisis HUS Pregnancy associated HUS Familial HUS (e.g. autosomal recessive, autosomal dominant, factor H deficiencies) Malignant hypertension induced HUS (malignant nephrosclerosis) Idiopathic HUS Anti-Phospholipid Antibody Syndrome (APS)* Primary APS Secondary APS (e.g. secondary to systemic lupus erythematosus, rheumatoid arthritis) Toxemia of pregnancy Preeclampsia Eclampsia HELLP syndrome Thrombotic Thrombocytopenic Purpura (TTP) predominant Familial/recurrent TTP (e.g. defects in or absence of vWF multimerase/ADAMTS13) Sporadic TTP (e.g. autoantibodies to vWF multimerase/ADAMTS13) Drug-induced TTP (e.g. ticlopidine, clopidogrel) Idiopathic TTP Disseminated Intravascular Coagulation (DIC)** Bacterial sepsis with DIC (e.g. gram negative sepsis) Viral hemorrhagic fever with DIC (e.g. Dengue, Marburg, Ebola) Neoplasia-induced DIC (e.g. pancreatic carcinoma-induced DIC) Pregnancy-related DIC (e.g. with retained dead fetus, placental separation) Venom-induced DIC (e.g. snakebite) * thrombus formation noted in large and small vessels including veins ** thrombus formation without mucoid intimal thickening or "onion-type scarring" Table 7: Predominant pathologic features and major pathogenic events in the thrombotic microangiopathies and coagulopathies. (modified from D'Agati V, Jennette JC, Silva FG: Non-Neoplastic Kidney Diseases Fascicle Atlas of nontumor pathology, American Registry of Pathology, Washington , D.C. , 2005)   Predominant pathology Major pathogenic event Hemolytic uremic syndrome Glomerular capillary subendothelial expansion, arteriolar fibrinoid necrosis, arterial edematous intimal expansion, vascular thrombosis Endothelial injury caused by many different etiologies, predominantly in renal vessels, causing focal occlusion, thrombosis and renal ischemia Thrombotic thrombocytopenic purpura Platelet-rich thrombi in many organs Enhanced platelet aggregation, e.g. caused by abnormal vWF-cleaving and metalloproteinase activity Disseminated intravascular coagulation Fibrin-rich thrombi in many organs Enhanced coagulation and fibrinolysis, e.g. caused by increased circulating tissue factor Anti-phospholipid antibody syndrome Thrombosis in veins, arteries, arterioles and glomerular capillaries; subendothelial expansion and GBM remodeling Antibodies to phospholipids or phospholipid-binding proteins to foster thrombosis Preeclampsia / Eclampsia Glomerular capillary endothelial swelling (endotheliosis) Abnormal placentation with generation of humoral factors that injure maternal endothelial cells Selected References (Section B) Alving BM: Diagnosis and management of patients with the antiphospholipid syndrome. J Thromb Thrombolysis 2001; 12 (1): 89 Bader R, Bader H, Grund KE, Mackensen-Haen S, Christ H, Bohle A: Structure and function of the kidney in diabetic glomerulosclerosis: correlations between morphological and functional parameters. Pathol Res Pract 1980; 167:204 Bertani T, Gambara V, Remuzzi G: Structural basis of diabetic nephropathy in microalbuminuric NIDDM patients: a light microscopy study. Diabetologia 1996; 39:1,625 Bianchi V, Robles R, Alberio L, Furlan M, Lammle B. Von Willebrand factor-cleaving protease (ADAMTS13) in thrombocytopenic disorders: a severely deficient activity is specific for thrombotic thrombocytopenic purpura. Blood. 2002;100(2):710. Bohle A, Wehrmann M, Bogenschutz O, Batz C, Muller CA, Muller GA. The pathogenesis of chronic renal failure in diabetic nephropathy: investigation of 488 cases of diabetic glomerulosclerosis. Pathol Res Pract 1991; 187:251 D'Cruz DP. Renal manifestation of the antiphospholipid syndrome. Lupus 2005; 14: 45 Dember LM. Emerging treatment approaches for the systemic amyloidoses. Kid Int 2005; 68: 1377 Egan JA, Bandarenko N, Hay SN, Paradowski L, Goldberg R, Nickeleit V, Brecher ME. Differentiating thrombotic microangiopathies induced by severe hypertension from anemia and thrombocytopenia seen in thrombotic thrombocytopenic purpura. J Clin Apheresis 2004; 19 (3): 125. Falk RH, Raymond LC, Skinner M. The systemic amyloidoses. N Engl J Med 1997; 337:899. Fioretto P, Mauer M, Brocco E, Velussi M, Frigato F, Muollo B, Sambataro M, Abaterusso C, Baggio B, Crepaldi G, Nosadini R: Patterns of renal injury in NIDDM patients with microalbuminuria. Diabetologia 1996; 39:1569 Furlan M, Robles R, Galbusera M, Remuzzi G, Kyrle PA, Brenner B, Krause M, Scharrer I, Aumann V, Mittler U, Solenthaler M, Lammle B. Von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome. N Engl J Med 1998;339 (22):1578. Gambara V, Mecca G, Remuzzi G, Bertani T: Heterogeneous nature of renal lesions in type II diabetes. J Am Soc Nephrol 1993; 3:1458 Hanley JC: Antiphospholipid syndrome: an overviw. CMAJ 2003; 168 (13): 1675 Horton TM, Stone JD, Yee D, et al. Case series of thrombotic thrombocytopenic purpura in children and adolescents. J Pediatr Hematol Oncol 2003; 25:336-339. Kimmelstiel P, Wilson C: Intercapillary lesions in glomeruli of kidney. Am J Pathol 1936; 12:83 Kisilevsky R, Young ID. Amyloidosis. In Anderson's Pathology, 10th edition 1996, pg 448-459. Kyle RA, Gertz MA. Primary systemic amyloidosis: clinical and laboratory features in 474 cases. Sem Hematol 1995; 32: 45-59. Laszik Z, Silva FG. Hemolytic-uremic syndrome, thrombotic thrombocytopenic purpura, and systemic sclerosis (systemic scleroderma). In Heptinstall's Pathology of the Kidney, 5th edition 1998, pg 1003--1058, Lippincott Raven publish. Markowitz GS. Dysproteinemia and the kidney. Adv Anat Pathol 2004; 11: 49-63. Mauer SM, Staffes MV, Ellis EN, Sutherland DE, Brown DM, Goetz FC: Structural-functional relationships in diabetic nephropathy. J Clin Invest 1984; 74:1143 Mauer SM, Goetz FC, McHugh LE, Sutherland DE, Barboas J, Najarian JS, Steffes MW: Long-term study of normal kidneys transplanted into patients with type I diabetes. Diabetes 1989; 38:516 Merlini G, Bellotti V. Molecular mechanisms of amyloidosis. N Engl J Med 2003; 349: 583 Moake JL. Thrombotic microangiopathies. N Engl J Med 2002; 347:589-600. Moake JL. Thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome. Arch Pathol Lab Med 2002; 126:1430. Moll S, Nickeleit V, Mueller-Brand J, et al. A new cause of renal thrombotic microangiopathy: yttrium 90-DOTATOC internal radiotherapy. Am J Kidney Dis 2001; 37:847-851. Musio F, Bohen EM, Yuan CM, et al. Review of thrombotic thrombocytopenic purpura in the setting of systemic lupus erythematosus. Semin Arthritis Rheum 1998; 28:1-19. Olson J: Diabetes mellitus. In Heptinstall's Pathology of the Kidney, 5th edition 1998, pg 1247-1286, Lippincott Raven publish. Osterby R, Gundersen HJ, Horlyck A, Kroustrup JP, Nyberg G, Westberg G: Diabetic glomerulopathy: structural characteristics of the early and advanced stages. Diabetes 1983; 2:79 Osterby R: Structural changes in the diabetic kidney. J Clin Endocrinol metab 1986; 15:733 Osterby R, Gall MA, Schmitz A, Nielsen FS, Nyberg G, Parving HH: Glomerular structure and function in proteinuric type-2 (non-insulin-dependent) diabetic patients. Diabetologia 1993; 36:1064 Paul SN, Sangle SR, Bennett AN et al. Vasculitis, antiphospholipid antibodies, and renal artery stenosis. Ann Rheum Dis 2005; 64: 1800 (letter) Rand JH. The antiphospholipid syndrome. Annu Rev Med 2003; 54: 409 Roecken C, Shakespeare A. Pathology, diagnosis and pathogenesis of AA amyloidosis. Virchows Arch 2002; 440: 111 Saracino A, Ramunni A, Pannarale G, Coratelli, P. Kideny disease associated with primary antiphospholipid syndrome: clinical signs and histopathological features in an experience of five cases.Clin Nephrol 2005; 6: 471 Schwartz M M: The dysproteinemias and amyloidosis. In Heptinstall's Pathology of the Kidney, 5th edition 1998, pg 1321-1369, Lippincott Raven publish. Selby JV, Fitzsimmons SC, Newman JM, Katz PP, Sepe S, Showstack J: The natural history and epidemiology of diabetic nephropathy: implications for prevention and control. JAMA 1990; 263:154 Seifter JL, Nickeleit V. A 67-year-old woman with vomiting, bloody diarrhea and azotemia. N Engl J Med 1997; 336 (22): 1587-1594. Shah NT, Rand JH. Controversies in differentiating thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Mt Sinai J Med. 2003 Oct;70 (5):344. Strom EH, Fogazzi GB, Banfi G, Pozzi C, Mihatsch MJ: Light chain deposition disease of the kidney – morphological aspects in 24 patients. Virch Archiv 1994; 425: 271 Tsai HM. Advances in the pathogenesis, diagnosis, and treatment of thrombotic thrombocytopenic purpura. J Am Soc Nephrol 2003; 14:1072 Tsai HM, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med. 1998;339(22):1585 Tsirpanlis G, Moustakas G, Sakka E et al. Catastrophic antiphospholipid syndrome in a 14-year-old child. Pediatr Nephrol 2005; 20: 519