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RENAL PATHOLOGYHistorically, the introduction of electron microscopy has been invaluable in renal pathology for the study of glomerular structure and ultrastructural detail of glomerular capillary walls and glomerular basement membranes. In addition, electron microscopy has confirmed the existence of the mesangium and defined many of its characteristics. NAZMOON LAILA
Normal Renal Histology • The normal renal cortex contains • glomeruli • other vessels • tubules and • interstitium • Features in H&E : • overall cellularity of the glomerulus • the symmetry of the glomerulus • the thickness of the capillary walls
The renal cortex • renal cortex is easily identified even at low magnification by the presence of renal corpuscles, which are absent in the renal medulla. However, the bulk of the cortex is occupied by the proximal and distal convoluted tubules. The arcuate arteries and veins help to demarcate the cortex from the medulla.
These micrographs compare the appearances of the proximal and distal convoluted tubules. The proximal convoluted tubule (PCT) is a coiled tube measuring approximately 14mm in length and random sections of PCT thus occupy most of the renal cortex. Approximately 65% of the glomerular filtrate is reabsorbed from the PCT, a function reflected in the structure of the epithelial lining • As seen in micrograph (c), distal convoluted tubules DCT may be differentiated from proximal convoluted tubules PCT by the absence of a brush border, a larger more clearly defined lumen, more nuclei per cross-section (since DCT cells are smaller than PCT cells) and paler cytoplasm (due to fewer organelles). In addition, sections of DCT are less numerous than sections of PCT since the DCT is much shorter than the PCT. In micrograph (d) the prominent brush border of the PCT is contrasted with the lack of brush border in the DCT
The renal medulla : • renal medulla consists of closely packed tubules of two types: the loop of Henle and the collecting tubules and ducts as well as the vasa recta. The loop of Henle is a continuation of the proximal convoluted tubule. It dips down into the medulla, where it loops back on itself and returns to the cortex to it's own renal corpuscle, becoming the first part of the distal convoluted tubule.
Acute nephritic syndrome is a glomerular syndrome dominated by the acute onset of usually grossly visible hematuria (red blood cells in urine), mild to moderate proteinuria, and hypertension; it is the classic presentation of acute poststreptococcal glomerulonephritis. • The nephrotic syndrome is characterized by heavy proteinuria (more than 3.5 gm/day), hypoalbuminemia, severe edema, hyperlipidemia, and lipiduria (lipid in the urine). • Asymptomatic hematuria or proteinuria, or a combination of these two, is usually a manifestation of subtle or mild glomerular abnormalities. • Acute renal failure is dominated by oliguria or anuria (reduced or no urine flow), with recent onset of azotemia. It can result from glomerular, interstitial, or vascular injury or acute tubular necrosis. • Chronic renal failure, characterized by prolonged symptoms and signs of uremia, is the end result of all chronic renal parenchymal diseases.
Renal tubular defects are dominated by polyuria (excessive urine formation), nocturia, and electrolyte disorders (e.g., metabolic acidosis). They are the result of either diseases that directly affect tubular structure (e.g., medullary cystic disease) or defects in specific tubular functions. The latter can be inherited (e.g., familial nephrogenic diabetes, cystinuria, renal tubular acidosis) or acquired (e.g., lead nephropathy). • Urinary tract infection is characterized by bacteriuria and pyuria (bacteria and leukocytes in the urine). The infection may be symptomatic or asymptomatic, and it may affect the kidney (pyelonephritis) or the bladder (cystitis) only. • Nephrolithiasis (renal stone) is manifested by renal colic, hematuria, and recurrent stone formation. • Urinary tract obstruction and renal tumors represent specific anatomic lesions with often varied clinical manifestations
Glomerular Diseases • constitute some of the major problems in nephrology; indeed, chronic glomerulonephritis is one of the most common causes of chronic renal failure in humans. • Glomeruli may be injured by a variety of factors and in the course of a number of systemic diseases. • Systemic immunologic diseases such as systemic lupus erythematosus (SLE), vascular disorders such as hypertension and polyarteritis nodosa, metabolic diseases such as diabetes mellitus, and some purely hereditary conditions such as Fabry disease often affect the glomerulus. These are termed secondary glomerular diseases to differentiate them from disorders in which the kidney is the only or predominant organ involved. • The latter constitute the various types of primary glomerulonephritis or, because some do not have a cellular inflammatory component, glomerulopathy. • However, both the clinical manifestations and glomerular histologic changes in primary and secondary forms can be similar.
Various types of glomerulonephritis are characterized by one or more of four basic tissue reactions. • Some inflammatory diseases of the glomerulus are characterized by an increase in the number of cells in the glomerular tufts. This hypercellularity is characterized by one or more combinations of the following: • Cellular proliferation of mesangial or endothelial cells • Leukocytic infiltration, consisting of neutrophils, monocytes, and, in some diseases, lymphocytes • Formation of crescents. These are accumulations of cells composed of proliferating parietal epithelial cells and infiltrating leukocytes. The epithelial cell proliferation that characterizes crescent formation occurs following an immune/inflammatory injury (see later). Fibrin, which leaks into the urinary space, often through ruptured basement membranes, has been long thought to be the molecule that elicits the crescentic response. such as interleukin-1, tumor necrosis factor, and interferon-γ.
Basement Membrane Thickening. By light microscopy, this change appears as thickening of the capillary walls, best seen in sections stained with periodic acid-Schiff (PAS). • By electron microscopy, such thickening can be resolved as one of two alterations: (1) deposition of amorphous electron-dense material, most often immune complexes, on the endothelial or epithelial side of the basement membrane or within the GBM itself. Fibrin, amyloid, cryoglobulins, and abnormal fibrillary proteins may also deposit in the GBM; or (2) thickening of the basement membrane proper, as occurs in diabetic glomerulosclerosis. • .
Hyalinization and Sclerosis. • Hyalinization, or hyalinosis, as applied to the glomerulus, denotes the accumulation of material that is homogeneous and eosinophilic by light microscopy. • By electron microscopy, the hyalin is extracellular and consists of amorphous substance, made up of plasma proteins that have exuded from circulating plasma into glomerular structures. • This change contributes to obliteration of capillary lumina of the glomerular tuft (a feature of sclerosis). Hyalinosis is usually a consequence of endothelial or capillary wall injury and typically is the end result of various forms of glomerular damage. Additional alterations include intraglomerular thrombosis or accumulation of lipid or other metabolic materials
All the glomerular capillaries should be about the same thickness, which is very thin (almost wispy). • With normal cellularity, cell nuclei are not clustered or overlapping. Clusters of cells, especially away from the hilum, indicate abnormal hypercellularity. Increased cells within the lumens of capillaries indicate leukocyte infiltration or, rarely, an angiotrophic lymphoma.
In the cortex but not the medulla, the tubules should be almost back to back, i.e. the tubular basement membranes should be almost touching. There is very little interstitium in the cortex, therefore, if there is space between the tubules, there is something wrong in the tubulointerstitial compartment (e.g. edema or fibrosis).
Intrarenal arteries have very little intima, i.e. there is little or no space between the endothelium and the muscularis. Pathologic processes expand the arterial intima, e.g. collagen in arteriosclerosis and proteinaceous insudate in an acute thrombotic microangiopathy.
(green = epithelial cells, yellow = endothelial cells, red = mesangial cells).
Visceral epithelial cells line the capillary walls. Parietal epithelial cells line Bowman's capsule, and are continuous with the proximal tubular epithelial cells. Endothelial cells line capillary lumens. Mesangial cells are in the middle (meso) between the capillaries (angis). The mesangial cells are modified smooth muscle cells that are continuous with the vascular smooth muscle cells in the hilar arterioles. As such, they have a contractile capability and can tug on the edges of the capillaries and thus control blood flow through the glomerulus
Mesangial cells also produce a variety of cytokines when stimulated, and are capable of phagocytosis. There is a route for trafficking of debris through the mesangium that begins in the subendothelial zone and enters the mesangium and then passes through physiologic if not actual channels through the matrix to the hilum.
PAS Trichrome H&E Jones silver stains
The silver stain accentuates collagenous structures, e.g., in the glomerulus, the mesangial matrix and the glomerular basement membrane. The PAS stain also accentuates matrix and basement membrane constituents, as does the blue or green component on the trichrome stain. In certain circumstances the trichrome stain demonstrates immune deposits as fuchsinophilic (red) structures.
The peripheral endothelial cell cytoplasm, which has pores through it, looks like a little string of sausages on cross section. This allows recognition of the lumenal side of the capillary wall. The visceral epithelial cells, or podocytes, have foot processes that are intact in normal glomeruli and often effaced in proteinuric conditions.
The glomerular basement membrane has 3 ultrastructural zones that can be disturbed in various glomerular diseases: the lamina densa in the middle, the lamina lucida (rara) externa and the lamina lucida (rara) interna.
The glomerular basement membrane does not completely enclose the lumen, unlike the endothelial basement membrance in most vessels, but rather splays out over the mesangium to become the paramesangial basement membrane. This leaves a functional gap where materials from the capillary lumen or subendothelial zone (having passed through the endothelial pores) can directly enter the mesangium without traversing the basement membrane. This explains why the mesangium is a preferential sequestration point for some types of debris, including immune complexes.
The endothelial cell nucleus sits over the origin of the mesangium, which is where it is usually found. A few pores through the endothelial cytoplasm can be seen. The glomerular basement membrane lamina lucida externa is the thin lucent zone just under the foot processes of the visceral epithelial cell. The bulk of the basement membrane is the lamina densa.
The pores through the endothelial cell are below the basement membrane. The thickness of the glomerular basement membrane lamina densa is about 5-6 times thicker than the lamina lucida externa in this particular electron micrograph. The lamina lucida externa thickness is a useful landmark that can be used to assess the normal thickness of the glomerular basement membrane. The thickness of the lamina densa is important in making the diagnosis, for example, of thin basement membrane nephropathy and diabetic glomerulosclerosis.
Another internal reference point for basement membrane thickness is an intact foot process. If you average the width of intact foot processes and then turn that 90 degrees, that is about the normal thickness of the laminar densa. Therefore, if you compare the thickness of the lamina densa to that of the lamina lucida externa or to an intact foot process, you can determine whether the basement membrane is normal or abnormal thickness.
Minimal Change Glomerulopathy • There are many synonyms for minimal change glomerulopathy, e.g., minimal change disease, lipoid nephrosis, nill disease. • No abnormality. • Sometimes there may be a little bit of mesangial hypercellularity in a few segments. Otherwise, any scarring, any infiltration of leukocytes, any necrosis, or any other substantial structural changes in glomeruli rule out a diagnosis of minimal change glomerulopathy
Slide 13 is a representative immunofluorescence micrograph of the immunohistology of minimal change glomerulopathy, i.e., background staining. There are occasional specimens that will have small amounts of exclusively mesangial immunoglobulin (especially IgM) or complement accumulation that can still be designated minimal change glomerulopathy. A little bit of mesangial IgM and/or C3 without ultrastructural evidence for electron dense deposits is tolerable for a diagnosis of minimal change glomerulopathy. When groups of patients with absolutely no immunofluorescence findings have been compared to those that have low levels of IgM dominant mesangial deposits without electron dense deposits, they act no differently with respect to their clinical response to steroids and long term outcomes. Well defined mesangial electron dense deposits, however, worsen the prognosis for response to steroids or spontaneous remission. Thus, if there are electron dense deposits, minimal change glomerulopathy is not an appropriate diagnoses.
Effacement of visceral epithelial foot processes and epithelial microvillous transformation. Microvillous transformation of epithelial cytoplasm often accompanies effacement. The effacement of foot processes and microvillous transformation are not specific for minimal change glomerulopathy.
Foot process effacement is characteristic for minimal change glomerulopathy and is required for the pathologic diagnosis of this disease; however, this same change is present in any patient with substantial proteinuria of any cause. Therefore, the diagnosis of minimal change glomerulopathy is one of exclusion, i.e., these ultrastructural changes should be present in the absence of light microscopic, immunohistologic or other ultrastructural features of any other cause of proteinuria.
almost complete effacement of the visceral epithelial foot processes. There is condensation of the epithelial cytoskeleton near the basement membrane. If you don't know what this is, you can mistake it for subepithelial electron dense deposits, suggesting membranous glomerulopathy. It is actin condensation that takes place inside of visceral epithelial cytoplasm when there is effacement of foot processes, suggesting that there is movement of cytoplasmic structures during the effacement event.
Membranous Glomerulopathy • Membranous glomerulopathy is the most common cause for the nephrotic syndrome in adults, whereas, minimal change glomerulopathy is the most common cause for the nephrotic syndrome in children. Even though membranous glomerulopathy is the most common cause in adults, it only accounts for about 1/3 of adults with nephrotic syndrome in my renal biopsy population. The frequency of membranous glomerulopathy in other series ranges from around 20% to around 50%, and most series are under 50%. Thus, in an adult with the nephrotic syndrome, if you guess membranous glomerulopathy every time, you are going to be wrong about 2/3 of the time. Therefore, in adults with nephrosis, most nephrologists will biopsy to identify the underlying disease • From age 16 to 65, membranous glomerulopathy is rather frequent, but its highest frequency is in the 40-60 year old age group.
early stage: If you don't have a good internal reference as to the thickness of capillary loops, it is hard to look at a membranous glomerulopathy biopsy and be sure there is something wrong by light microscopy, especially during early stages to the disease
a late stage membranous glomerulopathy with markedly thickened capillary walls.
the very thick capillary wall of an overt case of membranous glomerulopathy can be recognized. On a trichrome stained section (middle panel), if you have a good stain and if the stage of the disease is just right and there are big deposits, you can see the subepithelial immune complex deposits as fuchsinophilic (red) granular deposits. The blue basement membrane is beneath the deposits and there are projections of blue between them. On a silver stained section, and sometimes on a well- stained PAS stained section, as shown in the panel on the right, you can see the so-called spikes of basement membrane that project between the deposits in certain stages of membranous glomerulopathy, in particular stage II.
