Introduction to Kidney Biopsy and Basic Glomerular Pathology

Lihong Bu

Anthony Chang

KIDNEY BIOPSY INDICATIONS AND SPECIMEN HANDLING

Kidney biopsy is the gold standard to diagnose glomerular diseases. Common indications for native kidney biopsies include kidney dysfunction, hematuria, or proteinuria. Percutaneous kidney biopsy for the detection of primary kidney disease is generally not pursued in certain settings (Table 2.1).

During the biopsy procedure, sample adequacy may be assessed by visualizing the tissue under a dissecting microscope on dampened wax paper or a bright-field microscope with the specimen on a glass slide, although the smartphone camera may serve as an alternative to either microscope. Glomeruli look like small red globules, but this appearance is less reliable with increasing degrees of glomerulosclerosis; in contrast, the medulla is characterized by the presence of a reddish vascular network and absence of glomeruli (Figure 2.1).

The ideal biopsy evaluation requires glomeruli in the three tissue samples submitted for light microscopy (LM), immunofluorescence (IF), and electron microscopy (EM). The sample submitted in formalin is the most critical because it can be processed for both IF and EM, albeit with less sensitivity for the former and preservation/processing artifact for the latter. In general, the specimen is most optimal when at least 1.0 cm of renal cortex is obtained using a 16-gauge needle. Ideally, there should be at least 10 to 15 glomeruli in the LM sample. Generally, two to three tissue cores are obtained. A larger tissue sample increases the likelihood that the biopsy accurately reflects the patient’s kidney pathology (disease activity and chronicity) and decreases the probability of missing focal lesions. No minimum number of glomeruli has been defined for specimen adequacy because it varies for different glomerular diseases. For example, 15 to 20 glomeruli may be insufficient to diagnose focal segmental glomerulosclerosis, but one intact glomerulus in the IF sample may be enough to diagnose immunoglobulin (Ig) A or membranous nephropathy. The absence of glomeruli in one or more of these three modalities decreases the chance of reaching a precise and definitive diagnosis.

The tissue is divided into three parts with a minimum of 0.1 to 0.2 cm of cortex fixed in 2.5% glutaraldehyde for EM, a minimum of 0.5 cm cortex in Zeus solution for IF, and the remaining amount of tissue fixed in 10% neutral buffered formalin for LM. The division of the tissue cores should be performed quickly. In the absence of adequacy assessment, separate cores can be placed in three appropriate media when generous tissue is obtained. If the tissue is limited, it should be divided and triaged according to the clinical differential diagnosis.¹

TABLE 2.1 Contraindications to Percutaneous Kidney Biopsy for the Detection of Primary Kidney Disease

Small hyperechoic kidneys (generally <9 cm in length by ultrasound but adjusted for height, weight, and/or body mass index), which are generally indicative of chronic irreversible disease

Solitary native kidney (relative contraindication)

Multiple, bilateral cysts or a kidney tumor (relative contraindication)

Uncorrectable bleeding diathesis

Severe hypertension (ie, systolic blood pressure >170 mm Hg) that cannot be controlled with antihypertensive medications

Hydronephrosis

Active renal or perirenal infection

Anatomic abnormalities of the kidney that may increase risk (such as polycystic kidney disease or horseshoe kidney)

Skin infection over the biopsy site

An uncooperative patient

When a skilled operator (nephrologist or interventional radiologist, experienced in the performance of the procedure) or appropriate pathology support is not available

FIGURE 2.1: The lower half of the left core shows striations typical of the medulla. The upper half of the left core and the right core show pale glomeruli (look white), illustrating that they are sometimes difficult to visualize.

When the IF sample does not contain any intact glomeruli, IF microscopy can be performed on the formalin-fixed, paraffin-embedded tissue submitted for LM after antigen retrieval with pronase (or other similar proteases) digestion, but this salvage technique generally has lower sensitivity and specificity for the majority of Igs and Ig fragments compared to frozen tissues, and, in particular, C3 staining is unreliable.² When the tissue for EM does not contain glomeruli, the formalin-fixed, paraffin-embedded tissue for LM and unfixed frozen sample for IF may be reprocessed for ultrastructural studies for glomerular basement membrane (GBM) morphology and immune complex deposits, but the unfixed IF tissue sample will have poor tissue preservation and the LM sample will artifactually thin the GBM if precise measurements are needed to assess for thin basement membrane nephropathy.

GENERAL APPROACH TO MORPHOLOGIC EVALUATION

Serial sections of 2 to 3 µm in thickness are stained with hematoxylin and eosin, periodic acid-Schiff (PAS), Jones methenamine silver, and Masson trichrome (Figure 2.2). The extracellular matrix material, which includes the mesangium, Bowman capsules, and glomerular and tubular basement membranes, is best visualized on PAS and silver stains. Thrombi and fibrosis are better seen on trichrome stain. The four compartments (glomeruli, tubules, interstitium, and vessels) of the kidney are systematically evaluated, and the most severely affected component may represent the primary injury, but this determination may not be possible in advanced disease when all the kidney compartments are severely affected. When scarring of the glomerular compartment occurs, the subsequent
reduction in blood flow that normally supplies the peritubular capillary network results in interstitial fibrosis and tubular atrophy. Conversely, severe tubulointerstitial or vascular injuries also negatively affect the glomeruli. The major lesions involving the glomeruli are the focus of this chapter.

FIGURE 2.2: Normal glomerular histology. A, Periodic acid-Schiff. B, Jones methenamine silver. C, Masson trichrome. D, Hematoxylin and eosin. Original magnification 40×.

Normal Morphology of Glomerulus

A normal glomerulus consists of four types of cells. The endothelial cells line the glomerular capillaries as well as the arterioles and arteries. The glomerular endothelial cells are fenestrated and aid in the filtration of blood to form urine. Mesangial cells are part of the reticuloendothelial system and reside in the mesangia between the capillary loops. They also have muscle-like contractile properties. The parietal epithelial cells line the inside of the Bowman capsules and form the outer limit of the Bowman space. The visceral epithelial cells (podocytes) line the outer aspect of the GBMs. Podocytes are terminally differentiated, specialized epithelial cells that lack mitotic capability. Ultrastructurally, podocytes have long cytoplasmic processes that divide into interdigitating foot processes and come into direct contact with the lamina rara externa of the GBM. Together, the endothelial cells, mesangium, podocytes, and the GBM form the permselective filter (Figure 2.3). The GBM is an acellular membrane with a major component of type IV collagen. Ultrastructurally, the GBM is composed of a dense central layer, the lamina densa, and more electron-lucent inner and outer layers, the lamina rara interna and lamina rara externa. In healthy adults, the mean thickness of GBM is approximately 300 to 350 nm.³ The GBM is slightly thicker in men than in women and thinner in children, who achieve adult thickness by age 10 to 12 years.³

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