5.1 Background and development
The urinary system of the mouse comprises the paired kidneys, urinary bladder, paired ureters and urethra. The murine kidney demonstrates similar structures to those of other mammalian species and many mouse models of human kidney disease have been developed by researchers.
Mesodermal cells immediately adjacent to the intermediate mesoderm form the urogenital system, comprising the gonads, the sex ducts and the kidneys (Kispert and Gossler 2004). The development of the mouse kidney is characterised by the formation of three paired embryonic kidney structures called the pronephros, mesonephros and metanephros. These three paired kidney structures develop from mesodermal cells. The pronephros and mesonephros degenerate leaving the metanephros which develops into the adult kidney. The definitive adult kidney develops through a series of inductive interactions between the uteric bud and the surrounding metanephric mesenchyme (Ohse et al. 2009). The tubules of the mesonephros do not form part of the permanent kidney. The Wolffian duct (also known as mesonephric duct) is a paired organ found in mammals during embryogenesis. It connects the primitive kidney Wolffian body (or mesonephros) to the cloaca and serves as the foundation for certain male reproductive organs. The ureter buds from the Wolffian duct and the urinary bladder is formed partly from the endodermal cloaca and the Wolffian ducts.
The metanephric mesenchyme develops into the renal vesicle, which forms the functional kidney. Collecting tubules (Figure 5.1) and primitive glomeruli can be recognized from about embryonic day 14, at which stage the kidney starts to produce urine. Well differentiated cortex and medulla containing glomeruli and nephrons can be distinguished from about embryonic Day 16 (Kaufman and Bard 1999).
Figure 5.1 Metanephric development at embryonic day E14.5, with the start of differentiation of nephrons and the collecting duct system.
The kidney continues to mature anatomically and functionally over the first five to six weeks of life in the mouse. Regression of glomeruli occurs over the first two to three weeks of life (Zhong et al. 2012) but occasional immature glomeruli may be retained even in mature animals (Figure 5.2).
5.2 Sampling techniques
Both kidneys should be harvested at necropsy and it may be useful to make a longitudinal incision in the left kidney and a transverse incision in the right kidney at necropsy if it is important to distinguish left and right kidneys from one another. Both kidneys should be fixed for 24 hours (minimum) in 10% neutral buffered formalin before cutting either longitudinal or transverse sections of kidney and placing the tissue in a cassette. At necropsy it should be possible to visualize the outer zone of the kidney, the cortex, as well as the inner zone of the kidney called the medulla and the pelvis, which is the structure created from the expanded upper portion of the ureter (Pakurar and Bigbee 2009). The easiest way to ensure that all zones of the kidney are consistently sampled is by taking transverse sections. Longitudinal sections may be justified if there is suspicion that a lesion may only occur at the poles—which is unusual but may occur if a tumour is present.
The ureters are harvested by gently lifting the adipose tissue between the kidneys and the bladder and exposing the left and right ureters by removing the surrounding adipose tissue. The bladder is located at the base of the uterus (in female mice) and above the prostate gland in male mice. The bladder should be sectioned as close to the underlying organs (uterus in female mice and prostate in male mice) as possible. Infiltration of the bladder with formalin may be necessary in order to remove folds in the inner urothelium.
5.3 Artefacts
Artefacts in the kidney include the extrusion of renal cortical epithelial cells into the Bowman’s space due to excessive handling of the kidney at necropsy. Kidneys are also particularly susceptible to autolysis and this can be difficult to distinguish from acute tubular necrosis. Autolysed renal tissue is generally characterised by the presence of pale eosinophilic material and cuboidal epithelium within the cortical tubules, particularly epithelial cells without distinct borders and indistinct nuclei (Figure 5.3). Kocovski and Duflou (2009) reported that autolysis is extremely difficult to distinguish from acute tubular necrosis in the kidney and that only the presence of epithelial whorls within the tubules is always characteristic of acute tubular necrosis. Periodic Acid-Schiff (PAS) staining may be helpful in identifying autolysis in the kidney as a reduction in PAS positive staining has been reported to correlate with the onset of autolysis (Zdravkovic et al. 2006).
Figure 5.3 Autolysed renal tissue is generally characterised by the presence of pale eosinophilic material and cuboidal epithelium within the cortical tubules, epithelial cells without distinct borders or obvious nuclei (black arrow) or pyknotic nuclei (white arrow).
5.4 Background lesions
Background lesions are seen in urinary tract of young and old mice and have been reviewed recently (Taylor 2011 and Frazier et al. 2012). Familiarity with these lesions is important so that they can be differentiated from induced lesions. The investigator should also be aware that changes in incidence or severity of these lesions may be induced and therefore may represent a genuine phenotype.
Hydronephrosis (pelvic dilatation) (Goto et al. 1984) (Figure 5.4) and cysts (Figure 5.5) within the cortex or medulla of the kidney are common background lesions in the mouse kidney. Hydronephrosis may be congenital or acquired (in the case of obstruction in the ureters, bladder or urethra) and may be unilateral or bilateral.
Figure 5.4 Hydronephrosis or pelvic dilatation may be congenital or acquired (in the case of obstruction in the ureters, bladder or urethra) and may be unilateral or bilateral.
Figure 5.5 Cysts within the cortex or medulla of the kidney are common background lesions in the mouse kidney.
Chronic progressive nephropathy (CPN) is observed commonly in ageing mice (Figure 5.6) and is characterised by thickening of the glomerular basement membranes, obliteration of the glomeruli, mononuclear cell infiltration, basophilic cortical tubules and fibrosis of the interstitium (Percy and Barthold 2007). Even in mice as young as five weeks, the early changes of chronic progressive nephropathy may be present and these include lymphocyte aggregates in the interstitial cortex and cortical epithelial cells with blue staining cytoplasm referred to as basophilic tubules (Figure 5.7).
Figure 5.6 Chronic progressive nephropathy (CPN) is observed commonly in ageing mice and is characterised by thickening of the glomerular basement membranes, obliteration of the glomeruli, mononuclear cell infiltration, basophilic cortical tubules and fibrosis of the interstitium.
Figure 5.7 Cortical epithelial cells with blue staining cytoplasm, referred to as basophilic tubules in the mouse kidney.
Other changes that are commonly seen in tubules are vacuolation (Figure 5.8) and pigmentation as a result of accumulations of lipid and lipofucsin respectively (Frazier et al. 2012).
Interstitial changes include adipose aggregates (lipomatosis, lipomatous metaplasia), inflammation (particularly lymphocytes) (Figure 5.9) and mineralization (Figure 5.10), which may occur in the cortical, medullary or papillary regions of the mouse kidney (Seely 1999).
Figure 5.10 The collecting tubule drains urine from the distal convoluted tubules in the cortex and descends into the medulla where it joins other collecting tubules (*) in the papilla. Small foci of mineralization are commonly found in the papilla (arrows) as well as other regions of the kidney.
Refluxed seminal colloid plugs in the bladder (Figure 5.11) and urethral plugs (copulatory plugs) occur occasionally in mice. Ejaculation results in the formation of a copulatory plug and this often occurs agonally after euthanasia (Percy and Barthold 2007). The yellow plug is composed of eosinophilic, proteinaceous material within which spermatozoa may be embedded (Taylor 2011). Urolithiasis or the formation of urinary stones may be distinguished from colloidal reflux by the presence of inflammatory cells in the lumen and the adjacent epithelium and the mineralized concentric layered appearance of genuine uroliths.
Figure 5.11 Refluxed seminal colloid plugs in the bladder are composed of eosinophilic, proteinaceous material within which spermatozoa may be embedded.
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