Assessing the application of tissue microarray technology to kidney research

Abstract

Tissue microarray (TMA) is a new high-throughput method that enables simultaneous analysis of the profiles of protein expression in multiple tissue samples. TMA technology has not previously been adapted for physiological and pathophysiological studies of rodent kidneys. We have evaluated the validity and reliability of using TMA to assess protein expression in mouse and rat kidneys. A representative TMA block that we have produced included: (1) mouse and rat kidney cortex, outer medulla, and inner medulla fixed with different fixatives; (2) rat kidneys at different stages of development fixed with different fixatives; (3) mouse and rat kidneys with different physiological or pathophysiological treatments; and (4) built-in controls. As examples of the utility, immunostaining for cyclooxygenase-2, renin, Tamm Horsfall protein, aquaporin-2, connective tissue growth factor, and synaptopodin was carried out with kidney TMA slides. Quantitative analysis of cyclooxygense-2 expression in kidneys confirms that individual cores provide meaningful representations comparable to whole-kidney sections. These studies show that kidney TMA technique is a promising and useful tool for investigating the expression profiles of proteins of interest in rodent kidneys under different physiological and pathophysiological conditions.

Figure 1

Design of the kidney tissue microarray (TMA). Cores with missing tissues or with tissues less than 10% of core area are indicated by boxes.

Figure 2

Characterization of cyclooxgenase-2 (COX-2) immunostaining in kidney TMA. COX-2 immunostaining in neonate rat kidney fixed with FPAS (A), GPAS (B), paraformaldehyde (C), or formalin (D). Cortical COX-2 expression in postnatal day 14 (P14) rat (E), P28 rat (F), adult rat (G), and old rat (H). Renal cortical COX-2 expression increased after treatment with salt restriction (I), adrenalectomy (J), captopril (K), or candesartan (L), compared with control rats (G). (M) COX-2 immunostaining in FPAS-fixed outer medulla of adult rat. (N) Cortical COX-2–immunofluorescent staining in low-salt–treated rat. (O) Magnification of boxed area in N. (P) Synaptopodin-immunofluorescent staining in normal adult rat. Bars: A–N = 200 μm; O = 50 μm; P = 32 μm.

Figure 3

Tamm Horsfall protein immunostaining in rat kidney cortex (A) and medulla (B). (C,D) Aquaporin-2 immunostaining was found in cortical collecting duct principal cells but not in intercalated cells of normal adult rat kidney. Arrows in D indicate intercalated cells. (E,F) Aquaporin-2 immunostaining in rat medulla. Renal renin expression was higher in candesartan-treated (H) or low-salt diet–treated (I) mice than in control mice (G). Connective tissue growth factor expression in juxtaglomerular cells from control mouse (J) and candesartan-treated mouse (K). Bars: A–C,E,G–J = 200 μm; D,F = 50 μm; K,L = 80 μm.

Figure 4

(A) Topographical maps of COX-2 immunostaining in kidneys from P14 and P28 rats. Magnifications are equivalent. (B) Ratios of COX-2 immunostaining area vs core area. n=4 in each group.