TGR5 expression in normal kidney and renal neoplasms

With Institutional Review Board approval at Rhode Island Hospital, sixty-one renal cortical neoplasms were identified from the archives of the Department of Pathology between 2000 and 2014. These included 27 clear cell RCCs and 11 papillary RCCs, 8 of which were type 1 and 3 of which were type 2. The remainder of the cases consisted of 8 papillary urothelial carcinomas including one poorly differentiated case involving the cortex, 6 chromophobe RCCs, 5 oncocytomas, 2 clear cell papillary RCCs, 1 metanephric adenoma, and 1 poorly differentiated RCC with sarcomatoid features. Formalin-fixed paraffin-embedded tissue sections and blocks were retrieved. The corresponding hematoxylin-eosin slides were reviewed for confirmation of diagnosis and adequacy of material by a specialist in genitourinary pathology (AA). Multiple representative sections of each case were examined. Corresponding normal kidney control sections separate from the tumors were also reviewed for all cases. Snap-frozen tissue from normal kidney, clear cell RCCs, papillary RCCs, chromophobe RCC and oncocytomas was obtained from the Rhode Island Hospital tissue bank.

Paraffin blocks containing areas of carcinomas were identified on the hematoxylin and eosin-stained sections. Areas of the tumor were identified and marked on the source block. The source block was cored using a 1-mm needle and the tissue was transferred to a recipient “master block” using a Beecher Tissue Microarrayer (Beecher Instruments, Silver Spring, MD). Three to six cores of each tumor were arrayed per specimen. Additionally, a core of normal kidney tissue was also sampled when present [12].

Immunohistochemistry for TGR5 was performed on 4-μm paraffin sections of the tissue microarray or whole tissue sections. Slides were stained with TGR5 antibody (rabbit anti-human, polyclonal, 1:1000; Sigma-Aldrich, St. Louis, MO) using the DAKO Envision + Dual Link System and the DAKO Liquid 3,3′-Diaminobenzidine Substrate Chromogen System (DAKO North America, Carpinteria, CA). Bile ducts from liver tissue were used as positive controls. Negative controls were included by replacement of the primary antibody with non-reacting antibodies derived from the same species. The specificity of TGR5 antibody has previously been confirmed by Western blot analysis in our lab [12].

Tissue microarray was employed to study normal kidney and 44 cases of renal neoplasms, including all clear cell RCCs, 1 metanephric adenoma, 8 papillary urothelial carcinomas, 4 type 1 papillary RCCs, 3 oncocytomas, and 1 chromophobe RCC.

Immunohistochemistry was also performed on whole tissue sections for 17 cases including 4 papillary RCC (4 type 1 papillary RCC and 3 type 2 papillary RCC), 5 chromophobe RCC, 2 oncocytomas, 2 clear cell papillary RCC, and 1 poorly differentiated RCC with some sarcomatoid features.

Immunohistochemical results were evaluated semi-quantitatively within neoplastic tissue. Cells displaying strong intensity staining for TGR5 were scored as 3+, moderate staining as 2+, and weak staining as 1+. The extent of staining was scored as follows: 0 (< 5%), 1+ (5–25%), 2+ (26–50%), 3+ (51–100%). A combined score of intensity and extent was calculated and assigned as follows: weak staining 1–2, moderate staining 3–4, and strong staining 5–6. At least three cores were scored per case. Analysis of three cores per case in this fashion has been shown to be comparable to whole tissue sections [13]. All sections were independently scored by two pathologists (WC and CZ) blinded to clinicopathological features and outcome.

TGR5 mRNA was measured using real-time PCR analysis as we have previously described [14]. The primers used were as follows: TGR5 sense (5′- CTGGCCCTGGCAAGCCTCAT-3’), TGR5 antisense (5′- CTGCCATGTAGCGCTCCCCGT-3’), 18S sense (5’-CGGACAGGATTGACAGATTGATAGC-3’), and 18S antisense (5’-TGCCAGAGTCTCGTTCGTTATCG-3’). Reactions were carried out in an Applied Biosystems StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) for one cycle at 94 °C for 5 min followed by 40 cycles at 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s, 1 cycle at 94 °C for 1 min, and 1 cycle at 55 °C for 30 s. Fluorescence values of SYBR Green I dye, representing the quantity of product amplified at that point in the reaction, were recorded in real time at both the annealing and extension steps of each cycle. The C_t, defined as the point at which the fluorescence signal becomes statistically significant above background, was calculated for each amplicon for each sample using StepOne software (Applied Biosystems, Foster City, CA). The transcript level of each specific gene was normalized to 18S amplification.

In normal kidney, the proximal tubular cells are more columnar and eosinophilic than cells of the distal tubules on H&E slides. The ascending limb has no brush border and the cells are more cuboidal than adjacent proximal tubular cells [15]. According to this criterion, TGR5 was strongly expressed in the distal convoluted tubules, thin loop of Henle, and collecting ducts (Fig. 1a). In contrast, the proximal convoluted tubular cells showed absent or focal weak staining (Fig. 1b). TGR5 expression was not observed in the glomerular tuft, but focal immunoreactivity was identified in parietal epithelial cells (Fig. 1c).

The expression of TGR5 in renal cell neoplasms is summarized in Table 1. Notably, among 25 cases of clear cell RCCs, 92% (25/27) of cases were negative for TGR5 staining (p < 0.001, Fig. 2a). The remaining 2 cases of clear cell RCCs (2/27) exhibited TGR5 focal staining in areas with papillary features. One of these two TGR5-positive cases also exhibited sarcomatoid differentiation. Both cases of clear cell papillary RCCs (2/2) displayed strong TGR5 staining (Fig. 2d).

All papillary RCCs (11/11) demonstrated TGR5 staining. The TGR5 staining in Type 1 papillary RCCs (8 cases) were variable from weak to strong staining (Fig. 2b). All 3 cases of type 2 papillary RCCs showed only focal weak staining (Fig. 2c). There were no statistically significant differences between type 1 and type 2 papillary RCCs (p = 0.152).

All chromophobe RCCs (6/6) exhibited weak to moderate TGR5 staining (Fig. 2e). The oncocytomas all (5/5) showed weak TGR5 expression (Fig. 2f). No statistically significant differences between chromophobe RCCs and oncocytomas (p = 0.082) were identified. The metanephric adenoma (0/1; Fig. 2g) and poorly differentiated RCC with sarcomatoid features (0/1) were negative for TGR5 staining.

Urothelial carcinomas all uniformly (8/8) expressed TGR5 (Fig. 2h) including the case of poorly differentiated sarcomatoid case (Fig. 2i).

TGR5 mRNA was significantly diminished in clear cell RCC (N = 5, p < 0.001, fig. 3a) in comparison to normal non-neoplastic renal tissue. TGR5 mRNA expression in papillary RCCs, chromophobe RCCs, and oncocytomas did not significantly differ from normal renal tissue (fig. 3b).