Identification of uromodulin deposition in the stroma of perinephric fibromyxoid nephrogenic adenoma by mass spectrometry

Nephrogenic adenoma (NA) is an epithelial lesion that usually occurs in the mucosa of the urinary tract. Rare cases of deep infiltrative or perinephric lesions have also been reported. Recently, NA with characteristic fibromyxoid stroma (fibromyxoid NA) has been proposed as a distinct variant. Although shedding of distal renal tubular cells due to urinary tract rupture has been postulated as the cause of NA in general, the mechanism underlying extraurinary presentation of NA and fibromyxoid stromal change in fibromyxoid NA remains unknown. In this study, we performed mass spectrometry (MS) analysis in a case of perinephric fibromyxoid NA of an 82‐year‐old man who underwent right nephroureterectomy for distal ureteral cancer. The patient had no prior history of urinary tract injury or radiation. Periodic acid‐Schiff staining‐positive eosinophilic structureless deposits in the stroma of fibromyxoid NA were microdissected and subjected to liquid chromatography/MS. The analysis revealed the presence of a substantial amount of uromodulin (Tamm−Horsfall protein). The presence of urinary content in the stroma of perinephric fibromyxoid NA suggests that urinary tract rupture and engraftment of renal tubular epithelial cells directly cause the lesion.


INTRODUCTION
Nephrogenic adenoma (NA) is a rare benign epithelial lesion that can occur in any part of the urinary tract, that is, from the renal pelvis to the urethra. 1 NA occurs mostly in the bladder, followed by the urethra and ureter, but may also be seen in the renal pelvis and prostate. 1The morphological features of NA include epithelial cells arranged in tubular, papillary, flat, microcystic, signet-ring cell-like, and vessel-like structures. 2,35][6] Mazal et al. have shown that NAs originate from renal tubular cells by studying those arising in kidney transplantation recipients. 7Later, other researchers have demonstrated that the epithelium of NA is characterized by PAX8 immunopositivity.The results gave further support for the renal distal tubular origin of the lesion, 8,9 and the disease is now considered to be caused by the leakage of exfoliated epithelial cells from the prior injury site in the urinary tract.
Fibromyxoid NA is a rare variant characterized by epithelial cells surrounded by characteristic fibromyxoid stroma. 10,11A recent study by Li et al. showed that this variant of NA occurs predominantly in the bladder (80%), followed by the urethra (15%) and ureter (5%). 10he extent of fibromyxoid stromal change varies between cases, with 65% showing mixed classical and fibromyxoid NA morphology and the remaining 35% showing pure fibromyxoid morphology. 10PAX8 immunoreactivity, which is an immunohistochemical hallmark of classical NA, was also observed in the epithelial cells of fibromyxoid NA. 10 The characteristic background matrix of fibromyxoid NA was reported to be strongly periodic acid-Schiff (PAS)-positive but negative for amyloid (Congo red) and reticulin staining. 11There have been few studies of the substance comprising the background matrix of fibromyxoid NA, and mass spectrometry (MS) has not been applied to address this issue.
Although NA is usually limited to the lamina propria, rare cases showing infiltrative growth into the perinephric adipose tissue (perinephric NA) have also been reported.Focal fibromyxoid morphology was noted in one of the three cases of perinephric NA documented by Diolombi et al. 12 In a series of fibromyxoid NAs, Li et al. also reported a case with involvement of perinephric fat. 10 Biopsy-associated perforation and extensive disruption due to hemorrhage or mechanical obstruction have been postulated as possible causes of these deep infiltrative NAs.However, the precise mechanism underlying the development of NA in extraurinary connective tissue remains unknown.
We recently encountered a rare case of perinephric fibromyxoid NA that extended to the periureteral region.To investigate the possible involvement of urinary leakage in its pathogenesis, we performed liquid chromatography/ MS (LC/MS) analysis of the stromal component of NA.

Case and histopathology
An 82-year-old man underwent full-body computed tomography for workup of an anterior mediastinal tumor, which unexpectedly revealed a right ureteral tumor, proximal ureteral dilatation, and right hydronephrosis.He had no prior history of urinary tract disease, radiotherapy, or Bacillus Calmette−Guérin therapy.Retrograde ureterography revealed a defect area in the lower ureter, suggesting lower ureteral cancer.The urine cytology was positive for cancer cells.Biopsy was not performed prior to surgery.Retroperitoneoscopic right nephroureterectomy was performed.
A total of 37 sections were made from the kidney and ureteral tissues of the nephroureterectomy specimen.The ureter and the surrounding tissue was sectioned in toto, in order to evaluated the extent of fibromyxoid NA which was present in the regions proximal to the ureteral cancer (Figure 1).
All procedures were performed in accordance with the tenets of the Declaration of Helsinki.The study was approved by the institutional ethical committee of Kanazawa University (approval number: 12644-2).

Double staining with cytokeratin immunostaining and PAS staining
To assess the relation between the intensity of eosinophilic deposits and distribution of epithelial cells, we performed double staining with cytokeratin immunostaining and PAS.Immunohistochemical staining was performed using a Ventana BenchMark XT system (Roche Diagnostics International AG) with anticytokeratin antibody (mouse monoclonal antibody, clone AE1/AE3; Dako Corp.) (dilution 1:1).Following cytokeratin staining, the slides were stained with PAS.

Proteomics analysis by LC/MS
Formalin-fixed paraffin-embedded tissue of perinephric tissue containing fibromyxoid NA was cut into 10-µmthick sections that were stained with PAS.Areas where PAS-positive eosinophilic deposits were present were microdissected using a laser microdissection device (Leica LMD6; Leica Microsystems).The background perinephric adipose tissue without deposits was also microdissected as a control.Microdissected tissue was collected into 0.5 mL microcentrifuge tubes containing 35 μL of a mixture of 10 mM Tris, 1 mM EDTA, and 0.002% Zwitterget.The protein was then eluted at F I G U R E 1 Gross picture of nephroureterectomy specimen and its cut surfaces.A polypoid urothelial carcinoma was present in the distal ureter (yellow arrow heads).The proximal ureter and renal pelvis were dilated (hydronephrosis and hydroureter).The areas of stromal deposition of eosinophilic material are shown in areas circled in red; they were located mainly in the adipose tissue of the renal hilum, and in perinephric and periureteral regions.In most of the deposition areas, scattered epithelium was present.The lesion represented fibromyxoid nephrogenic adenoma.
98°C for 90 min with occasional vortex mixing and sonicated for 60 min in a water bath.MS was performed as described previously. 13

Pathological findings
Macroscopically, a grayish white polypoid mass measuring 23 × 10 × 10 mm obstructing the right distal ureter was observed.The proximal ureter and right renal pelvis were dilated.Irregularly distributed brownish areas were found in the renal hilar adipose tissue and periureteral tissue.
Histologically, the ureteral tumor was classified as noninvasive low-grade urothelial carcinoma.Amorphous eosinophilic material deposits, stromal fibrosis, mild mononuclear cell infiltration, and hemorrhagic changes were observed in the background adipose tissue of the proximal periureteral and perinephric regions.Areas of eosinophilic deposition were mapped on the gross picture of the resected specimen (Figure 1).The deposited areas were located proximal to the ureteral tumor and were not found distally.The deposited areas were multifocal, and their contours were irregular (Figure 2a).The intensity of deposition varied between areas; some were dense and hyaline-like (Figure 2b,c), whereas others were pale and pinkish.The hyaline-like deposits were positive on PAS staining (Figure 2d), and negative on Congo red and phosphotungstic acid-hematoxylin staining.The pattern of elastic-Van Gieson staining was different from those of collagen and fibrin.Scattered nests and tubules of epithelial cells were identified in the background of the stromal fibroblasts (Figure 2e,f).Some nests consisted of compressed cells with short-spindled nuclei.The tubular structures were generally small and atrophic.The epithelial cells had bland nuclei and mitoses were not found.The distribution of epithelial cells in the background of eosinophilic deposits was widespread, extending from the renal hilus to the periureteral adipose tissue.The intensity of deposits surrounding the epithelium was evaluated on cytokeratin-PAS double-stained slides.Deposition was not always centered around the epithelium (Figure 2g,h).Immunohistochemically, the epithelial cells were positive for CK7, AMACR, and PAX8 and negative for CK20, GATA-3, PSA, and estrogen receptor -(Figure 3).Based on the histopathological, histochemical, and immunohistochemical findings, a diagnosis of perinephric fibromyxoid NA was made.

MS of hyaline-like deposits around fibromyxoid NA
To comprehensively explore deposits around fibromyxoid NA, we performed MS on the microdissected PAS-positive eosinophilic deposits found in the stroma of fibromyxoid NA (Figure 4a,b), in addition to the background adipose tissue (Figure 4c,d).We identified 426 and 490 proteins with medium to high confidence according to the false discovery rate in the eosinophilic deposits and background periureteral adipose tissue, respectively.After subtracting the background signal, 159 proteins were detected specifically in the deposited area (Figure 4e) and uromodulin (UMOD) was the most abundant protein in the stroma of fibromyxoid NA (Table 1).

Validation of MS with uromodullin and periostin immunohistochemistry
MS revealed the proteins contained in PAS-positive deposits, and immunohistochemistry was used to verify whether the top two proteins, UMOD and periostin (POSTN), were contained in the deposits.
The results showed that PAS-positive deposits (Figure 5a) were positive for UMOD and POSTN (Figure 5b,c).

DISCUSSION
UMOD, also known as Tamm−Horsfall protein, is a high molecular weight glycoprotein synthesized exclusively in the ascending loop of Henle by distal tubular cells in the normal kidney. 9UMOD is the most abundant protein in urine. 14Mutation of the UMOD gene causes autosomal dominant tubulointerstitial kidney disease (ADTKD-UMOD), which is histologically characterized by abnormal accumulation of UMOD in renal tubular cells that can be identified by PAS staining. 15In several pathogenic conditions, UMOD deposits are found in the renal parenchyma, perirenal soft tissue, renal hilar lymph node, or wall of the urinary bladder. 9Histologically, UMOD deposition typically shows a "waxy" eosinophilic appearance.When the deposition amount is large, diagnosis is relatively straightforward even by H&E staining.In this study, we analyzed the substance deposited in the background stroma in a rare case of fibromyxoid NA involving perinephric and periureteral tissue.PAS-positive eosinophilic material, the morphology of which raised suspicion of UMOD deposition, was present.However, UMOD deposition had not been documented previously in fibromyxoid NA.In a series of eight cases of fibromyxoid NA, Hansel et al. performed immunohistochemistry for UMOD in three cases, all of which were negative. 11In a series of 18 cases of urinary bladder UMOD deposition, Truong et al. reported the coexistence of NA in two cases. 9However, apart from the areas of UMOD deposition, it is unclear whether these were cases of fibromyxoid NA or NA.As there were a number of candidates for the deposited substances, we applied a proteomics approach (LC/MS) to comprehensively investigate the proteins comprising the deposits.The results confirmed UMOD deposition in fibromyxoid NA.The results of our MS analysis were validated by immunohistochemistry for UMOD and POSTN.A larger study of fibromyxoid NAs by combination of MS and immunohistochemistry is warranted to examine whether UMOD deposition is a common phenomenon or not.
Several factors have been implicated in the pathogenesis of NA, including genitourinary trauma, chronic inflammation, prior surgery, renal calculi, repeated instrumentation, irritation anatomical anomalies, and pelvic radiation. 4,6In early 2000's, Mazal et al. analyzed NA in a total of 24 kidney transplant patients and reported that NA were derived from tubular cells of the kidney graft rather than from metaplastic proliferation of the recipient's bladder urothelium. 7It is now becoming increasingly common to view NA as a heterotopic autograft resulting from renal tubular cells spilling into the urinary tract due to minor injury. 12ased on the perirenal and periureteral stromal deposition of UMOD, along with the presence of epithelium of distal renal tubular lineage (i.e., positive for PAX8), we concluded that the extravasation of urinary contents is the key factor underlying the development of perinephric and/or fibromyxoid NA.In our case, eosinophilic deposits and scattered epithelium were observed on the proximal side of the ureteral carcinoma, but not in the distal ureter.Hydroureter and hydronephrosis were present.As no maneuvers that could cause mechanical damage to the urinary tract had been performed prior to the surgery, we assumed that increased luminal pressure due to obstruction by ureteral cancer led to urinary tract failure and leakage of urine into the surrounding stroma.Another possible mechanism of perinephric fibromyxoid NA formation is local production of UMOD by preexisting distal tubular epithelium in perinephric adipose tissue.In our case, however, there were areas with deposition only, that is, without epithelium.Furthermore, the results of cytokeratin-PAS double staining demonstrated that deposition was not centralized around the epithelium.Therefore, we considered it unlikely that such processes (epithelium followed by UMOD deposition) had taken place.
POSTN was another protein detected exclusively in the deposition areas in this study.7][18] POSTN has also been reported to be present in neoplastic tissues, such as breast and colon cancers. 19,20Kikuchi et al. showed that POSTN is produced by cancer-associated fibroblasts in colon cancer. 21In our case, stromal fibroblasts of fibromyxoid NA may have produced POSTN in response to infiltrating epithelium.Interestingly, other abundant proteins in the list, such as ceruloplasmin, fibrinogen, and ITIH4, are produced in the liver and secreted into the serum.Their presence in the stroma of fibromyxoid NA may have been due to leakage of serum contents, that is, a result of hemorrhage.Further proteomics studies in larger NA case series are necessary to elucidate the complex architecture of the stroma and evaluate the frequency and distribution of UMOD deposition.
Perinephric fibromyxoid NA is a diagnostic challenge for surgical pathologists, especially if they are unaware of the lesion.Invasive adenocarcinoma is one of the differential diagnoses because the epithelium is scattered in the perinephric adipose tissue and often shows an infiltrative pattern.However, in fibromyxoid NA, the nuclei of the epithelium are completely bland, and mitoses are hardly seen.The absence of desmoplastic stromal reaction may be another finding excluding invasive cancer.A variety of benign lesions, such as remnant of Müllerian ducts, 22 mesonephric duct derivatives, 23 duplicate ureters, 23 and ectopic prostatic glands, 24 are also considered as differential diagnoses.Furthermore, it has also been reported that there is a subgroup of urothelial carcinomas with a myxoid stroma, 25,26 and it is crucial to distinguish them from fibromyxoid NA.][29][30][31] In conclusion, we reported a rare case of perinephric fibromyxoid NA.Proteomics analysis suggested the involvement of urinary leakage.3][34][35] This study indicated the potential of MS for clarifying the pathogenesis of the disease.It is most effective when

F I G U R E 2
Microscopic findings of perinephric fibromyxoid nephrogenic adenoma (NA).(a) Low-power view of perinephric adipose tissue with irregularly distributed areas of eosinophilic deposition and stromal fibrosis (arrow heads).Focal hemorrhagic changes were seen.(b) Area with eosinophilic deposition.Intervening fibrous tissue and vascular structures were present.Mild mononuclear cell infiltration was observed in the surrounding stroma.(c) High-power view of amorphous and hyaline-like eosinophilic deposition.(d) Strong PAS positivity of the deposited material.(e) Epithelial component interspersed in the background of eosinophilic deposition.The epithelial cells were bland, with round to ovoid nuclei, and formed small nests and glands.(f) Glandular structure of fibromyxoid NA.A monolayer of epithelial cells lined the lumen.(g) Cytokeratin-PAS double staining of NA.Cytokeratin-positive epithelium was identified in the background of PAS-positive stroma.The intensity of PAS positivity varied between areas and deposition was not centered around the epithelium.(h) Cytokeratin-PAS double staining of NA.Epithelium was also present in the area without eosinophilic deposition.PAS, periodic acid-Schiff.

F I G U R E 4
Proteomic comparison of eosinophilic deposits and background adipose tissue.The area of perinephric eosinophilic deposits without epithelium (a) prior to microdissection and (b) after microdissection.The area of background adipose tissue without deposition (c) prior to microdissection and (d) after microdissection.(e) Venn diagram showing the number of proteins identified in eosinophilic deposits with background adipose tissue.A total of 426 significant proteins were identified in the eosinophilic deposits and 490 were identified in the background.We detected 195 proteins exclusively in the deposited material, which included uromodulin.
T A B L E 1 Top 20 proteins identified exclusively in the eosinophilic deposits of fibromyxoid nephrogenic adenoma.