Wu, Xue‐Ru

Expression of Tamm-Horsfall protein (THP or uromodulin) is highly restricted to the kidney thick ascending limb (TAL) of loop of Henle. Despite the unique location and recent association of THP gene mutations with hereditary uromodulin-associated kidney disease and THP single nucleotide polymorphisms with chronic kidney disease and hypertension, the physiological function(s) of THP and its pathological involvement remain incompletely understood. By studying age-dependent changes of THP knockout (KO) mice, we show here that young KO mice had significant salt and water wasting but were partially responsive to furosemide, due to decreased luminal translocation of Na-K-Cl cotransporter 2 (NKCC2) in the TAL. Aged THP KO mice were, however, markedly oliguric and unresponsive to furosemide, and their NKCC2 was localized primarily in the cytoplasm as evidenced by lipid raft floatation assay, cell fractionation, and confocal and immunoelectron microscopy. These aged KO mice responded to metolazone and acetazolamide, known to target distal and proximal tubules, respectively. They also had marked upregulation of renin in juxtaglomerular apparatus and serum, and they were hypertensive. Finally, the aged THP KO mice had significant upregulation of Na-coupled urate transporters Slc5a8 and Slc22a12 as well as sodium-hydrogen exchanger 3 (NHE3) in the proximal tubule and elevated serum uric acid and allantoin. Collectively, our results suggest that THP deficiency can cause progressive disturbances in renal functions via initially NKCC2 dysfunction and later compensatory responses, resulting in prolonged activation of the renin-angiotensin-aldosterone axis and hyperuricemia.

Abstract Background The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively; yet, much less is known about full-length ERβ (ERβ1) and even less about its 4 variant forms (ERβ2-5). We have recently implicated a role for ERβ1 in sensitizing ERα expressing breast cancer cells to anti-estrogens. However, the ability of ERβ2-5 to modulate ERα and ERβ1 activity, and their association with cancer development, progression, and response to estradiol (E2) and anti-estrogens are not well understood. Here, we provide evidence that the presence of ERβ variants may be of diagnostic and clinical relevance for breast cancer patients and describe the development and characterization of a novel, highly specific monoclonal antibody (MC10) that is able to detect their expression in tumor biopsies. Methods: Transient transfection and luciferase assays were used to determine the transcriptional activity of ERβ2-5 in response to E2 and anti-estrogens alone or in combination with ERα and ERβ1. A novel monoclonal antibody targeting all ERβ variants (MC10) was developed and characterized. The sub-cellular localization of ERβ2-5 was determined via confocal microscopy. Finally, the MC10 antibody was used to assess ERβ positivity in breast tumors and was compared to that of another monoclonal antibody which only detects ERβ1. Results: Unlike ERβ1, ERβ2-5 do not activate an estrogen response element (ERE) in response to E2 and instead, slightly repress the activity of this reporter construct. Expression of ERβ2-5 does not significantly alter the transcriptional activity of ERβ1 following E2 treatment. However, ERβ2, 3 and 5, but not ERβ4, significantly enhance the E2-induced transcriptional activity of ERα. Interestingly, expression of ERβ3, 4 and 5, but not ERβ2, enhance the ability of anti-estrogens to block ERα mediated transcriptional activity. Confocal microscopy revealed that ERβ1 and 2 are almost exclusively localized to the cell nucleus. However, ERβ3-5 exhibit significant cytoplasmic and peri-nuclear localization. Immunohistochemistry of breast cancer biopsies using the MC10 antibody revealed multiple staining patterns including tumors which exhibit primarily nuclear staining and others primarily cytoplasmic, both in the presence and absence of ERα. These results are in contrast to the almost exclusive nuclear staining obtained on the same tumors with an ERβ1-specific antibody. Conclusions: ERβ variants exhibit variable sub-cellular localization patterns and can influence the function of ERα, both in response to E2 and anti-estrogens. Therefore, the differential expression of ERβ variants and their cellular localization may influence breast cancer progression and/or therapeutic responses. The use of ERβ antibodies which do not detect all ERβ variants, or the use of a single ERβ antibody which does not discriminate between ERβ1 and its variants, is unlikely to reveal the complete biological significance of total ERβ expression in breast cancer and may in part explain the conflicting studies which have been reported for ERβ in the field. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-02-03.

Mammalian urine contains a range of macromolecule proteins that play critical roles in renal stone formation, among which Tamm-Horsfall protein (THP) is by far the most abundant. While THP is a potent inhibitor of crystal aggregation in vitro and its ablation in vivo predisposes one of the two existing mouse models to spontaneous intrarenal calcium crystallization, key controversies remain regarding the role of THP in nephrolithiasis. By carrying out a long-range follow-up of more than 250 THP-null mice and their wild-type controls, we demonstrate here that renal calcification is a highly consistent phenotype of the THP-null mice that is age and partially gene dosage dependent, but is gender and genetic background independent. Renal calcification in THP-null mice is progressive, and by 15 mo over 85% of all the THP-null mice develop spontaneous intrarenal crystals. The crystals consist primarily of calcium phosphate in the form of hydroxyapatite, are located more frequently in the interstitial space of the renal papillae than intratubularly, particularly in older animals, and lack accompanying inflammatory cell infiltration. The interstitial deposits of hydroxyapatite observed in THP-null mice bear strong resemblances to the renal crystals found in human kidneys bearing idiopathic calcium oxalate stones. Compared with 24-h urine from the wild-type mice, that of THP-null mice is supersaturated with brushite (calcium phosphate), a stone precursor, and has reduced urinary excretion of citrate, a stone inhibitor. While less frequent than renal calcinosis, renal pelvic and ureteral stones and hydronephrosis occur in the aged THP-null mice. These results provide direct in vivo evidence indicating that normal THP plays an important role in defending the urinary system against calcification and suggest that reduced expression and/or decreased function of THP could contribute to nephrolithiasis.