We previously showed that supplementation with 17beta-estradiol (E2) from the onset of diabetes attenuates the development of diabetic renal disease. The aim of the present study was to examine whether E2 can also attenuate the disease process once it has developed. The present study was performed in nondiabetic and streptozotocin-induced diabetic Sprague-Dawley rats. E2 supplementation began after 9 wk of diabetes and continued for 8 wk. Diabetes was associated with an increase in urine albumin excretion, glomerulosclerosis, tubulointerstitial fibrosis, renal cortical collagen type I and IV, laminin, plasminogen activator inhibitor-1, tissue inhibitors of metalloproteinase-1 and -2, transforming growth factor (TGF)-beta, TGF-beta receptor type I and II, Smad2/3, phosphorylated Smad2/3, and Smad4 protein expression, and CD68-positive cell abundance. Decreases in matrix metalloproteinase (MMP)-2 protein expression and activity and decreases in Smad6 and Smad7 protein expression were also associated with diabetes. E2 supplementation completely or partially attenuated all these changes, except Smad4 and fibronectin, on which E2 supplementation had no effect. These data suggest that E2 attenuates the progression of diabetic renal disease once it has developed by regulating extracellular matrix, TGF-beta, and expression of its downstream regulatory proteins. These findings support the notion that sex hormones in general, and E2 in particular, are important regulators of renal function and may be novel targets for the treatment and prevention of diabetic renal disease.

We investigated the role of Lef1, one of the four transcription factors that transmit Wnt signaling to the genome, in the regulation of bone mass. Microcomputed tomographic analysis of 13- and 17-week-old mice revealed significantly reduced trabecular bone mass in Lef1+/− females compared to littermate wild-type females. This was attributable to decreased osteoblast activity and bone formation as indicated by histomorphometric analysis of bone remodeling. In contrast to females, bone mass was unaffected by Lef1 haploinsufficiency in males. Similarly, females were substantially more responsive than males to haploinsufficiency in Gsk3β, a negative regulator of the Wnt pathway, displaying in this case a high bone mass phenotype. Lef1 haploinsufficiency also led to low bone mass in males lacking functional androgen receptor (AR) (tfm mutants). The protective skeletal effect of AR against Wnt-related low bone mass is not necessarily a result of direct interaction between the AR and Wnt signaling pathways, because Lef1+/− female mice had normal bone mass at the age of 34 weeks. Thus, our results indicate an age- and gender-dependent role for Lef1 in regulating bone formation and bone mass in vivo. The resistance to Lef1 haploinsufficiency in males with active AR and in old females could be due to the reduced bone turnover in these mice.

Krox20/EGR2, one of the 4 early growth response genes, is a highly conserved transcription factor implicated in hindbrain development, peripheral nerve myelination, tumor suppression, and monocyte/macrophage cell fate determination. Here, we established a novel role for Krox20 in postnatal skeletal metabolism. Microcomputed tomographic analysis of 4-and 8-week-old mice revealed a low bone mass phenotype (LBM) in both the distal femur and the vertebra of Krox20 ؉/؊ mice. This was attributable to accelerated bone resorption as demonstrated in vivo by increased osteoclast number and serum C-terminal telopeptides, a marker for collagen degradation. Krox20 haploinsufficiency did not reduce bone formation in vivo, nor did it compromise osteoblast differentiation in vitro. In contrast, growth and differentiation were significantly stimulated in preosteoclast cultures derived from Krox20 ؉/؊ splenocytes, suggesting that the LBM is attributable to Krox20 haploinsufficiency in the monocytic lineage. Furthermore, Krox20 silencing in preosteoclasts increased cFms expression and response to macrophage colony-stimulating factor, leading to a cell-autonomous stimulation of cell-cycle progression. Our data indicate that the antimitogenic role of Krox20 in preosteoclasts is the predominant mechanism underlying the LBM phenotype of Krox20-deficient mice. Stimulation of Krox20 expression in preosteoclasts may present a viable therapeutic strategy for high-turnover osteoporosis. (Blood. 2010; 116(19):3964-3971) IntroductionThe family of early growth response (EGR) genes consists of 4 members, EGR1/Krox24, EGR2/Krox20, EGR3, and EGR4. 1 Their expression is rapidly induced by serum and growth factors, such as nerve growth factor, epidermal growth factor, and plateletderived growth factor. [2][3][4] The 4 EGR proteins are transcription factors that share a highly conserved DNA-binding domain composed of 3 zinc fingers, which recognize G:C-rich DNA motifs 1,[5][6][7] in the promoters of target genes such as Hox-1.4, 8 Hoxa-2 and Hoxb-2, 9 thymidine kinase, 8,10 and synapsin I and II. 11,12 The transactivation activity of EGR factors is modulated by coactivators such as host cell factor C1 13 and corepressors such as NAB1, 14 NAB2, 15 and Ddx20. 16 Early studies highlighted the role of EGR genes in cell proliferation. 4 In cancer cells of various origins, Krox20 plays a role downstream of the PTEN tumor suppressor, and its deletion is associated with cancer cell proliferation. 17,18 Krox20 knockout mice have severe abnormalities in hindbrain development [19][20][21] and impaired peripheral nerve myelination 22 attributable to the role of Krox20 in regulating Schwann cells growth and differentiation. [22][23][24] Krox20 has also been implicated in fate determination in the myeloid lineage. Specifically, it inhibits neutrophil-and activates macrophage-specific genes such as c-fms, encoding the macrophage colony-stimulating factor (M-CSF) receptor. 25,26 Krox20 interacts with PU.1, a master transcription factor critical in macrophage dif...

Background/Aims: Our previous studies have shown that supplementation with 17β-estradiol (E₂) from the onset of diabetes attenuates diabetic nephropathy. However, E₂ is accompanied by feminizing effects as well as adverse side effects on other organs. The current study examined the renoprotective effects of a selective estrogen receptor modulator, raloxifene (RAL), in an experimental model of diabetic nephropathy. RAL activates estrogen receptors and estrogen-receptor-mediated cellular events without the side effects of E₂. Methods: The study was performed in Sprague-Dawley nondiabetic (ND), streptozotocin-induced diabetic (D) and streptozotocin-induced D + RAL rats (n = 6/group). Results: After 12 weeks of treatment, D was associated with increased urine albumin excretion (ND: 4.2 ± 0.4; D: 41.3 ± 9.0 mg/day), glomerulosclerosis [glomerulosclerotic index; ND: 0.26 ± 0.04; D: 1.86 ± 0.80 arbitrary units (AU)], tubulointerstitial fibrosis (tubulointerstitial fibrosis index; ND: 0.37 ± 0.05; D: 2.12 ± 0.50 AU), increased collagen type I [ND: 1.31 ± 0.07; D: 4.65 ± 0.09 relative optical density (ROD)], collagen type IV (ND: 0.64 ± 0.03; D: 1.37 ± 0.11 ROD) and transforming growth factor beta (TGF-β) protein expression (ND: 0.65 ± 0.08; D: 1.25 ± 0.10 ROD), increased density of CD68-positive cells (ND: 1.37 ± 3.02; D: 29.2 ± 1.74 cells/mm²) and increased plasma levels of interleukin-6 (ND: 14.8 ± 5.0; D: 51.3 ± 14.0 pg/ml). Treatment with RAL partially or fully attenuated these processes (urine albumin excretion: 21.0 ± 5.0 mg/day; glomerulosclerotic index: 0.40 ± 0.06 AU; tubulointerstitial fibrosis index: 0.20 ± 0.04 AU; collagen type I: 2.55 ± 0.49 ROD; collagen type IV: 0.70 ± 0.09 ROD; TGF-β: 0.91 ± 0.08 ROD; CD68: 6.03 ± 2.38 cells/mm²; interleukin-6: 31.2 ± 5.0 pg/ml). Conclusions: Our data indicate that treatment with RAL attenuates albuminuria and renal structural changes associated with diabetes.

Level III, retrospective comparative series.