1. Volume 93, Issue 1, Pages 147-158 (January 2018)
The activin receptor is stimulated in the skeleton, vasculature, heart, and kidney during chronic kidney disease 
Matthew J. Williams, Toshifumi Sugatani, Olga A. Agapova, Yifu Fang, Joseph P. Gaut, Marie-Claude Faugere, Hartmut H. Malluche, Keith A. Hruska 
Kidney International 
Volume 93, Issue 1, Pages (January 2018)
DOI: /j.kint
Copyright © 2017 International Society of Nephrology Terms and Conditions

2. Figure 1
Bone histomorphometry in the groups of mice: wild-type (white bars), vehicle-treated Alport (black bars), and RAP-011–treated Alport (gray bars). (a) Eroded perimeter/bone perimeter (E.Pm/B.Pm). (b) Osteoclast number (N.Oc/BL [100 mm]). (c) Osteoclast perimeter/B.Pm (Oc.Pm/B.Pm). (d) Osteoblast number (N.Ob/BL [110 mm]). (e) Osteoblast perimeter/B.Pm (Ob.Pm/B.Pm). (f) Mineral apposition rate (MAR). (g) Bone formation rate/B.Pm (BFR/B.Pm). (h) BFR/osteoblast (BFR/Ob). (i) Mineralization lag time (MLT). (j) Osteoid area/bone area (O.Ar/B.Ar). (k) Osteoid perimeter/B.Pm (O.Pm/B.Pm). (l), Osteoid width (OW). (m) B.Ar. (n) Double label perimeter (dL.Pm). Alport mice had increased bone resorption and osteoclast stimulation, which were prevented by RAP-011 treatment. Alport mice had increased N.Ob and osteoblast surface but MAR and BFR/B.Pm were not increased. RAP-011 did not further increase N.Ob but increased MAR and BFR/B.Pm. Alport mice had increased O.Pm, which was corrected by RAP-011 treatment. ND, not different; n = 13 for wild type (WT), n = 8 for Alport, and n = 12 for RAP-011; see Materials and Methods for histomorphometry techniques. Data are represented as mean ± SEM.
Kidney International  , DOI: ( /j.kint )
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3. Figure 2
Vascular calcification in Alport mice and aortic activin receptor type IIA (ActRIIA) signaling. (a) Deposits of calcium phosphate (black patches) were detected by microscopic computed tomography in the aortic adventitia and media adjacent to the adventitia of Alport mice. (b) Deposits of calcium phosphate were detected by von Kossa staining of the aortic adventitia and media adjacent to the adventitia of Alport mice (Bar = 20 μm). (c) Aortic calcium levels in the groups of mice. Alport mice had significantly elevated aortic calcium (Ca) levels, which were reduced by RAP-011 treatment. (d) Analysis of ActRIIA signaling in aortic homogenates. Westerns blots of aortic homogenates from 3 wild-type (WT), 3 vehicle-treated Alport, and 3 RAP-011–treated Alport mice. (e) Quantification of the Western blots in (d) Activin A and phospho-Smad2 (p-Smad2) levels were increased in homogenates of vehicle-treated Alport mice, and they were reduced in homogenates of RAP-011–treated Alport mice. sm22α (transgelin) and smooth muscle actin (SMA) were increased in the homogenates of the vehicle-treated Alport mice aortas. SMA was decreased in the homogenates of the RAP-011–treated Alport mice aortas compared to vehicle-treated Alport mice aortas. Runx2 and osterix (Osx) were induced in the homogenates of vehicle-treated Alport mice aortas and were reduced by RAP-011 treatment. n = 6 for each group in (e). ALP, Alkaline phosphatase; IB, immunoblot. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

4. Figure 3
Cardiac weights in the groups of mice. Cardiac hypertrophy developed in the vehicle-treated Alport mice, and this was prevented in RAP-011–treated Alport mice. WT, wild type.
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

5. Figure 4
Western blot analysis of renal klotho. (a) Klotho levels in renal homogenates from 6 wild-type mice, 5 vehicle-treated Alport mice, and 6 RAP-011-treated Alport mice. (b) Quantification of the klotho levels in (a). Data are reported as mean ± SEM. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IB, immunoblot; WT, wild type. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

6. Figure 5
Kidney function in Alport mice. (a) Inulin clearances in 150-day-old groups of mice. The 150-day-old vehicle-treated Alport mice had a significantly reduced glomerular filtration rate (GFR) compared to wild-type (WT) littermates. RAP-011–treated mice had better preserved GFR than vehicle-treated mice. (b) Serum blood urea nitrogen (BUN) in 200-day-old mice of the 3 groups. BUN of 200-day-old Alport mice was consistent with severe chronic kidney disease (CKD) and GFR of ≤10% to 15% of that in WT littermates. RAP-011 treatment decreased BUN at 200 days of life. (c) Urine albumin-to-creatinine ratios in the groups of mice. Consistent with the delayed progression of RAP-011–treated Alport mice compared to vehicle-treated Alport mice, the urinary albumin-to-creatinine ratio was not different between the 2 groups of mice. The “n” for the groups in (a–c) were as follows: WT mice = 6, vehicle-treated Alport mice = 7, and RAP-011–treated Alport mice = 7; P < 0.05.
Kidney International  , DOI: ( /j.kint )
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7. Figure 6
Photomicrographs of trichrome-stained cortical sections (Bar = 50 μm). Sections were obtained from (a) a 200-day-old wild-type littermate mouse; (b) a 200-day-old vehicle-treated Alport mouse showing severe interstitial fibrosis and glomerulosclerosis; and (c) a 200-day-old RAP-011–treated mouse showing less interstitial fibrosis compared to the vehicle-treated mouse and retention of more normal glomerular morphology. (d) Quantification of interstitial inflammation and fibrosis: the results are expressed as mean ± SD; n = 6 kidneys per group. Five fields from each kidney were measured to obtain the interstitial volume of that kidney. The blue line passing through the bars is the upper limit of the normal interstitial volume. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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8. Figure 7
Analysis of activin receptor type IIA (ActRIIA) signaling in renal homogenates. (a) Western blots of kidney homogenates of 6 wild-type (WT), 5 vehicle-treated Alport, and 6 RAP-011–treated Alport mice. Phospho-Smad2 (p-Smad2) and Smad2/3 levels were increased in homogenates of vehicle-treated Alport mice, and they were reduced in homogenates of RAP-011–treated Alport mice. ActRIIA was induced in Alport mice, and the target fibronectin (FN) and monocyte chemoattractant protein-1 (MCP-1) were also induced in Alport mice. RAP-011 treatment lowered FN and MCP-1 levels. (b) Quantification of Western blots in (a). Data are reported as mean ± SEM. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IB, immunoblot; SMA, smooth muscle actin. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions