1. Parathyroid hormone–related protein protects renal tubuloepithelial cells from apoptosis by activating transcription factor Runx2 
Juan A Ardura, Ana B Sanz, Alberto Ortiz, Pedro Esbrit 
Kidney International 
Volume 83, Issue 5, Pages (May 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
Parathyroid hormone-related protein (PTHrP)(1–36) and PTH(1–34) increase total Runx2 expression in cultured proximal tubular mouse cortical tubule (MCT) and human kidney-2 (HK-2) cells in a dose-dependent manner. (a) Representative autoradiogram that corresponds to western blot analysis of Runx2 expression in osteoblast-like MG-63 cells (used as Runx2-positive control) and MCT and HK2 renal tubuloepithelial cells. Bands of 55kDa that correspond to the predicted size of Runx2 are shown. (b) Runx2 type I mRNA levels were analyzed by real-time PCR after 6h of 100nmol/l PTHrP(1–36) or PTH(1–34) stimulation in MCT and HK-2. (c–f) Densitometric values of Runx2 protein levels stimulated for 24h with different concentrations of PTHrP(1–36) in MCT (c) and HK-2 (d) cells or stimulated with different concentrations of PTH(1–34) in MCT (e) and HK-2 (f) cells, respectively. Representative autoradiograms are shown. Mean±s.e.m. of three independent experiments. *P<0.05 vs. control (0nmol/l).
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

3. Figure 2
Parathyroid hormone-related protein (PTHrP)(1–36) promotes the nuclear localization of Runx2 in murine tubuloepithelial mouse cortical tubule (MCT) cells. (a) Western blot analysis of nuclear extracts of MCT cells stimulated with 10nmol/l PTHrP(1–36) for different times. Densitometric values correspond to the mean±s.e.m. of three independent experiments. *P<0.05 vs. control (0h). (b) Nuclear Runx2 localization analyzed by confocal microscopy in MCT cells treated with 100nmol/l PTHrP(1–36) for various times. Runx2 staining is shown in green and propidium iodide (PI, nuclei marker) in orange (magnification × 320). Images are representative of three independent experiments.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

4. Figure 3
Parathyroid hormone-related protein (PTHrP)(1–36)- and PTH(1–34)-dependent protection from folic acid– or serum deprivation–induced cell death in mouse cortical tubule (MCT) cells or in HK-2 cells is mediated by Runx2. MCT and human kidney-2 (HK-2) cells were transfected with siRunX2 (a–d) or dnRunx2 (e, f) for 24h, and subsequently exposed to 100nmol/l PTHrP(1–36) or PTH(1–34) for 2h and 10mmol/l folic acid (FA) for 24h (a–e) or serum deprivation for 48h (f). Apoptosis was assessed by flow cytometry of DNA content (hypodiploid cells) in MCT (a) or by measurement of Annexin V/7-amino-actinomycin (7-AAD) (b–d) and cell viability by trypan blue staining (e, f) in MCT and HK-2 cells, as described in Materials and Methods. (d) Representative dot plot of Annexin V/7-AAD staining in HK-2 cells. Results are the mean±s.e.m. of three independent experiments. *P<0.05 vs. control; aP<0.05 vs. death stimulus alone or in the presence of siRunX2 or dnRunx2. FBS, fetal bovine serum.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

5. Figure 4
Runx2 mediates parathyroid hormone-related protein (PTHrP)(1–36) stimulation of Bcl-2 protein expression in murine tubuloepithelial mouse cortical tubule cells. Cells were transfected during 24h with dnRunx2, followed by exposure to 100nmol/l PTHrP(1–36) for 24h. Western blot analysis and representative images of Bcl-2 (a), Bax (b), and Bcl-xL (c) changes are shown. Bcl-2/Bax (d) and Bcl-xL/Bax (e) protein ratios are also shown. Results correspond to the mean±s.e.m. of three independent experiments. *P<0.05 vs. basal; **P<0.01 vs. PTHrP(1–36).
Kidney International  , DOI: ( /ki )
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6. Figure 5
Runx2 mediates parathyroid hormone-related protein (PTHrP)(1–36) and PTH(1–34) stimulation of Bcl-2 protein expression in human tubuloepithelial human kidney-2 cells. Cells were transfected during 24h with dnRunx2 or siRunx2, followed by exposure to 100nmol/l PTHrP(1–36) or PTH(1–34) for 24h. Western blot analysis and representative images of Bcl-2 (a) and Bax (b) changes are shown. Bcl-2/Bax protein ratios (c) are also shown. Results correspond to the mean±s.e.m. of three independent experiments. *P<0.05 vs. basal.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

7. Figure 6
Runx2 mediates parathyroid hormone-related protein (PTHrP)(1–36) oversecretion of osteopontin. Mouse cortical tubule (MCT) cells were transfected or not during 24h with siRunx2, followed by 100nmol/l PTHrP(1–36) stimulation. Supernatants from MCT cells were lyophilized and osteopontin secretion was tested by western blot for 1–24h (a) or at 24h (b). Equal loading was confirmed by Ponceau S staining (not shown). Results represent the mean±s.e.m. of three independent experiments. *P<0.05 vs. basal; **P<0.01 vs. PTHrP(1–36).
Kidney International  , DOI: ( /ki )
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8. Figure 7
Increased renal Runx2, ostepontin, and Bcl-2 expressions in parathyroid hormone-related protein (PTHrP)-TG mice. Kidneys from wild-type (WT) and PTHrP-TG mice were studied. (a) Runx2 mRNA levels were analyzed by real-time PCR. (b) Representative renal Runx2 immunostaining images from six animals per group are shown. Negative control images represent incubations without primary antibody. Numbers on top of images represent magnification values. (c) Osteopontin mRNA levels were analyzed by real-time PCR. (d) Representative renal osteopontin immunostaining images from six animals per group are shown. (e) Bcl-2 protein expression was analyzed by western blot. Representative autoradiograms correspond to western blot analysis. *P<0.05 vs. WT.
Kidney International  , DOI: ( /ki )
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9. Figure 8
Increased renal Runx2 mRNA expression in folic acid–treated and unilateral ureteral obstructed (UUO) kidneys from wild-type mice. (a) Runx2 mRNA expression from mouse kidney samples after 72h of vehicle (V) or folic acid (FA) injection is shown. (b) Runx2 mRNA expression in mouse kidney samples from sham-operated (Sham) and 4-day UUO wild-type mice were studied. *P<0.05 vs. V or Sham.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions