Figure 1.
Functional protection against AKI by TUDCA.
Daily administration of 400 mg/kg of TUDCA protected against ischemic AKI. Renal function was determined by daily measurement of blood urea. Rats that received TUDCA (squares) had significantly less elevation in blood urea levels on day 1 and day 2 following ischemia-reperfusion injury as compared to rats that received vehicle (circles) (p<0.01). Similarly, on days 3–5, blood urea levels continued to remain lower in the TUDCA-treated rats; however, the difference was not statistically significant. Results are expressed as mean ± standard deviation of a least 3 different animals in each group. *p<0.001 and §p<0.01 from day 0.
Figure 2.
Protection against AKI-induced histological damage by TUDCA.
A, Representative PAS stained images from deep cortex from animals that received vehicle control (a) or TUDCA (b). B, Animals that received TUDCA as compared to controls, showed significantly less damage in the deep cortex where the S3 segment is located. Results are expressed as mean ± standard deviation of a least 3 different animals in each group. *p<0.001 and †p<0.05 from vehicle-injected controls.
Figure 3.
Protection against AKI-induced apoptosis by TUDCA.
A, Representative images from cortico-medullary junction from vehicle (a; control) and TUDCA-treated (b) groups. Brown staining and arrows identify TUNEL-positive cells. B, There were significantly less TUNEL-positive cells in the TUDCA-treated group as compared to the vehicle-treated (control) group in the cortex (p<0.05) and outer strip of the outer medulla (p<0.001). Results are expressed as mean ± standard deviation of a least 3 different animals in each group. *p<0.001 and †p<0.05 from vehicle-injected controls.
Figure 4.
TUDCA treatment significantly blocked activation of caspase-9 following AKI as compared to vehicle treatment (rats 1, 2, and 3) (top panel). There was no difference in the activation of caspase-8 and caspase-12 between the TUDCA- and vehicle-treated rats. Densitometry analysis of cleaved caspase-9 normalized for β-actin (lower panel). When densitometry results for caspase-9 were compared between the TUDCA- and vehicle-treated groups, there was significantly less (p<0.01) activation of caspase-9 in the TUDCA group. Results are expressed as mean ± standard deviation of a least 3 different animals in each group. §p<0.01 from vehicle-injected controls.
Figure 5.
TUDCA treatment upregulated ERK1/2 following ischemia-reperfusion injury to the kidney in rats (top panel); however, there was no statistical significant difefrence as compared to the vehicle-treated rats (top panel). There was no difference in JNK and p38 proteins between the TUDCA and vehicle groups. Densitometry analysis of ERK1/2 in each rat in the TUDCA group were compared with those in the vehicle group (lower panel); the difference was not significant (p = 0.29). Results are expressed as mean ± standard deviation of a least 3 different animals in each group.
Figure 6.
Cytotoxicity studies of TUDCA in primary human RPTE cells.
Cells were treated with either vehicle (control) or 15 to 1200 µM of TUDCA for 24 hours. A, TUDCA was not cytotoxic in concentrations from 15 to 600 µM. Significant cytotoxicity was seen only with 1200 µM of TUDCA, as compared to the vehicle (p<0.05). B, TUDCA did not decrease cell viability in all the tested concentrations from 15 µM to 1200 µM. Results are expressed as mean ± standard deviation. All experiments were performed in triplicate. †p<0.05 from vehicle-treated control.
Figure 7.
Caspase activation following cryoinjury and treatment with TUDCA in primary human RPTE cells.
A, Caspase-3 activity following cryoinjury in RPTE cells treated with either vehicle or different concentrations of TUDCA. Caspase-3 activity in cryoinjured RPTE cells was compared with that in uninjured RPTE cells. There was signficant activation of caspase-3 following cryoinjury (p<0.05), which was significantly inhibited by 150–600 µM of TUDCA in a dose-dependent fashion. 15 µM of TUDCA did not inhibit activation of caspase-3 following cryoinjury. B, Caspase-9 activity following cryoinjury in RPTE cells treated with either vehicle or different concentrations of TUDCA. There was statistically significant increased caspase-9 activity in cryoinjured cells as compared to uninjured cells (p<0.05). Both 100 and 150 µM of TUDCA significantly decreased caspase-9 activity. C, Caspase-8 and caspase-12 analysis following cryoinjury in RPTE cells treated with either vehicle or different concentrations of TUDCA. There was no difference in the amount of caspase-8 and procaspase-12 between the uninjured and cryoinjured cells treated with vehicle or TUDCA. Results are expressed as mean ± standard deviation. All experiments were performed in triplicate. †p<0.05 from vehicle-treated control. Cryo, cryoinjury.
Figure 8.
Survival pathway analysis following cryoinjury and treatment with TUDCA in primary human RPTE cells.
Phosphorylated ERK1/2 protein in cryoinjured cells that were treated with either vehicle (control) or 100 µM or 150 µM TUDCA as compared to uninjured cells (top panel). There was no difference in the amount of phosphorylated ERK1/2 between uninjured and cryoinjured cells treated with vehicle. Densitometry analysis of phosphorylated ERK1/2 (lower panel). Results are expressed as mean ± standard deviation. All experiments were performed in triplicate. §p<0.01 and †p<0.05 from vehicle-treated control. Cryo, cryoinjury.