Fig 1.
Design of experiments to determine the effects of the 5/6 Nx animal model on cognitive function, NF-kB activity, cytokines levels and KLOTHO levels.
For the behavioral experiments, the open field and Inhibitory avoidance behavior or novel object recognition tests were performed thirty days after renal ablation and repeated once a month for more than three months. For the biochemical experiments, 24 hours after the end of the behavioral studies, animals were killed by decapitation with CSF, hippocampal and frontal cortex samples then collected.
Table 1.
Clinical Parameters of CKD in sham and Nx groups after 60 and/or 120 days of the surgical procedure.
Fig 2.
Histological kidney analysis in sham and Nx groups at 121 days after the surgical procedure.
(A,B). The photomicrographs are representative samples of glomerular sclerosis observed in sham and Nx kidneys that were stained with periodic acid-Schiff (SPADES). The results showed a significant increase in the number of glomeruli with sclerosis (yellow arrow) in the Nx group (B) (n = 8) when compared to the sham group (A) (n = 5). (C,D) The photomicrographs are representative samples of cortical interstitial area expansion observed in Nx (D) (yellow arrow) when compared to sham (C) that were stained with SPADES. The analysis showed a significant increase in cortical interstitial area in the Nx group (n = 8) when compared to the sham group (n = 5). (E) The graphical representation of the percent of glomerular sclerosis (A,B) and (F) of cortical interstitial area (C,D), respectively. *p<0.05 versus sham—Student's t test.
Fig 3.
Inhibitory avoidance test analysis in sham and Nx.
(A) The first exposure of animals to the inhibitory avoidance equipment and the test performed 24h after the training session. Sham and Nx groups entered the dark space in short time, showing no difference concerning the preference for this ambient but a significant difference between both groups behavior was observed in the test performed 24h after the training session. (B) All the animals were tested at 30, 60, 90 and 120 days following the 24h-test day. During the observational time, 24h-test was considered the 0 (zero) time. The latency time to cross from the light to the dark side of the chamber was measured and used as a reference to distinguish if the rats learned from a fear conditional stimulus. Animals from Sham group maintained the memory of the task and this was significantly higher than the memory of the Nx group [F(1,129) = 5.2, P < 0.05]. Nx group showed worse performance during all observational time (B). *: P < 0.05; a: P < 0.001. The group was divided into Nx-M group (latencies above the median level) and Nx-Cl group (latencies under the median level) (C).
Fig 4.
Novel Object Recognition Test analysis in sham and Nx.
All the animals were tested at 30, 60, 90 and 120 days following the training day. Kruskal-Wallis one-way ANOVA (22.67, p<0.001) with Müller-Dunn post-tests: Nx-90, Nx-120 vs Nx-30, p<0.005. Nx group was divided into two groups: those animals that remember the task (Nx-M group, n = 5) and those animals that did not remember the task (Nx-Cl, n = 7) (red boxes). Student's t test, p <0.05. Both tests showed that Nx induced memory impairment.
Fig 5.
Locomotor activity in sham and Nx.
Total distance travelled during 300s of Sham (n = 20) and Nx (n = 28) groups at 30 and 120 days—Kruskal-Wallis one-way ANOVA with Müller-Dunn post-test, F(1,3) = 0.17, NS.
Fig 6.
Changes in NF-κB activity in rat hippocampus and frontex cortex at 121 days in sham and Nx (Nx-M and Nx-CI) groups.
(A) Nuclear proteins (15 μg) were extracted from frontal cortex and hippocampus of sham, Nx-M and Nx-CI groups. (B) Densitometric analysis (arbitrary units, A.U.) of the NF-κB bands (complexes 1 and 2) are presented in panel A. Results are expressed as mean ± S.E.M. from three individual experiments (n = 9). *p<0.05 versus sham group and **versus Nx-M group—one-way ANOVA followed by Newman-Keuls test. Frontal Cortex: F (2,6) = 31.76, p<0.001; Hippocampus F(2,6) = 106.7, p<0.0001. The positions of specific NF-κB/DNA and non-specific (NS) binding complexes are indicated.
Fig 7.
Changes in TNF-α, IL-10, Il-1β, IL-6, IFN-γ, CRP and GC in CSF at 121 days in sham and Nx (Nx-M and Nx-CI) groups.
Values are the mean ± S.E.M. (n = 21). *p<0.05 versus sham group and ** versus Nx-M group—one-way ANOVA followed by Newman-Keuls test. TNF-α F(2,6) = 38.75, p<0.001; IL-10 F(2,7) = 200.50, p<0.0001; GC F(2,6) = 30.75, p<0.001; IL1-β F(2,6) = 38.75, p<0.001; IL1-β F(2,11) = 1.62, p<0.001; IL6 F(2,9) = 2.78, NS; IFNγ F(2,8) = 2.75, NS; CRP F(2,3) = 1.83, NS.
Fig 8.
Changes in TNF-α in hippocampus and frontal cortex at 121 days in sham and Nx (Nx-M and Nx-CI) groups.
Values are the mean ± S.E.M. (n = 21). *p<0.05 versus sham group and ** versus Nx-M group—one-way ANOVA followed by Newman-Keuls test. TNF-α Frontal cortex: F(2,14) = 39.43, p<0.001; TNF-α hippocampus: F(2,14) = 9.24, p<0.05.
Fig 9.
Correlation linear and regression between values of latency time measured in the Inhibitory avoidance test analysis at 120 days of observation and TNF-α in frontal cortex at 121 days in Nx (Nx-M and Nx-CI) groups.
Analyses were performed based on [70] by using GraphPad Prism Software. Pearson correlation coefficient r = −0.67, p < 0.001; linear regression, r2 = 0.67, F = 24.7, p < 0.001.
Fig 10.
Changes in soluble KLOTHO levels in rat (A) frontal cortex and (B) hippocampus at 121 days in sham and Nx (Nx-M and Nx-CI) groups.
Values are the mean ± S.E.M. (n = 21)—*p< 0.05 vs control and Nx-M—one way ANOVA followed by Newman-Keuls test. Frontal cortex: F(2,18): 23.23, p<0.0001; Hippocampus: F(2,12): 3.79, NS.
Fig 11.
Correlation linear and regression between values of TNF-α and KLOTHO levels in frontal cortex at 121 days in Nx (Nx-M and Nx-CI) groups.
Analyses were performed based on [70] by using GraphPad Prism Software. Pearson correlation coefficient r = −0.70, p < 0.001; linear regression, r2 = 0.70, F = 28.3, p < 0.001.