Inhibition of TRPC6 channels ameliorates renal fibrosis and contributes to renal protection by soluble klotho

Fibrosis is an exaggerated form of tissue repair that occurs with serious damage or repetitive injury and ultimately leads to organ failure due to the excessive scarring. Increased calcium ion entry through the TRPC6 channel has been associated with the pathogenesis of heart and glomerular diseases, but its role in renal interstitial fibrosis is unknown. We studied this by deletion of Trpc6 in mice and found it decreased unilateral ureteral obstruction-induced interstitial fibrosis and blunted increased mRNA expression of fibrosis-related genes in the ureteral obstructed kidney relative to that in the kidney of wild-type mice. Administration of BTP2, a pyrazol derivative known to inhibit function of several TRPC channels, also ameliorated obstruction-induced renal fibrosis and gene expression in wild-type mice. BTP2 inhibited carbachol-activated TRPC3 and TRPC6 channel activities in HEK293 cells. Ureteral obstruction caused over a 10-fold increase in mRNA expression for TRPC3 as well as TRPC6 in the kidneys of obstructed relative to the sham-operated mice. The magnitude of protection against obstruction-induced fibrosis in Trpc3 and Trpc6 double knockout mice was not different from that in Trpc6 knockout mice. Klotho, a membrane and soluble protein predominantly produced in the kidney, is known to confer protection against renal fibrosis. Administration of soluble klotho significantly reduced obstruction-induced renal fibrosis in wild-type mice, but not in Trpc6 knockout mice, indicating that klotho and TRPC6 inhibition act in the same pathway to protect against obstruction-induced renal fibrosis. Thus klotho and TRPC6 may be pharmacologic targets for treating renal fibrosis.Fil: Wu, Yueh-Lin. University of Texas Southwestern Medical Center; Estados Unidos. Taipei Medical University Hospital; China. Taipei Medical University; ChinaFil: Xie, Jian. University of Texas Southwestern Medical Center; Estados UnidosFil: An, Sung-Wan. University of Texas Southwestern Medical Center; Estados UnidosFil: Oliver, Noelynn. Boehringer Ingelheim Pharmaceuticals Inc.; Estados UnidosFil: Barrezueta, Nestor X.. Boehringer Ingelheim Pharmaceuticals Inc.; Estados UnidosFil: Lin, Mei-Hsiang. Taipei Medical University; ChinaFil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Pontificia Universidad Católica Argentina ; Argentina. Research Triangle Park; Estados UnidosFil: Huang, Chou-Long. University of Texas Southwestern Medical Center; Estados Unido

Tau overexpression impacts a neuroinflammation gene expression network perturbed in Alzheimer's disease.

Filamentous inclusions of the microtubule-associated protein, tau, define a variety of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). To better understand the role of tau-mediated effects on pathophysiology and global central nervous system function, we extensively characterized gene expression, pathology and behavior of the rTg4510 mouse model, which overexpresses a mutant form of human tau that causes Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We found that the most predominantly altered gene expression pathways in rTg4510 mice were in inflammatory processes. These results closely matched the causal immune function and microglial gene-regulatory network recently identified in AD. We identified additional gene expression changes by laser microdissecting specific regions of the hippocampus, which highlighted alterations in neuronal network activity. Expression of inflammatory genes and markers of neuronal activity changed as a function of age in rTg4510 mice and coincided with behavioral deficits. Inflammatory changes were tau-dependent, as they were reversed by suppression of the tau transgene. Our results suggest that the alterations in microglial phenotypes that appear to contribute to the pathogenesis of Alzheimer's disease may be driven by tau dysfunction, in addition to the direct effects of beta-amyloid

Upregulation of neuroinflammation markers in the rTg4510 brain.

<p>(A) <i>Gfap</i> and <i>Spp1</i> (which encodes for osteopontin) were upregulated at the mRNA level, as measured on Affymetrix microarrays, in rTg4510 hippocampus. <i>Gfap</i> expression increased progressively across the 3 age groups, whereas <i>Spp1</i> increased between 1.9 and 4.7 months of age, but did not differe significantly between 4.7 and 6.1 months of age. Expression levels are normalized to 6.1 month old tTA animals. Significance was determined using a one-way ANOVA followed by the Dunnett multiple comparison test. *p<0.05, ***p<0.001 compared to 1.9 month-old rTg4510. ^###p<0.001 compared to tTA. (B) GFAP and osteopontin protein levels, as measured by ELISA, were significantly highers in the cortex of rTg4510 animals at 4.6 and 6.1 months. Significance was determined using a one-way ANOVA followed by the Dunnett multiple comparison test. ****p<0.0001 compared to tTA of the respective ages. Error bars indicate SEM.</p

Inflammatory genes upregulated in the rTg4510 frontal cortex.

<p><i>C4b</i>, <i>Gfap</i> and <i>Spp1</i> mRNA expression levels were higher in rTg4510 animals compared to all other genotypes in the frontal cortex, as determined by qRT-PCR. mRNA expression levels are all normalized to tTA. **p<0.01, ***p<0.001 compared to tTA using the Dunnett multiple comparison test. Error bars indicate SEM.</p

Spatial working memory and object recognition memory in rTg4510 mice.

<p>(A) At 2 and 4 months of age, rTg4510 displayed cognitive deficits in spatial working memory compared to DN (1-way ANOVA followed by Tukey’s post-hoc ***p<0.001; student’s t-test *p<0.05). By 6 months of age, spatial working memory was confounded by stereotypic behavior observed in rTg4510 mice. n = 10–12/group. (B) At 2 months of age, all 3 genotypes displayed a preference for the novel object over the familiar object (2-way ANOVA, Bonferroni’s post-hoc **p<0.01, ***p<0.001). By 4 months of age, there was no significant difference in the amount of time rTg4510 mice explored the novel object compared to the familiar object, and the percent of time that rTg4510 explored the novel object was significantly less than the time spent by DN animals (2-way ANOVA, Bonferroni’s post-hoc *p<0.05, ***p<0.001). By 6 months of age, both rTg4510 and tTA animals explored the novel object less than DN animals (2-way ANOVA, Bonferroni’s post-hoc *p<0.05, ***p<0.001). n = 12–15/group. Error bars indicate SEM.</p

Heirarchical clustering of age-dependent gene expression changes in rTg4510 mice.

<p>Heirarchical clustering of the 165 probe sets that showed age-dependent changes in rTg4510 mice (A) using all ages and genotyopes analysed, or (B) only 6.1 month old animals, illustrates probe sets that were either downregulated or upregulated with age in rTg4510 animals. Standardization was achieved by shifting the expression values to a mean of zero and scaling to a standard deviation of one.</p

Overview of gene expression changes identified in microdissected hippocampal subfields.

<p>(A) Venn diagram of number of differentially expressed genes in the CA1, CA3 and DG microdissected samples. Most of the gene expression changes were observed in the CA1 region, with only modest overlap with the other two regions. (B) Venn diagram of the number of differentially expressed genes in the hippocampal subfields with the whole hippocampus.</p

Upregulation of the complement system in rTg4510 mice.

<p>The complement cascade, shown here adapted from the IPA library, is the most significant pathway overrepresented by genes with age-dependent changes in expression in rTg4510 hippocampus. All gene expression changes in this pathway were increases in expression. The degree of shading in red corresponds to the amount of upregulation in rTg4510 hippocampus compared to tTA at 6.1 months of age, with the numbers indicating the fold-change.</p

Suppression of tau expression leads to reversal of inflammation.

<p>(A) Administration of doxycycline (Dox) to rTg4510 mice resulted in a decrease in total human tau (HT7) in the cortex and hippocampus. The most dramatic reduction was observed in cortical layer IV (arrowhead). Doxycycline administration also resulted in a decrease of Iba-1 and GFAP staining in the cortex. Scale bar, 200 µm. (B) Doxycycline resulted in a decrease in human tau and <i>Gfap</i> gene expression measured by qRT-PCR after 1 week of treatment. Further reductions in <i>Gfap</i> gene expression were observed with progressively longer treatments prior to 5.3 months of age. (C) Quantitation of GFAP levels by ELISA reveal a 3-fold reduction in rTg4510 mice with 6 weeks of doxycycline treatment, but no change in tTA mice. (D) Plasma levels of the monocyte marker, CD40, were higher in rTg4510 animals than tTA controls, and were suppressed to tTA levels after 6 weeks of doxycycline administration. **p<0.01, ***p<0.001 using the Dunnett multiple comparison test. Error bars indicate SEM.</p