Abstract
The role and precise mechanism of TLR4 in mitochondria-related oxidative damage and apoptosis of renal tubules in diabetic kidney disease (DKD) remain unclear. We examined the expression of TLR4 in renal biopsy tissues. Db/db diabetic mice and HK-2 cells cultured under high glucose (HG) were used as in vivo and vitro models. Real-time RT-PCR, Western blot, and immunohistochemistry were performed to examine the mRNA and protein levels of TLR4, NF-κΒ, PGC-1α, cytochrome C, and cleaved caspase-3. ATP level, activity of electron transport chain complex III, and antioxidant enzymes were investigated for mitochondrial function. Electron microscopy (EM) and MitoTracker Red CMXRos were used for mitochondrial morphology alteration. DHE staining and TUNEL assay were detected for ROS accumulation and apoptosis. PGC-1α plasmids were used for the overexpression of PGC-1α in HK-2. TAK242 and parthenolide were used as TLR4 and NF-κB blockers, respectively. Results showed that TLR4 was extensively expressed in the renal tubules of DKD patients and db/db diabetic mice, which was positively related to the tubular interstitial damage score and urinary β-NAG levels. In diabetic mice, inhibition of TLR4 could reverse the decreased expression of PGC-1α, increased expression of cytochrome C and cleaved caspase-3, mitochondrial dysfunction and deformation, increased accumulation of ROS, and activation of tubular cell apoptosis. In vitro, inhibition of TLR4 or NF-κB showed consistent results. PGC-1α overexpression could reverse the mitochondrial dysfunction, increased cleaved caspase-3, and apoptosis in HK-2 cells treated with HG. Data indicated that the TLR4/NF-κB signaling pathway might be the upstream pathway of PGC-1α and promote the tubular damage of DKD by modulating the mitochondria-related oxidative damage and apoptosis.
Highlights
Toll-like receptors (TLRs) are pattern recognition receptors and play a fundamental role in the activation of innate and adaptive immune responses [1, 2]
The mitochondrial changes in Electron microscopy (EM) were quantified (Figure 1(c)): mitochondrial length was measured in tubular cells to determine the percentage of cells that showed filamentous mitochondria less than 1% long (>2 μm). ∗P < 0 05 compared with the N-diabetic kidney disease (DKD) group
TLR4 has been previously described involving in hyperglycemia-induced inflammatory state of renal tubulus in vitro and vivo [15, 16], but its role in oxidative damage and apoptosis in renal tubular cells in DKD remains unclear
Summary
Toll-like receptors (TLRs) are pattern recognition receptors and play a fundamental role in the activation of innate and adaptive immune responses [1, 2]. HG was proved to induce the overexpression of TLR4 through NF-κB-dependent signaling and lead to the accumulation of ROS in podocytes [7], indicating the important role of TLR4 NF-κB signaling in the mechanism of DKD progression. According to our previous study, renal tubular oxidative stress injury and apoptosis play a key role in the progression of DKD in high glucose (HG) conditions [8]. These let us speculate that activation of the TLR4-NF-κB signaling pathway might be involved in mitochondrial dysfunction and mitochondriarelated oxidative damage of renal tubular epithelial cell (RTEC) in hyperglycemia, which is gradually to have an extremely important effect in the progression of DKD
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