Tubular Cell Senescence After Renal Injury Mediated By Epithelial Innate Immunity

Abstract

Acute kidney injury (AKI) is a common clinical disease with increasing incidence. Patients with severe acute kidney injury have a higher lifetime risk of interstitial fibrosis, chronic kidney disease, and end-stage kidney disease. Cell senescence is a persistent cell cycle arrest and change in gene expression patterns, which is caused by a variety of stressors. The number of senescent cells increases with age, and even in small numbers, these cells can cause chronic inflammation and fibrosis; In fact, it is important in several cases, including in the kidneys, that although Myd88 inactivation after injury improved fibrosis, it did not reduce damage to the tubules. In order to test the pharmacological effect of cistanche on kidney, the renal cell status of rats fed with and without cistanche extract was verified in rat experiments.

Introduction

Acute kidney injury (AKI) is a common clinical condition and an increasing cause of death. Although AKI has always been considered a self-limiting disease, recently an association has been recognized between recurrence, severity of AKI attacks, and subsequent development of chronic kidney disease. Tubular repair is a common event following kidney injury, but is often associated with interstitial inflammation and maladaptive processes that lead to fibrosis, which is a hallmark of all forms of kidney disease and a reliable predictor of progression to CKD. Renal fibrosis is also associated with the arrest of tubular epithelial cells in the G2/M phase of the cell cycle, suggesting that epithelial cells play a major role in the progression of renal disease. Cell cycle arrest is a common hallmark of cell aging and is caused by a variety of stressors, including oncogene activation, replication stress, replication failure, telomere dysfunction and oxidative stress. Senescent cells are resistant to apoptosis, and their persistent presence in tissues, even in small numbers, leads to chronic inflammation and fibrosis, an aging-related phenotype in multiple organs, including the kidney. Senescent cells communicate with neighboring tissues by secreting different kinds of molecules. Studies have shown that cistanche polysaccharide and D-mannitol can activate SOD and reduce lipofuscin accumulation, while total glucoside of phenylate can increase SOD activity and renal coefficient in vivo.

Our results suggest that TEC senescence is a common early event after kidney injury, and that the signaling pathway of the TLR/IL-1R pathway in epithelial cells controls this phenomenon in a cellular autonomic manner. Our findings also suggest that early intervention after injury may be needed to reduce organ damage after AKI. In addition, in contrast to published studies focusing on the role of innate immune signaling in pericytes, this study reveals a novel function of cistanche in controlling the onset of TEC senescence in a cellular autonomic manner and in non-cellular autonomic control of pericyte proliferation and cell fate, consistent with the concept that tubular epithelium induces kidney disease after injury and drives its progression.

Pic: Cistanche Extract

Results

AKI induced senescence of TECs cells. To test whether cellular senescence is a common event after kidney injury, we looked for two established aging features: increased activity of the age-related enzyme, galactosidase (SA-beta-gal), and decreased abundance of the protein B1 (LAMNB1) in the middle nuclear membrane. We used four mouse models of renal injury: folic acid induced nephrotoxicity, ischemia/reperfusion injury (IRI), cisplatin induced nephrotoxicity (cp), and kidney injury mice fed cistanches. We assessed aging at 28 days after the initial injury. In TECs of the first three injury models, SA-β-Gal activity increased and LAMNB1 levels decreased, but the opposite was true in cistanche fed mice. This suggests that cistanche has the effect of alleviating kidney injury.

Renal tubular cells senescence occurs early after renal injury. To further characterize the occurrence of tubular cell senescence after AKI, we used p16-3mr transgenic mouse strains. These mice were generated by cloning a gene encoding a three-mode reporter fusion protein containing the functional domain of a synthetic Renilla luciferase (LUC), a monomer red fluorescent protein (mRFP), and a cut off herpes simplex virus 1 (HSV-1) thymidine kinase (HSV-TK), Framed with the promoter of the tumor suppressor gene p16Ink4a, whose expression is activated in senescent cells. mRFP allows for the classification of expressing cells from tissues based on flow cytometry, and HSV-TK allows for their killing by ganciclovir (GCV), a nucleoside analogue with a high affinity for TK from HSV but a low affinity for its mammalian counterpart (cell TK). HSV-TK converts GCV into a toxic DNA strand terminator that induces mitochondrial DNA breakage in undivided senescent cells and leads to death through apoptosis (26). For the experiments described below, we chose not to use CP or IRI damage models because CP induces DNA damage and aging, while IRI induced damage is not limited to tubular cells. Therefore, we focused on the FA model where the use of a single injection of FA is toxic to rodent TECs because it causes intracavitary precipitation of folic acid crystals and oxidative stress. Surprisingly, quantification of mRFP fluorescence in transplanted kidneys showed that in the case of FA injury, senescence levels were significantly higher than in the vehicular injection control group as early as 3 days after FA injury, and that senescence levels increased slowly at subsequent points in time. SA-β-Gal staining confirmed senescent cells in the tubules from day 2 to day 3 after injury, indicating that TEC senescence was an early event after injury.

Pic: Effects of cistanche：improve kidney function

Epithelial-specific Myd88 deletion reduces senescence of tubule cells after injury. Considering that TLR/ IL-1-dependent NF-κB activation leads to paracrine effects in senescent cells, and senescent cells induce secondary senescence in neighboring cells, we asked whether epithelial inactivation of Myd88 could inhibit the spread of renal senescence. Indeed, 28 days after injury, the absence of Myd88 in tubular cells resulted in decreased SA-β-Gal activity, increased LAMNB1 abundance, and proliferation of tubular cells. These results suggest that cistanche can open the epithelial TLR/IL-1R signaling pathway to control the occurrence of renal tubular senescence, and inhibition of this signaling pathway is sufficient to limit the accumulation of senile tubular cells after renal injury.

Discussion

Here we report our examination of the occurrence of cellular senescence after renal injury in mice and whether senescence contributes to the progression of renal tubule injury and fibrosis after AKI. The positivity of cell senescence markers in four different models of kidney injury suggests that cell senescence is a common event independent of the type of injury. Using transgenic report labeling of senescent cells, we show that senescence occurs primarily in proximal tubule cells based on the localization and morphology of this marker. An interesting and unexpected finding is that tubular cell senescence occurs relatively early, independent of the type of injury, and senescent cell burden increases slowly over time. At the same time, cistanche can reduce the apoptosis rate of kidney cells and play a good protective effect on the kidney.

Our results also highlight the limited efficacy of anti-aging drugs that induce apoptosis in senescent cells to limit kidney damage. Our experiments with p16-3mr transgenic mice and FOXO4-DRI peptide have produced mixed results in preventing fibrosis. These findings suggest that different types of senescent cells may coexist and that subpopulations of these cells may have different functions in damaged tissues. Importantly, in both sets of experiments, eliminating senescent cells, at least in the early stages after injury, did not affect kidney damage and repair. This is contrary to what has been reported for natural or pathological aging, in which removal of senescent cells improves tissue homeostasis. The reasons for these different results are unclear. One possibility is that, given the higher burden of senescent cells in the context of acute injury, widely induced apoptosis in tubule cells may offset the benefits of reduced pro-inflammatory stimuli, such as those produced by the slow accumulation of senescent cells in aging tissues, leading to increased tubule damage and the initiation of maladaptive processes. However, the elimination of both tubules and non-epithelial senescent cells by anti-aging drugs may also impair the post-injury preparation process.

Pic: Reduce the apoptosis of your kidney cells

Conclusion

We have identified tubular cell senescence as an early central mechanism that leads to further accumulation of senescent cells after kidney injury and advances in the treatment of kidney injury and fibrosis by cellular autonomic activation of innate immune signals and cistanche. Importantly, the partial effect of eliminating senescent cells after injury in reducing inflammation and fibrosis, but not tubule damage, suggests that cell aging may also contribute to tissue repair after AKI.

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