Klotho Deficiency Aggravated Diabetes-induced Podocyte injury

Background: Diabetic nephropathy (DN) is a progressive disease, the main pathogeny of which is podocyte injury inducing glomerular filtration barrier and proteinuria. The occurrence and development of DN could be partly attributed to the reactive oxygen species (ROS) generated by mitochondria. However, researches on how mitochondrial dysfunction (MtD) ultimately causes DNA damage is poor. Methods: We generated streptozotocin (STZ)-induced diabetic mice with wild-type(C57BL/6J) or Klotho deficiency mice ( KL +/- ) and treated podocytes with high glucose (HG) to investigated the function of Klotho on HG-induced podocyte injury in vivo and in vitro . Results: The absence of Klotho aggravated diabetic phenotypes indicated by podocyte injury accompanied by elevated urea albumin creatinine ratio (UACR), creatinine, urea nitrogen. Then, Klotho deficiency could significantly aggravate DNA damage by increasing 8-OHdG and reducing OGG1. Finally, Klotho deficiency may promote MtD to promote 8-OHdG-induced podocyte injury. Conclusions : Klotho deficiency may promote diabetes-induced podocytic MtD and aggravate 8-OHdG-induced DNA damage by affecting OOG1.


Background
The incidence and prevalence of diabetes mellitus (DM) have grown significantly throughout the world due to the overall increase in type 2 diabetes.
As one of the most frequent complications of DM, diabetic kidney disease (DKD) defined as a syndrome, is the most common metabolic disease in the world that is the leading cause of end-stage renal disease (ESRD) comprising about 40% of patients with CKD 1,2 . The onset of clinically overt DKD is defined as persistent proteinuria that is most closely 3 associated with podocytopathies that results from damage to the glomerular filtration barrier at the level of the highly differentiated glomerular podocyte cells 3 − 5 . Despite the status quo, the factors that precipitate the development and progression of diabetic kidney disease (DKD) remain to be fully elucidated. Moreover, the molecular mechanisms leading to proteinuria and podocyte effacement are poorly understood. DM is characterized by increased levels of reactive oxygen species (ROS) leading to high levels of adenosine triphosphate (ATP) 6 . Nevertheless, mitochondria are the energy powerhouses of cells by ATP synthesis through oxidative phosphorylation (OXPHOS) and play a key role in apoptosis. Mitochondria also attaches great importance to renal function, of which dysfunction is becoming increasingly recognized as contributing to renal glomerular and tubular diseases 7 − 11 . Besides, tubular epithelial cells are mitochondrial rich. Mitochondria dysfunction (MtD) and reactive oxygen species(ROS)induced damage is also well reported in glomerular podocytes, as they are highly specialized and terminally differentiated epithelial cells 12 . As limited replicative capability of regeneration, podocytes injury may represent the major mechanism of progressive renal damage. Remarkably, mitochondrial electron transport chain is identified as the major non-enzymatic source of diabetes-induced ROS in podocytes, that are believed to cause the onset of albuminuria followed by progression to renal damage through podocytes depletion 12 . MtD is usually accompanied by a reduction in the efficiency of the DNA repair capacity and antioxidant defense, consequently leading to the accumulation of cellular damage 13,14 . DNA continually exposed to exogenous and endogenous stressors will lead to DNA breaks, damage or improperly repair of which can activate pro-apoptotic pathways, or induce cellular senescence 14,16 . The most frequently formed oxidative DNA damage is 8-hydroxydeoxyguanosine , that could be repaired by base-excision repair (BER) system. 8-oxo-deoxyguanosine DNA glycosylase 1 (OGG1), an enzyme that is involved in the process, is associated with the DNA repair activity and decreased risk for some oxidative stress-related diseases 17,18 . Cells have developed complex DNA damage signaling and repair mechanisms, that were collectively called DNA damage response (DDR) 15,16 . DNA damage also plays an important role in the development of DM and its complications 19,20 , but deep molecular mechanisms remains to be studied. α-Klotho, also known as Klotho, which predominantly produced in renal tubular epithelial cells, regulates ageing-related processes existing in membrane-bound and soluble forms 21   to enhance expression of the T antigen, and cells were cultivated at 33℃ (permissive conditions). To induce differentiation, podocytes were cultured on type I collagen at 37℃ without γ-interferon for at least 14 days. Podocytes were cultured in 6-well plates and then preincubated with Klotho 48 h before HG induction. In parallel, podocytes incubated with mannitol (30 mmol/l) for same time were taken as negative control.

Histology
Histological and morphometric analysis was carried out on paraffin sections (2 µm thickness) cut on a rotation microtome (Microm) and stained with hematoxylin-eosin and periodic acid-schiff, respectively.

Urine Micro Albuminuria
Albumin concentration in spot urine samples was measured with a commercially available competitive enzyme-linked immunosorbent assay following the instructions of the manufacturer (TP0100-1KT, Sigma, USA) and was normalized to urine creatinine. 6 ELISA Assay of Serum Klotho The blood samples from mice were collected and centrifuged for 10 minutes at 3000 rpm (4 °C), and the serum Klotho was measured in duplicate using a mouse Klotho ELISA kit according to the manufacturer's protocol (Cusabio, Cologne, Germany).

Detection of ROS Production
The Immunofluorescence Assay Immunofluorescent staining and images were obtained by a LSM780 laser scanning confocal microscope (ZEISS, Germany) system. 7 Transmission electron microscopy (TEM) TEM images were analyzed using Image Pro plus 6.0. The GBM thickness, foot process width and the number of foot processes per µm of GBM were calculated using a curvimeter (SAKURAI CO., LTD, Tokyo, Japan).

Statistical Analyses
Data are expressed as means ± SE. Student's t-test was employed for comparisons between two groups. Multiple comparisons were performed using one-way ANOVA, followed by Bonferroni's post hoc test. P values < 0.05 were considered significant and are indicated in the figures by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001). Analyses were performed using Graph Pad Prism software (GraphPad Software Inc, version 7.0).

Klotho deficiency exacerbated diabetic nephropathy
To analyze the relationship between Klotho and phenotypes of diabetic nephropathy. As high embryonic mortality and failure rate of diabetes model in the Klotho homozygous mice, we selected Klotho heterozygous mice. We generated STZ-induced diabetic mice with wild-type(C57BL/6J) or Klotho deficiency mice (KL + /− ) aged 8 to 10w. The concentration of Klotho in serum did decrease in diabetic mice, especially in STZ-induced KL + /− mice (KL + /− STZ) (Fig. 1a). Further compared with normal mice, all diabetic mice' concentration of blood sugar was higher than 13.8 mmol/l and no downregulation was observed, but the levels further increased in KL + /− STZ (Fig. 1b). Urine volume in diabetic WT group (WT STZ) was obviously higher than the WT group but Klotho deficiency aggravated the phenomenon when compared to WT STZ (Fig. 1c). Diabetes also induced kidney weight increase and increased ratio of kidney weight to body weight but Klotho deficiency induced greater increase of those ( Fig. 1d and e). Meanwhile, we also measured 8 serum creatinine, blood urea nitrogen (BUN) and UACR, respectively. We compared different groups and found the further rise of creatinine, BUN, as well as UACR in KL + /− STZ when compared to WT STZ (Fig. 1f-h). To analyze the difference of glomerular morphology in different groups, we used hematoxylin-eosin staining and found mesangial matrix expansion in WT STZ. However, KL + /− STZ also induced mesangial matrix expansion that had more expansion compared with WT STZ (Fig. 1i). Using periodic acid schiff (PAS) staining to observe glycogen deposition, we found there are glycogen deposition in glomerular of both WT STZ and KL + /− STZ. Moreover, compared with WT STZ, glomerular accumulated more glycogen in KL + /− STZ (Fig. 1i). All these data indicated Klotho deficiency may aggravate diabetic renal dysfunction.

Klotho deficiency exacerbated STZ-induced podocyte injury
Whether Klotho deficiency induces podocyte injury, we examined the expression of WT1, podocyte's marker, by immunohistochemistry (IHC). We found diabetes inhibited the expression of WT1 (Fig. 2a) and promoted expression of Caspase3 (Fig. 2a). It's worth noting that Klotho deficiency pushed the pathological process (Fig. 2a), as was further confirmed by immunofluorescence (Fig. 2b). Podocyte was injury as evidenced by glomerular basement membrane (GBM) thickening, podocyte foot process broadening or effacement in WT STZ, that were significantly lower than KL + /− STZ (Fig. 2c). Nephrin, another podocyte's marker, and Peroxiredoxin 2 (PRDX2), an antioxidant enzyme detoxifying reactive oxygen species, were also subjected to western blot analysis. We found Klotho deficiency further inhibited the expression of Nephrin and partially suppressed PRDX2 in KL + /− STZ as compared to WT STZ (Fig. 2d). The results suggested Klotho deficiency further exacerbated STZ-induced podocyte injury.
Klotho deficiency aggravated HG-induced MtD of podocytes 9 As glucose metabolism is related to energy metabolism, mitochondria play an important role in this process. To wonder whether Klotho deficiency affected MtD of podocytes, transmission electron microscope (TEM) was first performed. The results showed mitochondrial hypertrophy appeared in podocytes of WT STZ when compared to that of WT (Fig. 3a). Meanwhile, numerical density of damaged mitochondria increased significantly in WT STZ as compared to WT Veh (Fig. 3a). However, slight mitochondrial contraction was observed in non-diabetic KL + /− mice but this phenomenon was obviously aggravated in KL + /− STZ (Fig. 3a). Moreover, lower numerical density of damaged mitochondria was observed in KL + /− STZ when compared to WT STZ (Fig. 3a). Further analysis by immunofluorescence suggested 8-hydroxydeoxyguanosine (8-OHdG), a marker of DNA damage, increased with diabetes, especially in KL + /− STZ, which was inversely related to the expression of Nephrin (Fig. 3b). The results indicated Klotho deficiency may be related to MtD of podocyte.

Klotho deficiency affected OGG1 in HG-treated podocytes
Having found the relationship between Klotho and 8-OHdG, we wonder whether DNA damage repair enzymes were suffered interference. We first detected 8-oxoguanine DNA glycosylase (OGG1), a DNA repair enzyme, by westernblot and found the Klotho deficiency was accompanied with low expression of OGG1 in diabetic mice (Fig. 4a). Further experimental confirmation by immunofluorescence showed STZ-induced diabetes suppressed the expression of both synapotopodin,a marker of podocyte and OGG1. Klotho deficiency further inhibited the expression (Fig. 4b). Meanwhile, we analyzed the role of reactive oxygen species (ROS) in podocytes when treated with HG. We found a sharp rise of ROS in HG-induced podocytes (Fig. 4c). When applied with human Klotho recombinant protein or N-acetyl-L-cysteine (NAC), HG-treated ROS was ameliorated, respectively ( Fig. 4c). We also demonstrated the expression of WT1, Podocin, OGG1 as well as Caspse3, respectively. Both Klotho and NAC could inhibit the reduction of WT1 and Podocin and reduce the expression of Caspase3 in HG-treated podocytes (Fig. 4d). Moreover, OGG1 was ameliorated by Klotho or NAC (Fig. 4d). The above results indicated that HG activated OGG1 which was disturbed by Klotho deficiency leading podocyte injury.

Discussion
Diabetic nephropathy (DN) is a progressive disease that affects about one third of diabetic patients, the main pathogeny of which is podocyte injury that leads to glomerular filtration barrier and proteinuria 23,24 . Klotho, anti-aging protein with critical roles in protecting kidney, is expressed predominantly in the kidney and secreted in the blood. Nonetheless Podocyte is nonrenewable and vulnerable to a variety of injuries as is highly specialized, terminally differentiated epithelial cells that line the outer surface of the glomerular basement membrane 26 − 28 . Enhanced production of reactive oxygen species (ROS) has been recognized as the major determinant of age-related endothelial dysfunction 29,30 . p66SHC induced by HG could mediate mitochondrial dysfunction 12,31,32 . However, our study focused on diabetes-induced DNA damage that may be the root cause of podocyte damage. As previously reported, we also confirmed that HG induced the production of ROS and leaded MtD. However, the correlation of researches between Klotho and ROS in diabetes mainly in ROS-induced podocyte apoptosis 12 . In this study, we studied the relationship between Klotho and DNA damage in diabetes nephropathy (DN). We found Klotho deficiency promoted 8-OHdG to induce DNA damage as inhibiting OGG1 expression in HG-treated podocytes.
As we have demonstrated the role of Klotho on diabetes-induced DNA damage of podocyte, how Klotho regulates OGG1 to inhibit 8-OHdG remains to be further studied.
OGG1 is the major DNA glycosylase in human cells for removing 8-OHdG, one of the most frequent endogenous base lesions formed in the DNA of aerobic organisms. OGG1 could be phosphorylated in vivo on a serine residue and is subject to protein kinase C (PKC)mediated phosphorylation in vitro, suggesting that PKC is responsible for the phosphorylation event 33 . Nevertheless, Protein kinase C (PKC) transducing signals is mediated by diacylglycerol (DG) or the second messengers Calcium ion 34 . Klotho as a regulator of calcium homeostasis inhibits transient receptor potential channel 6 (TRPC6)mediated Ca 2+ influx in cultured mouse podocytes and ameliorates albuminuria and renal fibrosis 35,36 . In summary, Klotho may be as a regulator of calcium to inhibit PKC and then activate OOG1, but further molecular mechanisms need further study.

Conclusions
Our results imply that Klotho deficiency may aggravate podocyte MtD by affecting ROS and 8-OHdG-induced DNA damage by affecting OOG1 in diabetes.

Ethics approval and consent to participate
Experiments were performed in accordance with the Animal Research Institute Committee 12 of University of Chinese Academy of Sciences for the Care and Use of Laboratory Animals.

Consent for publication
Written informed consent for publication was obtained from all participants.

Availability of data and materials
All data generated or analyzed during this study are available from the corresponding author on reasonable request.

Competing interests
The author declares that they have no competing interests.

Funding
This work was supported by grants from the science and technology project of Chongqing Yuzhong district (20180135) and the Natural Science Foundation of Chongqing cstc2019jcyj-msxmX0248 .

Authors' contributions
ZC wrote the article and edited the manuscript; QZ and CL were responsible for the statistical analysis; YPZ and SLY revised the manuscript. All authors read and approved the final manuscript.