Research Article - Biomedical Research (2018) Volume 29, Issue 8
Accepted date: August 07, 2017
Neurologic involvement is the most threatening complication of uremic syndrome. Currently, the main treatment of complicated neuropsychic symptom in end-stage uremia is depended on the advent of dialysis and transplant programs, which may associated with many other complications. Effective therapy for this serious and debilitating condition is needed. This study was aimed to elucidate the roles of exogenous α-Klotho (α-KL) on the recovery of injured sciatic nerve induced by uremia. Hematoxylineosin staining was carried out to observe histopathologic changes between the groups. Neurocytes activity was assessed by measuring ATPase, Na+, K+-ATPase, Succinate Dehydrogenase (SDH) activity levels, and NADH as well as NADPH concentrations. NF-kBp65 was assessed to indicate inflammatory. Caspase-3 level was evaluated to indicate the apoptotic pathway. We found that in a damaged nerve induced by uremia, obvious decreased ATPase and Na+, K+-ATPase were observed demonstrating the dysfunction of nerve system conduction, and significant decreased SDH activity, while increased NADH and NADPH levels were observed demonstrating the mitochondrial ROS generation that directly associated with oxidative stress injury and apoptosis, moreover the activation of NF-KBp65 and Caspase-3 singling pathways were observed demonstrating that inflammatory and apoptosis were involved after sciatic nerve being injured. α-KL was effective in the recovery of nerve system conduction, played anti-oxidative stress, anti-inflammatory and anti-apoptosis roles in damaged sciatic nerve, besides promoted the regeneration of peripheral nerve at histopathologic levels. We therefore concluded that α-KL may serve as a potential therapeutic agent for uremia-induced sciatic nerve injury.
α-KL, Uremia-induced sciatic nerve injury, Physiology, Histopathology, Inflammatory, Apoptosis.
Advanced renal failure leads to uremia. The term “Uremia” refers to illness accompanying kidney failure, which cannot be attributed to derangements in extracellular volume, absence of known renal synthetic products, or inorganic ion concentrations . Some groups with uremia are more prone to associate with nervous system dysfunction [2-6]. Reasons of neuropsychic symptom in end-stage uremia were related to multiple factors such as uremia toxin storage, metabolic acidosis and electrolyte disturbance. Patients often become confused, due to being affected by cognitive impairment [7,8]; stroke ; somnolence, or seizures  and peripheral neuropathies [11,12]. Uremia has always been reported as serious problem that threatened the whole public health around the world.
Dialysis and kidney transplantation have revolutionized the outcome of patients with progressive renal disease. However, current treatment with dialytic therapy or kidney transplantation may even carry a high price and induce neurological complications or other related diseases. Dialysis therapy for uremic neuropathy has itself been associated with Central Nervous System (CNS) disorders such as dialysis disequilibrium syndrome, dialysis dementia, and progressive intellectual dysfunction . Moreover, patients undergoing dialysis have a “residual syndrome” including partially treated uremia; dialysis related effects, for example extracellular fluid volume fluctuation; exposure to biological incompatible materials and residual inorganic ion disturbances . As for kidney transplantation, there is less frequent use because of a short supply of donor. Besides, psychiatric or neurological disorders; infectious, gastrointestinal, vascular and urologic complications are common after renal transplantation [14-19]. Due to the shortcomings of the above two treatment methods, searching for other more effective therapeutic agents for the treatments of uremic neuropathy is very important and urgent.
Known as an anti-aging gene, α-Klotho (α-KL) is a protein mainly produced in the kidney and its circulating form (soluble α-KL) have been demonstrated to have a lot of therapeutic effects such as anti-oxidative stress, anti-senescence and antiapoptotic as well as vasculo protective effects [20-24]. Laboratory data suggest that α-KL expression is decreased in many experimental models of kidney disease including Chronic Kidney Disease (CKD) [25-27]. The enhancement of supplement of exogenous α-KL is a promising therapeutic strategy to prevent, retard, and decrease the comorbidity burden of kidney injury [28,29]. Although α-KL has been proved to have protective effects against kidney injury, its function on the pathology and histopathology of sciatic nerve injury induced by uremia has not been demonstrated. In the current study, to explore the roles of α-KL in the recovery of uremia-induced sciatic nerve injury, we presents uremia rat models with recommendations for diagnosis and treatment, and investigated the effects of different concentrations of α-KL at the neurophysiological and histopathologic responses towards the injured sciatic nerve. Moreover, the regulatory mechanism of α-KL in uremia-induced sciatic nerve injury is studied in this article.
Uremia-induced sciatic nerve injury
Rats were numbered and randomly assigned to four experimental groups: a normal group (group A, with six rats in normal diet), a control group (group B, with six adenineinduced uremia rats), and α-KL-treated group (groups C and D, with six rats in each subgroup). Adenine-induced uremia rat model was obtained by a continuous stomach injection of adenine (2.5%, suspension) at a dose of 250 mg/kg/d for 14 d, then every two days for 14 d, which resulted in progressive renal dysfunction, like human uraemic features (control group). The dosages of α-KL protein were 0.01 mg/kg/d and 0.02 mg/kg/d, administrated through intraperitoneal injection for 3 consecutive days in α-KL-treated groups (group C: uremia+α- KL 0.01 mg/kg/d and group D: uremia+α-KL 0.02 mg/kg/d). The dosages were chosen according to reported literatures [30-32]. At the same time, normal group and uremic group were given the equivalent volume of normal saline. Two weeks later, the rats were sacrificed after blood sampling and then renal and sciatic nerve tissues were obtained from all of the rats.
Renal histology analysis was carried as follows: after fixed in 10% neutral buffered paraformaldehyde, the biopsies were dehydrated in graded concentrations of ethanol, immersed in xylene, and then embedded in paraffin. The sections of 4 μm thickness were cut, stained with haematoxylin and eosin for assessments under light microscopy. Histopathologic changes were observed.
Sciatic nerves histological analysis: After two weeks, biopsies were collected from the L4-5 root ganglion, including the distal sciatic nerve segments of all rats. Operation method is the same as mentioned above.
Neurocytes activity studies
The total protein concentration, ATP hydrolysis (ATPase); sodium, potassium adenosine triphosphatase (Na+, K+-ATPase) and Succinate Dehydrogenase (SDH) activity levels were assayed using commercially available kits (Nanjing Jiancheng Bioengineering institute). Reduced Nicotinamideadenine Dinucleotide (NADH) and reduced Nicotinamide Adeninedinucleotide Phosphate (NADPH) concentrations were assayed according to the manufacturer’s protocol (AAT Bioquest, Inc.). The total protein concentration was expressed as microgram per milliliter, whereas ATPase, Na+-K+ ATPase, SDH activity was expressed as unit per milligram of protein, NADH and NADPH concentration were expressed as micromole per liter.
Western blot analysis
After α-KL-treated at the designed concentrations and time, sciatic nerve were harvested and cut into tiny pieces, which were lysed at 0°C in radio immunoprecipitation assay buffer (RIPA, 150-250 μl/20 mg, Solarbio Biotechnology, Shanghai, China) with freshly added 0.01% phosphatase and protease inhibitors (Sigma, Shanghai, China), followed by incubating on ice for 30 min. Cell lysis (12, 000X g) was centrifuged at 12,000 rpm for 15 min at 4°C in a microcentrifuge. The supernatant was collected in fresh tubes. The total protein concentration in the lysates was determined using a colorimetric Bicinchoninic Acid (BCA) assay kit (Thermo, Shanghai, China). The lysates were subsequently diluted with a reducing Laemmli buffer and stored at -80°C before being utilized for the assay. The supernatant (30 μg of protein) was run on SDS-PAGE gel, electrophoretically was transferred to a Nitrocellulose membrane (NC) (Millipore, Bredford, USA). The membranes were blocked with 5% skim milk and incubated with primary antibodies. Antibodies against NF-KB p65 and GAPDH were purchased from Cell Signaling Technology, Inc. (Beverley, MA), while antibodies against Caspase-3 were purchased from Fermentas (Vilnius, LT). Glyceraldehyde 3-phosphatedehydrogenase antibody (GAPDH) was used as an internal control. Blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime), the HRP signal was visualized by Chemiluminescence (ECL) system (Millipore). The experiment was repeated three times independently.
Each experiment was done four times in parallel. All data were expressed as mean ± Standard Error of the Mean (SEM). Oneway Analysis of Variance (ANOVA) followed by Student’s ttest was performed for statistical analysis. A level of Probability p<0.05 was considered significant.
Renal histology analysis
Hematoxylin-Eosin staining (H & E) analysis in renal tissues was performed to observe histopathologic changes in control group as compared with normal group to make sure a successful adenine-induced uremia rat model at the histological level (Figure 1). Normal renal structure was observed in group A demonstrating that the overall integrity of glomerulus was surrounded by bowman capsule as well as convoluted tubules. Compared to group A, group B showed significant morphologic changes such as interstitial edema and neutrophil accumulation, which demonstrated significant kidney damage, with obvious inflammation being observed . Besides, in all α-KL-treated subgroups (groups C and D), the kidney damage was markedly reduced in a dose-dependent manner with maximum reduction being observed in group D when compared with the control group, demonstrating a positive effect of α-KL in kidney damage reduction.
Sciatic nerves histological examination
H & E staining in sciatic nerves was performed to see histopathologic changes in the treat groups as compared with control group. In Figure 2, group A showed that normal rats’ sciatic nerve fibers showed neat and orderly arrangement. In group B, numerous wispy tissues were visible and nerve fibers were thinner, which demonstrated a significant sciatic nerve damage [34,35]. In all α-KL-treated subgroups (groups C and D), the sciatic nerve damage was markedly reduced in a dosedependent manner with maximum reduction being observed in group D when compared with the control group demonstrating a positive effect of α-KL on the regeneration of peripheral nerve.
Neurocytes activity studies
ATPase and Na+, K+-ATPase activity were measured to assess the pump function, which played a fundamental role in neuronal excitability. SDH activity was measured to assess mitochondrial metabolism, which represented the predominant cellular source of ATP and directly related with ROS production . NADH and NADPH, as reducing powers in many cellular reactions, were measured to evaluate ROS level. In Figure 3, group B (a control group) showed that significant decreased ATPase, Na+, K+-ATPase and SDH activity (p<0.05) while obvious increased NADH and NADPH concentrations were observed as compared with group A (a normal group), demonstrating the significant ion imbalance and mitochondrial damage in injured sciatic nerve. In all α-KL-treated subgroups (groups C and D), relative increased ATPase, Na+, K+-ATPase and SDH activity were observed as compared with group B (a control group), demonstrating the improved nerve conduction and mitochondrial metabolism. Meanwhile significant decreased NADH and NADPH concentrations were observed in a dose-dependent manner with maximum reduction being observed in group D (p<0.05) when compared with the control group, which showed that the level of mitochondrial ROS was significantly decreased, demonstrating that oxidative stress was markedly alleviated in neurocytes.
Figure 3: Effect of α-KL treatment on neurocytes activity studies. A and B. Quantified ATPase, Na+, K+-ATPase and SDH activities. C. Quantified NADH and NADPH concentrations. α-KL (0.01 mg/kg/d and 0.02 mg/kg/d) relatively promotes ATPase, Na+, K+-ATPase as well as SDH activity, and significantly decreases NADH and NADPH concentrations in a dose-dependent manner with maximum reduction observed in group D when compared with uremia group. *p<0.05 versus the normal group, #p<0.05 versus the uremia group.
NF-KB p65 and caspase-3 activity in sciatic nerve cells
Western blot analysis was carried out to detect the protein levels of NF-KBp65 and caspase-3. In Figure 4, group B (a control group) showed significant enhanced expressions of NFKB p65 (from 0.17 ± 0.07% to 0.48 ± 0.09% ) and caspase-3 (from 0.17 ± 0.10% to 0.77 ± 0.12%) as compared with group A (a normal group). In all α-KL-treated subgroups (groups C and D), significant decreased NF-KB p65 as well as caspase-3 expression levels were observed in a dose-dependent manner with maximum reduction being observed in group D (p<0.05) when compared with the control group. These results suggested that α-KL was specifically associated with lower levels of NF-κBp65 as well as caspase-3.
Figure 4: Effects of α-KL on the expression of NF-KB p65 as well as caspase-3 proteins. A. Representative Western blot results showing the levels of NF-KBp65 and caspase 3. GAPDH serves as an internal control. B. Quantification of Western blot results. All experiments were repeated three times. *p<0.05 versus the normal group, #p<0.05 versus the uremia group.
Uremia is still a serious public health problem around the world with high mortality and morbidity . Some groups with uremia are more prone to affect nervous system dysfunction. Dialysis and kidney transplantation has become two main treatment methods. However, current treatment with dialytic therapy or kidney transplantation may even carry a high price and induce neurological complications or other related diseases. A potential therapeutic agent is urgent.
Peripheral nerves become injured through a variety of mechanisms, and most of them contribute to the blocking of nerve system conduction  and the generation of mitochondrial ROS causing exposure to an inflammatory microenvironment [39-41]. Early inflammatory events in injured nerves could prevent the functional recovery  and promote apoptosis or necrosis [43,44]. Soluble α-KL has been reported to be involved in many biological activities such as the regulation of oxidative stress, senescence, apoptosis, and so on. Although α-KL has been proved to have protective effects against kidney injury, its function on the sciatic nerve injury induced by uremia has not been studied. In the current study, to explore the impact of α-KL on the recovery of uremia-induced injured sciatic nerve, we presented rat uremia models. Also, identification of the abnormalities in peripheral sensory fibers on histological level, and evaluation of the function in nerve system conduction and mitochondrial metabolism on the molecular levels were taken together into account in our present study.
The morphological changes in renal tissue and sciatic nerve were observed to evaluate the roles of α-KL at histological level. We found that uremia is a proinflammatory state, observed by H & E staining in renal tissue, while α-KL played a positive anti-inflammatory effect on uremic renal tissue. Moreover, the H & E staining of sciatic nerve showed that the internal environment of fibers changed with sciatic nerve injury, which directly inhabited the regeneration of peripheral nerves, while α-KL played a positive role in the regeneration of peripheral nerve.
Na+, K+-ATPase couples ATP hydrolysis (ATPase), discovered as an energy transducing ion pump, played an fundamental role in maintaining Na+ and K+ gradients and were involved in the release and uptake of neurotransmitters [38,45]. ATP depletion leading to a compromise of axonal ion concentrations may result in axon degeneration [46,47]. Therefore, we considered it pertinent to determine ATPase and Na+, K+-ATPase activity to assess the pump function, which directly associated with the neuronal excitability, and the normal operation of the axons.
In mitochondrial metabolism, Succinate Dehydrogenase (SDH) plays a central role. Low level of SDH will not only contribute to decreased ATP production, but also forming more powerful reactive oxygen species (Mitochondrial ROS) . ROS reacted with lipids, proteins and DNA that could result in mitochondrial and cellular dysfunction, causing oxidative stress, inflammation and cell death [48-50]. The redox couples of nicotinamideadenine dinucleotide (NAD+/NADH) as well as nicotinamide adeninedinucleotide phosphate (NADP+/ NADPH) determined redox state in the cell. NADH and NADPH have been reported to serve as reducing molecules in energy metabolism, reductive biosynthesis, and anti-oxidation reactions . So it becomes more important to know the SDH and NAD(P)H levels that directly associated with Mitochondrial ROS generation.
Above all, at biochemical aspects, the levels of ATPase, Na+, K+-ATPase, as indices of nervous system conduction, while the levels of SDH, NADH and NADPH, as indices of mitochondrial ROS generation, were assessed in our article. Our study showed that significant decreased ATPase, Na+, K+- ATPase SDH activity, while obvious increased NADH and NADPH concentrations were observed, which demonstrates the blocking of nervous system conduction and the high generation of mitochondrial ROS. α-KL could markedly alleviate nerve damage through modulating the enzymatic levels that associated with the nervous system conduction and mitochondrial ROS generation. α-KL was effective in the recovery of nerve system conduction, and played an antioxidative stress effect on injured sciatic nerve induced by uremia.
Nuclear Factor-kB (NF-kB) is a transcription factor, and its characteristic ability is to respond to extracellular signals and translocate to the nucleus to up regulate transcription of specific genes . NF-kBp65 (a subunit of NF-kB) is activated by free oxygen radicals resulting in inflammation. NF-kBp65 cascade is widely accepted as one of the key players for inflammatory response. Activation of NF-kBp65 promotes the levels of inflammatory mediators. Moreover, caspase-3 has been widely accepted to serve as primary executioner for apoptotic death. Whether inflammation-related NF-KBp65 or apoptosis-related protein cleaved-caspase-3 would involve in the treatment of α-KL for damaged sciatic nerve remains unknown. In our present study, NF-KBp65 and Caspase-3 protein levels were evaluated by Western blot. Our results showed that α-KL could activate both of the two downstream signaling pathways. We found that α-KL could exert positive anti-inflammation and anti-apoptosis effects on the damaged nerve by significantly attenuating the expression of NF-KBp65 and caspase-3. Besides, activated NF-kB can also induce genes that regulate Caspase function . Apoptosis mechanisms of α-KL involve a caspase signaling cascade, suggesting that α- KL may act through signal NF-kBp65/caspace-3 signal transduction pathway. α-KL is a potential therapeutic agent for sciatic nerve inflammation and apoptosis induced by uremia.
Taken together, our present study suggested that: α-KL could significantly reduce sciatic nerve damage observed in H & E staining; improve neurocytes activity by regulating the related enzymatic levels associated with nerve conduction and mitochondrial ROS generation; and prevent inflammatory as well as apoptosis through inhibiting both NF-kBp65 and caspase-3 signaling pathways. These results suggested that α- KL involved in the recovery of nerve system conduction, the regeneration of peripheral nerve fibers, undergoing antioxidative stress, anti-inflammatory and anti-apoptosis pathways in the uremia-induced sciatic nerve damage. α-KL may represent a new therapeutic approach for the recovery and regeneration of injured sciatic never.