Identification of proteins potentially associated with renal aging in rats

Establishing a young - old rat kidney transplant model

As shown in Table 1, following kidney transplantation, perioperative and postoperative mortality increased stepwise from the Y→Y to the O→O group. Nonetheless, this rat renal allograft transplantation model had fewer complications and an improved survival rate, thereby providing a more practical and reliable model for further experiments.

As shown in Figure 1B, there was no significant difference in renal function between the O control (O→O) and O→Y rats or between the Y control (Y→Y) and Y→O rats.

Figure 1. The histological examinations result from each group in this study. (A) Schematic diagram of kidney transplantation. Y→Y, young rat with kidney transplanted from young rat (young control); Y→O, old rat with kidney transplanted from young rat; O→Y, young rat with kidney transplanted from old rat; O→O, old rat with kidney transplanted from old rat (old control). (B) Renal functionality (serum creatinine, serum BUN, proteinurine) in each group. (C) HE staining of kidney tissue samples from each group (×200). (D) PAS staining of kidney tissue samples from each group (×200). (E) Masson staining of kidney tissue samples from each group (×200);

Histological examination

Histological examination was performed using hematoxylin and eosin (HE), periodic acid Schiff (PAS) and Masson staining (Figure 1C-E). HE staining revealed the morphological changes within the kidney tissue. Aged kidneys exhibited increased glomerular volume and swelling of the tubular epithelium. PAS staining revealed marked glycogen deposition in the aged kidneys, indicating tissue damage. Masson staining was used to assess renal fibrosis. In each case, there was no significant difference between the kidneys used for O→O and O→Y transplantation or those used for Y→Y and Y→O transplantation

Senescence-associated β-galactosidase staining and p16 expression

Senescent cells are characterized by growth arrest, enlarged and flat cellular morphology, and an expression profile characterized by expression of senescence-associated genes. The most commonly used marker to identify senescent cells is β-gal activity. As shown in Figure 2A, levels of β-gal expression were lower in the O→Y transplanted kidney tissue than in O→O transplanted kidneys. Conversely, β-gal expression was higher in Y→O transplanted kidney tissue than in Y→Y transplanted kidneys. A similar profile was seen with p16, another biomarker of senescence (Figure 2B).

Figure 2. Levels of aging biomarkers. (A) SA-β-gal staining in each group (*p < 0.05). (B) Western blot showing p16 expression levels (*p < 0.05). (C) Results of specific ELISAs showing expression levels of TNF-α and IL-1β (*p < 0.05).

Inflammatory cytokine expression

Because renal aging is associated with chronic inflammation, we assessed expression of two inflammatory cytokines, TNF-α and IL-1β, in the four groups. We found that levels of TNF-α and IL-1β were higher in the Y→O than the Y→Y group, and were lower in the O→Y than the O→O group (Figure 2C).

Identification and quantification of differentially expressed proteins

Compared with the Y→Y group, we detected 391 differentially expressed proteins (181 upregulated proteins, 210 downregulated proteins) in the Y→O group. Compared with the O→O group, we detected 380 differentially expressed proteins (169 upregulated proteins, 211 downregulated proteins) in the O→Y. Sixty-six proteins were associated with anti-aging (downregulated in the Y→O vs. Y→Y and upregulated in the O→Y vs. O→O), and 73 proteins were associated with pro-aging (up-regulated in the Y→O vs. Y→Y and downregulated in the O→Y vs. O→O).

Gene Ontology (GO) term enrichment analysis

We uploaded all differentially expressed proteins (DEPs) to the online software DAVID to identify overrepresented GO categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The GO analysis showed that DEPs were significantly enriched in biological processes, including cellular iron ion homeostasis, lipid metabolic process, and negative regulation of cholesterol biosynthetic process. For cell components, the DEPs were enriched in extracellular exosome, extracellular space, mitochondrion, and others. In addition, a GO molecular function analysis showed that the DEPs were significantly enriched for proton-transporting ATP synthase activity, rotational mechanism, poly(A) RNA binding, peptide binding, and protein binding (Table 2).

KEGG pathway analysis

Table 3 contains the most significantly enriched KEGG pathways of the analyzed DEPs. The DEPs were enriched in metabolic pathways, Huntington's disease, carbon metabolism, Alzheimer's disease, propanoate metabolism, pyruvate metabolism, and glutathione metabolism.

Module screening from the protein-protein interaction network

A protein-protein interaction (PPI) network was constructed using the STRING database (Figure 3A). Using information from the database, we screened for the top 10 high-degree hub nodes. These hub proteins were SOD1, ENO1, BCL2, ATP5A1, SOD2, CAT, GPX1, ACTBL2, LCHB, and ATP5H. Among them, SOD1 was the highest-degree (12) node. Moreover, a total of 73 nodes and 99 edges were analyzed using the MCODE plug-in. The top 3 significant modules were selected (Figure 3B), and the functional annotation of the genes involved in the modules was analyzed. Enrichment analysis of most of the proteins in modules 1-3 is shown in Table 4. GO molecular function analysis showed that DEPs were mainly involved in oxidative stress, while the GO cellular component analysis showed that DEPs were mainly located in the mitochondria, and the KEGG analysis showed that the genes were mainly associated with the peroxisome pathway.

Figure 3. Module screening of a PPI network of DEPs. (A) Protein-protein interaction network of DEPs (STRING). (B) PPI network modules screened using the Molecular Complex Detection plug-in. Three PPI network modules were screened (1, 2 and 3).

SOD1 and the Ac-NF-κB signaling pathway in renal aging

Because SOD1 was the highest-degree node in the PPI network of DEPs, we verified its expression in vivo. We found that SOD1 expression was lower in the Y→O than the Y→Y group, and was higher in the O→Y than the O→O group. In addition, we observed that acetylated (Ac)-NF-κB showed the opposite expression profile (Figure 4A). In vitro. Sod1 was broadly expressed in renal cells, especially in tubular epithelial cells. When renal tubular epithelial cells isolated from the young and old rats were cultured in the same culture medium under the same culture conditions, Sod1 expression was lower in aged tubular epithelial cells (Figure 4C). This finding was consistent with our earlier proteomics results summarized above.

Figure 4. Expression of SOD1 and NF-κB in vivo and in vitro. (A) Expression of SOD1, p16 and NF-κB in kidney tissue from each group (*p < 0.05). (B) SA-β-gal staining of young and old tubular epithelial cells. (C) Expression of SOD1 and p16 in young and old tubular epithelial cells (*p < 0.05 vs young group). (D) SA-β-gal staining of tubular epithelial cells after the indicated interventions (*p < 0.05 vs control; #p < 0.05 vs SOD1 overexpression). (D) Expression of NF-κB in tubular epithelial cells after the indicated interventions (*p < 0.05 vs control).

We also observed that Sod1 knockdown led to increased Ac-NF-κB expression and that overexpression of SOD1 had the opposite effect. In both cases, total NF-κB was unchanged. (Figure 4D). Thus, Sod1 knockdown appears to promote cell senescence, while Sod1 overexpression appears to inhibit it.

Animals

Male F344 rats were purchased from the Si Bei Fu Laboratory Animal Company (Beijing, China). Rats were housed under specific pathogen-free conditions in the Experimental Animal Center of the Chinese General Hospital of PLA: 22 ± 1°C, 40% humidity, 12/12-h light/dark cycle, with free access to water. Young (3 months) and old (20 months) rats were used in this study. As shown in Figure 1A, the rats were divided into six groups: young and old controls and kidney transplanted from young rat to young rat (Y→Y), from young rat to old rat (Y→O), from old rat to young rat (O→Y), and from old rat to old rat (O→O). Rats were sacrificed 16 weeks after transplantation, and the transplanted kidney was further studied. All experimental protocols were approved by the Animal Care Committee of Chinese General Hospital of PLA.

Establishment of the model

Rats were anesthetized using isoflurane (5%, induction of anesthesia; 2.2% 0.3-0.6 L/min, during surgery; the concentration was adjusted according to the depth of anesthesia). Transplantation was performed with one kidney. After separating the renal vessels, the rats were heparinized (500 U/ml, 2 ml), and a renal protection fluid was used for hypothermic irrigation of the transplanted kidney. The transplanted kidney, vessels, ureter and bladder disc were then excised from the donor rat, after which the kidney was transplanted to the donee through vascular and urinary reconstruction. A rat might be both a donor and a donee. After surgery, the rats were placed on an insulation blanket and were intraperitoneally administered penicillin (400,000 U).

Histopathological analysis

Kidney slices were fixed in 10% formalin solution overnight. After automated dehydration through a graded alcohol series, transverse kidney slices were embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin-eosin and periodic acid-Schiff (PAS). Histological examinations were performed independently in a blinded fashion by two observers. Quantitative analyses of kidney tissue were done using Image-Pro software (Media Cybernetics Inc., Silver Springs, MD, USA) with 20 randomly selected ×200 fields per rat.

Senescence-associated β-galactosidase staining

Cryostat sections (4 μm) were mounted on glass slides and fixed in 0.2% glutaraldehyde and 2% formaldehyde at room temperature for 15 min. The sections were then washed in PBS, incubated in freshly prepared senescence-associated β-galactosidase (SA-β-gal) staining solution overnight at 37°C, counterstained with eosin, and examined under a microscope. An investigator blinded to the sample identity performed the image analysis. Quantitative analysis of SA-β-gal positive-stained areas was performed using Image-Pro software with 20 randomly selected ×200 fields per rat.

Western blot analysis

Protein concentrations were determined using a Pierce BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Aliquots of protein (50-100 μg) were separated with 6-16% SDS-PAGE, transferred to nitrocellulose membranes, blocked with blocking buffer for 1 h at room temperature, and incubated with primary antibodies at 4°C overnight. Blots were subsequently incubated with secondary immunoglobulins conjugated with horseradish peroxidase. Immunoreactive bands were visualized using enhanced chemiluminescence, and densitometry was performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Band intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA).

Sample collection and protein extraction

Kidney sample collection and protein extraction were performed according to a standard procedure [22].

Enzyme-linked immunosorbent assay (ELISA)

After collecting tissue samples from the transplanted kidneys, TNF-α and IL-1β levels were determined by using ELISA kits (R&D Systems Inc, Minneapolis, MN) according to the manufacturer’s instructions. Levels of the cytokines were normalized to the protein concentration in the lysate.

LC-MS/MS analysis

Digested peptide mixtures were pressure-loaded onto a fused silica capillary column packed with 3-μm Dionex C18 material (RP; Phenomenex). The RP sections with 100 Å were 15 cm long, and the column was washed with buffer A (water, 0.1% formic acid) and buffer B (acetonitrile, 0.1% formic acid). After desalting, a 5-mm, 300-μm C18 capture tip was placed in line with an Agilent 1100 quaternary high-performance liquid chromatograph and analyzed using a 12-step separation. The first step consisted of a 5-min gradient from 0% to 2% buffer B, followed by a 45-min gradient to 40% buffer B. Next, buffer B flowed in a 3-min gradient from 40% to 80% and 10-min of 80% buffer B. After a 2-min buffer B gradient from 80% to 2%, approximately 100 µg of a tryptic peptide mixture was loaded onto the columns and was separated at a 0.5 µL/min flow rate using a linear gradient. As peptides were eluted from the microcapillary column, they were electrosprayed directly into a micrOTOF-Q II mass spectrometer (BRUKER Scientific) with application of a distal 180°C temperature source. The mass spectrometer was operated in the MS/MS (auto) mode. Survey MS scans were acquired in the TOF-Q II with the resolution set to a value of 20,000. Each survey scan (50-2,500) was followed by five data-dependent tandem mass (MS/MS) scans at a 2 Hz normalized scan speed. Tandem mass spectra were searched against the mascot 2.1 (Local Host) RAT protein database. The search results were then filtered using a cutoff of 1% for a peptide false identification rate. Peptides with a Z score < 4 or Delta-Mass > 5 ppm were rejected. Furthermore, the minimum number of peptides used to identify a protein was set to 1. The default parameters for the Profile Analysis 2.0 software were used throughout the analysis.

Bioinformatics analysis

To analyze DEPs at a functional level, gene ontology (GO) enrichment and KEGG pathway analyses were performed using the DAVID online tool (https://david.ncifcrf.gov/). To evaluate the interactive relationships among DEPs, we mapped them to STRING (www.string-db.org/) and experimentally validated the interactions. Those with a combined score > 0.4 were selected as significant. PPI networks were constructed using Cytoscape software. The plug-in Molecular Complex Detection (MCODE) was used to screen the modules of the PPI network in Cytoscape. The criteria were set as follows: MCODE scores > 3 and number of nodes > 4.

Cell culture and transfections

Isolation and culture of rat primary tubular epithelial cells were performed as described previously [23]. Young and aging renal tubular epithelial cells were respectively isolated from young and old rat kidneys and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. After three passages, the cells were collected for experimentation. Small interfering RNAs (siRNAs) targeting Sod1, which is highly expressed in tubular epithelial cells, and a RNAi negative control duplex were purchased from Santa Cruz Biotechnology. The RNAi oligonucleotide and RNAi negative control duplex were transfected into cells as instructed by the manufacturer.

Statistical analysis

All data analyses were performed using SPSS software (ver. 18.0; SPSS, Chicago, IL, USA). Data are expressed as the mean ± SD. Comparisons among groups were made using analysis of variance. Values of p < 0.05 were considered significant.