Assessment of Physiological Rat Kidney Ageing-Implications for the Evaluation of Allograft Quality Prior to Renal Transplantation

Abstract

Due to organ shortage and rising life expectancy the age of organ donors and recipients is increasing. Reliable biomarkers of organ quality that predict successful long-term transplantation outcomes are poorly defined. The aim of this study was the identification of age-related markers of kidney function that might accurately reflect donor organ quality. Histomorphometric, biochemical and molecular parameters were measured in young (3-month-old) and old (24-month-old) male Sprague Dawley rats. In addition to conventional methods, we used urine metabolomics by NMR spectroscopy and gene expression analysis by quantitative RT-PCR to identify markers of ageing relevant to allograft survival. Beside known markers of kidney ageing like albuminuria, changes in the concentration of urine metabolites such as trimethylamine-N-oxide, trigonelline, 2-oxoglutarate, citrate, hippurate, glutamine, acetoacetate, valine and 1-methyl-histidine were identified in association with ageing. In addition, expression of several genes of the toll-like receptor (TLR) pathway, known for their implication in inflammaging, were upregulated in the kidneys of old rats. This study led to the identification of age-related markers of biological allograft age potentially relevant for allograft survival in the future. Among those, urine metabolites and markers of immunity and inflammation, which are highly relevant to immunosuppression in transplant recipients, are promising and deserve further investigation in humans.

Keywords: ageing; inflammation; kidney; transplantation; urinary metabolites.

Figure 1

Urine analysis of 3- and 24-month-old rats. High levels of urinary albumin were detected in all eight 24-month-old rats by SDS-PAGE and silver staining (A), as well as by ELISA (B; p = 0.0002). The total protein level in urine was not significantly different at 3 and 24 months of age (B; p = 0.959). This might be explained by the presence of high levels of major urinary proteins (MUPs) in young male rats, as opposed to older rats (A), hence masking the detection of albuminuria in old rats in the BAC assay. ** p < 0.01 versus 3 months old rats.

Figure 2

Targeted profiling of urine metabolites of 3- and 24-month-old rats by NMR spectroscopy. All concentrations were normalized to creatinine [µmol/mmol]. p-values were determined by a two-sided Welch t-test. To account for multiple testing the false discovery rate was controlled at the 5% level. For all figures p-values ≤ 0.05, ≤0.01, and ≤0.001 are indicated by one, two or three asterisks, respectively.

Figure 3

(A). Fingerprinting analysis from urine of 3- and 24-month-old rats by 1D 1H NMR spectroscopy. Principal component analysis (PCA). Blue triangels and red dots denote young and old animals, respectively. (B). Heatmap of the 50 most regulated features. Metabolites up- and down-regulated relative to their respective means are shown in blue and red, respectively. Samples 1–8 originate from young animals (3 months), while samples 9–14 correspond to old animals (24 months). Metabolites that could be unambiguously assigned to a given feature are marked.

Figure 3

(A). Fingerprinting analysis from urine of 3- and 24-month-old rats by 1D 1H NMR spectroscopy. Principal component analysis (PCA). Blue triangels and red dots denote young and old animals, respectively. (B). Heatmap of the 50 most regulated features. Metabolites up- and down-regulated relative to their respective means are shown in blue and red, respectively. Samples 1–8 originate from young animals (3 months), while samples 9–14 correspond to old animals (24 months). Metabolites that could be unambiguously assigned to a given feature are marked.

Figure 4

Histomorphometric analyses on kidney sections of 3- and 24-month-old rats. (A) PAS-positive areas were significantly increased in 24-month-old rats (p = 0.007). (B) Sirius Red staining of collagen, was significantly increased in 24-month-old rats (p = 0.033). (C) Glomerular desmin staining was significantly increased in 24-month-old rats (p = 0.001). (D) The proportion of Ki-67-positive proliferating glomerular cells was significantly reduced in 24-month-old rats (p = 0.002). For all Figures p-values ≤ 0.05, ≤0.01, and ≤0.001 are indicated by one, two or three asterisks, respectively. Magnification x400.

Figure 5

Expression of TGF-β pathway-specific genes in kidneys of 3- and 24-month-old rats by RT-qPCR. Expression of TGF-β1, TGF-β2 and Smad2 was significantly higher in 24-month-old rats compared to 3-month-old rats, while expression of TGF-β3, Smad3, Smad4 and Smad7 was not significantly affected. Data was normalized with respect to the corresponding values determined in 3-month-old rats. For all figures p-values ≤ 0.05, and ≤0.01 are indicated by one or two, respectively.

Figure 6

Glomerular and interstitial infiltration of T cells in 3- and 24-month-old rats. (A) Immunohistochemistry with CD3 antibody (magnification ×200). Intraglomerular infiltration of CD3-positive T cells was significantly reduced in 24-month-old rats (B). In contrast periglomerular, peritubular, and periarterial infiltration of T cells was significantly increased in 24-month-old rats. Perivenous T cell infiltration was also increased in old rats, albeit not significantly. For all Figures p-values ≤ 0.05, ≤0.01, and ≤0.001 are indicated by one, two or three asterisks, respectively.

Figure 7

Expression of chemokines and adhesion marker genes in kidneys of 3- and 24-month-old rats by RT-qPCR. The expression of the chemokines CCL2 (MCP-1) and CCL5 (RANTES)(A) and the adhesion molecule ICAM1 (B) in the whole kidney of 24-month-old rats was significant increased, while expression of VCAM1 was comparable in 3- and 24-month-old rats (A,B). Data was normalized with respect to the corresponding values determined in 3-month-old rats. p-values ≤ 0.05, ≤ 0.01, and ≤0.001 are indicated by one, two or three asterisks, respectively.

Figure 8

Expression of endogenous TLR ligands and downstream signaling molecules. Data was normalized with respect to the corresponding values determined in 3-month-old rats. p-values ≤ 0.05 and ≤0.01 are indicated by one or two asterisks, respectively.

Figure 9

Expression of age-related renal metabolic markers in kidneys of 3- and 24-month-old rats by RT-qPCR expression of the anti-ageing kidney-secreted factor Klotho and of NFAT5, a transcription factor involved in the response to hypertonic and hypoxic stress in the kidney was not significantly altered in old rats. Expression of cysteine sulfinate decarboxylase (CSD) and cysteine dioxygenase (CDO), two enzymes involved in taurine biosynthesis, was significantly downregulated in 24-month-old rats. Similarly, expression of the taurine and betaine transporters, TauT and BGT1 respectively, was also reduced in old rats, albeit not significantly. Data was normalized with respect to the corresponding values determined in 3-month-old rats. p-values ≤ 0.05 are indicated by one asterisks.