The metabolic profile of a rat model of chronic kidney disease

Animals

Animal care and experiments were approved by the Institutional Animal Care and Use Committee of Kyoto University (MedKyo 14183) and conducted following the Guide for Care and Use of Laboratory Animals published by the United States National Institutes of Health. Inbred male DS rats (Japan SLC, Hamamatsu, Shizuoka, Japan) were fed a 0.3% NaCl (low-salt; LS) diet until the age of 6 weeks, after which they were fed an 8% NaCl diet (high-salt; HS) (Inoko et al., 1994; Kato et al., 2010; Kawamoto et al., 2015). The rats in the current study were the same animals used in our previous report (Tanada et al., 2015). Therefore, the characterization of hemodynamics in these rats has already been reported.

Protocols

DS rats fed only an LS diet (LS group) were used as controls, and DS rats fed an HS diet (HS group) were used as a model of renal failure. There were four groups of rats categorized by the amount of salt in the diet (LS or HS) and week of sacrifice (11 weeks or 21 weeks): the 11-week-old LS group (n = 8), 11-week-old HS group (n = 8), 21-week-old LS group (n = 12), and 21-week-old HS group (n = 11). Serial measurements of heart rate and blood pressure were performed every two weeks from the age of 11 weeks until sacrifice at 21 weeks.

Blood and tissue sampling

The rats were anesthetized with inhaled diethyl ether (Wako Pure Chemical Industries, Osaka, Japan) and sacrificed by decapitation without fasting. Blood samples were collected via the right atrium in tubes and centrifuged at 1,500 rpm for 10 min, and blood serum was taken from the supernatant. Measurement of serum creatinine and blood urea nitrogen (BUN) was entrusted to SRL Inc. (Kyoto, Japan). Both kidneys were rapidly removed, snap frozen in liquid nitrogen, and stored at −80°C or fixed with 4% paraformaldehyde (PFA).

Cardiac echocardiography

Transthoracic echocardiography was performed as previously reported (Kato et al., 2010). Rats were anesthetized briefly with inhaled diethyl ether (Wako Pure Chemical Industries, Osaka, Japan), and transthoracic echocardiography was performed using a Sonos-5500 echocardiograph (Agilent Technologies, Santa Clara, CA, USA) with a 15-MHz linear transducer. Intraventricular septal thickness (IVSd), left ventricular dimension in the diastolic phase (LVDd), and left ventricular dimension in the systolic phase (LVDs) were measured with M-mode echocardiography, and fractional shortening (FS) was calculated.

Quantitative reverse transcription-polymerase chain reaction

Total RNA was isolated from the kidneys in each group by the acid guanidinium thiocyanate-phenol-chloroform method. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed as in previous reports (Inuzuka et al., 2009; Niizuma et al., 2012). The oligonucleotide primers are listed in Table S1. The mRNA level of each gene was standardized using the expression level of the 18S ribosomal RNA as a control.

Fibrosis of the kidneys

The kidneys were fixed in 4% PFA, embedded in paraffin, and sectioned for histological evaluation. The fibrotic area was quantified in tissue sections with Sirius Red staining, as previously described (Okuda et al., 2013).

Metabolomic analysis of the kidneys

The kidneys were subjected to metabolomic analysis as described previously (Kato et al., 2010) using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) (Soga et al., 2003). Briefly, frozen kidney tissues were immediately plunged into methanol (0.5 ml) containing internal standards (300 μM each of methionine sulfate for cations and MES for anions) and homogenized for 3 min to inactivate enzymes. 200 μl of deionized water and 500 μl of chloroform were added, and the mixture was thoroughly mixed. The solution was centrifuged at 15,000 rpm for 15 min at 4°C and the 600 μl upper aqueous layer was filtered centrifugally through a Milipore-5 kDa cutoff filter to remove proteins. All CE-TOFMS was performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Santa Clara, CA). We measured the levels of metabolites in glycolysis and the tricarboxylic acid (TCA) cycle, and amino acids and their derivatives.

Western blotting

Western blotting of kidney tissues was performed as described previously (Tanada et al., 2015). The primary antibodies used for Western blotting were as follows: NDUFA9 (1:1,000, Santa Cruz, Dallas, TX, USA) and GAPDH (1:1,000; Cell Signaling, Danvers, MA, USA).

Statistical analysis

Values are expressed as means ± the standard error of the mean (SEM). ANOVA was used for comparisons multiple groups. In all tests, a value of p < 0.05 was considered statistically significant.

Dahl salt-sensitive rats fed a high-salt diet show a high heart rate, systolic blood pressure, and cardiac dysfunction

As we previously reported (Tanada et al., 2015), DS rats fed an HS diet (HS group) developed hypertension, showed cardiac dysfunction, and died from heart failure. DS rats fed an LS diet (LS group) did not develop hypertension, and were used as controls. The heart rate was significantly higher in the HS group compared to the LS group from 11 weeks of age to 19 weeks of age (Fig. S1A). Conversely, systolic blood pressure was significantly higher in the HS group compared to LS group from 11 weeks of age to 21 weeks of age (Fig. S1A). At 21 weeks of age, intraventricular septal thickness (IVSd) increased in the HS group on echocardiographic examination. In addition, left ventricular dimension in the diastolic phase (LVDd) increased, and fractional shortening (FS) decreased in the HS group compared to that of the LS group (Fig. S1B).

The HS group had a high concentration of serum creatinine and blood urea nitrogen at 21 weeks of age

There was no significant difference in the concentration of serum creatinine or BUN between the LS group and HS group at 11 weeks of age (Fig. S1B). However, the HS group showed a significantly higher concentration of both creatinine and BUN compared to the LS group at 21 weeks of age (Fig. S1C).

Fibrosis in the kidney and gene expression related to the glomerulus, renal tubules, and fibrosis

At 21 weeks of age, the area of fibrosis in the kidney was significantly increased in the HS group compared to the LS group. There was no difference between the HS and LS groups at 11 weeks of age (Figs. S2A and S2B).

Nephrin and podocin are extracellular components of the slit diaphragm coded by Nphs1 and Nphs2, respectively (Boute et al., 2000; Ruotsalainen et al., 1999). The expression of Nphs1 was significantly decreased in the HS group compared to the LS group at 21 weeks of age (Fig. 1A). Though the expression of Nphs2 decreased in the HS group at 21 weeks of age compared to 11 weeks of age, there was no difference between the HS and LS groups at 21 weeks of age (Fig. 1B).

The gene expression of kidney injury molecule-1 (Kim1) indicates the degree of interstitial fibrosis, renal tubular damage, and inflammation (Van Timmeren et al., 2006). Neutrophil gelatinase–associated lipocalin (NGAL) is also associated with renal tubular damage in ischemic or inflammatory responses (Mori et al., 2005). In the present study, the expression of Kim1 and Ngal was significantly higher in the HS group than the LS group at both 11 and 21 weeks of age (Figs. 1C and 1D). In addition, in the HS group, the expression of these genes was significantly increased at 21 weeks of age compared to 11 weeks of age (Figs. 1C and 1D).

The expression of the osteopontin gene increases in rat renal fibroblasts with aldosterone (Irita et al., 2008) and in the kidneys of hypertensive rats (Blasi et al., 2003). Similarly, the expression of type 1 collagen (collagen 1) and α-smooth muscle actin genes (αSMA) increases in renal fibrosis (Lekgabe et al., 2005; Wang et al., 2008). In the present study, expression of these genes was significantly higher in the HS group than the LS group at both 11 and 21 weeks of age (Figs. 1E–1G). The expression of these genes increased significantly at 21 weeks of age compared to 11 weeks in the HS group only (Figs. 1E–1G).

Gene expression related to mitochondrial function, glycolysis, and free fatty acid metabolism in the kidneys

We examined the expression level of some genes related to mitochondrial function, glycolysis, and fatty acid metabolism at 11 and 21 weeks of age. Gene expression related to glycolysis was measured, and the expression of hexokinase (Hk) 1 and Hk2, which catalyze the transfer from glucose to glucose-6-phosphate (G6P), significantly increased in the HS group compared to the LS group at 21 weeks of age. Conversely, the expression of Tp53-induced glycolysis and apoptosis regulator (TIGAR), which alters the concentration of fructose 2,6-bisphosphate (Bensaad et al., 2006), was significantly decreased in the HS group compared to the LS group at 21 weeks of age (Table 1).

Peroxisome proliferator-activated receptor γ coactivator1-α (PGC1-α) is a master regulator of mitochondrial biogenesis, though there was no change between the LS and HS group (Table 1 and Fig. 2A). Proliferator activated receptor α (PPARα), nuclear respiratory factor 1 (NRF1), and NRF2 are related to mitochondrial biogenesis. Pparα expression was significantly decreased and Nrf1 expression was significantly increased in the HS group compared to the LS group at 21 weeks of age. Otherwise, compared to that in the LS group, Nrf2 expression was significantly increased at 11 weeks of age and decreased at 21 weeks of age in the HS group (Table 1 and Fig. 2).

Subsequently, we measured the expression of genes related to oxidative phosphorylation. Though uncoupling protein 3 (UCP3) is known for uncoupling ATP synthesis from oxidative metabolism, its transcription is regulated by PPARα (Young et al., 2001). In this study, like Pparα expression, Ucp3 expression decreased significantly in the HS group compared to the LS group at 21 weeks of age. The expression levels of several genes related to the respiratory chain also changed in the HS group; NADH dehydrogenase 4 (Nd4), alpha-subcomplex 9 (αS9), succinate dehydrogenase b (Sdhb) and cytochrome-c (Cyt-c) expression significantly decreased in the HS group from that in the LS group at 21 weeks of age. Conversely, in the HS group, expression of cytochrome c oxidase 1 (Cox1) decreased at 11 weeks of age and increased at 21 weeks of age, and Cox4 increased significantly at 21 weeks of age (Table 1 and Fig. 2).

Medium-chain acyl-CoA dehydrogenase (MCAD) is an enzyme related to fatty acid oxidation, and its expression decreased significantly in the HS group compared to the LS group at 21 weeks of age. ATP-binding cassette sub-family A member 1 (ABCA1) has a critical role in cellular cholesterol and phospholipid efflux, and its gene expression was significantly increased in the HS group compared to the LS group at 21 weeks of age (Table 1).

Metabolomic profile of the kidney

To examine energy metabolism in renal damage, we performed a comprehensive metabolomic analysis. The results of the quantification showed metabolites related to glycolysis, including the TCA cycle, and amino acids and their derivatives (Fig. 3 and Table S3). By two-way ANOVA analysis, we assessed the significance (p value) for time (11 weeks and 21 weeks) and treatment effects (LS diet and HS diet). The metabolites that were not significantly different (p ≥ 0.05) regarding time or interaction effects are only representedby the bar plots of 21-week-old rats. Other metabolites are represented by bar plots of 11-week-old and 21-week-old rats (Fig. 3). The metabolites of kidney tissue significantly changed in the HS group from that in the LS group. Especially, the increased urea and creatinine levels at 21 weeks of age validated that the metabolome analysis was performed successfully.

The levels of 2,3-diphosphoglycerate (2,3-DPG) and phosphoenolpyruvate (PEP), intermediate metabolites of glycolysis, decreased in the HS group at 21 weeks of age. The levels of several amino acids and derivatives such as serine, betaine, alanine, histidine, citruline, proline, arginine and putrescine, increased in the HS group at 21 weeks of age. On the contrary, taurine, β-alanine, and glutamate were decreased in the HS group at 21 weeks of age. Levels of several metabolites in the TCA cycle also changed; the levels of citrate, cis-acotinate and isocitrate increased, and the levels of succinate and acetyl CoA decreased (Fig. 3 and Table S3).

Subsequently, we measured the gene expression of enzymes that are involved in the TCA cycle (Fig. 4): aconitase is encoded by Aco2, which catalyzes the reversible isomerization of citrate and isocitrate; 2-oxoglutarate dehydrogenase (OGDH) is an enzyme that catalyzes the oxidation of 2-oxoglutarate; succinyl-CoA synthetase (SCS) is encoded by the gene Suclg1, and is an enzyme that hydrolyzes succinyl-CoA into succinate and CoA-SH; fumarate hydratase is encoded by the Fh gene and is the enzyme that catalyzes the hydration of fumarate to malate. The gene expression of Aco2, Ogdh, Suclg1, and Fh was significantly decreased in the HS group compared to the LS group at 21 weeks of age (Fig. 4).

Furthermore, the amounts of nicotinamide adenine dinucleotide (NAD+) and NADH were measured on metabolome analysis. NAD+ plays a central role in cellular energy metabolism and energy production. The NAD+/NADH ratio is often considered as the key of mitochondrial function. Cytoplasmic NAD+/NADH ratios range between 60 and 700, and mitochondrial NAD+/NADH ratios are maintained at 7–8 (Stein & Imai, 2012). The reduction of NAD+/NADH ratio has been reported in renal injuries in a type 1 diabetic mouse model (Zhu et al., 2014). In the present study, both NAD+ and NADH levels significantly decreased at 21 weeks of age, while NAD+/NADH ratio showed no change between the HS group and LS group (Fig. S3B). It is well known that mitochondrial Complex I catalyzes the first step of NADH oxidation, and increases the NAD+/NADH ratio. Therefore, we measured the protein expression of NADH dehydrogenase ubiquinone 1 α subcomplex subunit 9 (NDUFA9), a subunit of mitochondrial Complex I, at 21 weeks of age, because the level of mRNA expression of NDUFA9 (αS9) was significantly decreased in the HS group (Table 1 and Fig. 2D). However, there was no significant change between the LS group and HS group (Fig. S3B).