3. Diseño metodológico
4.2 Entrevistas de profundidad
Three different mouse models of NAFLD were investigated with respect to their Agxt expression and their daily urinary oxalate excretion. The Table below summarizes some important results and differences among those models.
Table 4.1: Summarized information of three different mouse models of NAFLD compared to corresponding control mice. Numbers indicate the fold change compared to control mice. Data are shown in the appropriate
figures.
Fold change ob/ob db/db Western diet
Body weight 1.9 1.7 1.3
Liver to body ratio 1.7 ns 1.4
Agxt expression - 2.4 ns - 3.0
Hao1 expression - 1.6 ns ns
Urine volume 4.4 4.4 - 2.5
Hyperoxaluric Yes No No
ns: not significant
The comparison of these mouse models of NAFLD revealed important differences. Of the three models, the leptin deficient ob/ob mice (age: 15 – 18 weeks) was the only one showing a reduction in hepatic Agxt expression (fold change -2.4 ± 0.7, data not shown) accompanied by an increased daily excretion of urinary oxalate. Of note, the oxalate concentration in the ob/ob mouse urine was lower than that of ob/+ mice, but when the urinary volume, estimated by the creatinine level, was considered, oxalate excretion per day was significantly higher (Figures 3.31 D and 3.33 D). Surprisingly, Agxt expression was not downregulated in the liver of the db/db mice (age: 10 weeks). In contrast, mice fed a Western-type diet for 30 weeks (age: ca 40 weeks) showed a strong and significant downregulation of Agxt, but no increased daily excretion of oxalate.
The reason for the lack of downregulation of Agxt in the liver of db/db mice at the examined age of 10 weeks can only be speculated: The liver tissue of db/db mice showed only 3.5 fold more hepatic triglycerides than the corresponding controls; whereas, the ob/ob mice had 16 fold more triglycerides than their corresponding controls at the age of 10 weeks (Figure 3.1). This could indicate that Agxt downregulation is only triggered after a certain amount of lipid storage is reached. Likewise, db/db mice displayed no increased liver to body weight ratio, and thus no liver hypertrophy, in contrast to ob/ob mice, suggesting that the db/db mice exhibit a
milder phenotype than the ob/ob mice. It cannot be excluded that as they age, db/db mice display reduced Agxt expression. For example, in a proteomics study, 48 weeks old db/db mice were reported to have less hepatic mitochondrial Agxt compared to control mice (Nesteruk et al. 2014).
The mice on the Western diet already had reduced liver Agxt levels after 6 weeks on the diet, and this level stayed reduced until week 30 on the diet (Figure 3.40). Analysis of publically available data (GSE38141) from an earlier study also showed the downregulation of Agxt mRNA in mice after 20 weeks on the Western diet (Kobori et al. 2011).
Interestingly, despite a strong downregulation of Agxt in the liver after 30 weeks on the Western diet, these mice did not excrete higher levels of oxalate compared to their controls. Even though they had higher urinary oxalate concentrations – most likely due to lower urine volume - the daily excretion of oxalate was similar to mice on the control diet (Figure 3.42 D). Thus, in contrast to the ob/ob mouse model, downregulation of Agxt upon Western diet does not correlate with increased oxalate excretion. The reason for this discrepancy is not understood. Here, both RNA and protein levels of Agxt were measured, but not the (remaining) enzymatic activity. Despite the reduced Agxt expression, residual Agxt activity working together with the glyoxylate-detoxifier enzyme Grhpr may be enough to remove endogenous glyoxylate and, as a result mice on the Western diet with reduced Agxt expression do not excrete more oxalate. To investigate this further, urinary glycolate should be measured, which might be elevated in urine of mice on the Western diet due to increased glyoxylate detoxification by Grhpr.
In addition to Agxt, the expression of the glycolate oxidising enzyme Hao1 was only reduced at the RNA level (fold change -1.6 ± 0.2, data not shown) in ob/ob mouse livers, which is indicative of reduced endogenous peroxisomal glyoxylate production from glycolate in ob/ob mice. Hao1 plays an important role in endogenous glyoxylate production in mice, as shown in several publications (Dutta et al. 2016; Li et al. 2016; Martin-Higueras et al. 2016). Agxt knockout mice with impaired transcription of Hao1 excrete less oxalate than Agxt knockout mice with normal transcription of Hao1 (Dutta et al. 2016; Li et al. 2016). This observation supported an earlier report where ob/ob mice excreted less oxalate upon ethylene glycol challenge than ob/+ mice (Taguchi et al. 2015). The fact that inactivation of Hao1 rescues the
phenotype of the Agxt knockout mice highlights the importance of hepatic peroxisomal production of glyoxylate from glycolate in the pathogenesis of kidney stones.
In addition to oxalate excretion, the hepatic glycine content of ob/ob and ob/+ mouse livers was also analysed. As glyoxylate is transaminated by Agxt to glycine, the hepatic glycine concentration can partly indicate the functionality of Agxt. As shown by HR MAS 1HNMR, livers
of ob/ob mice had less glycine than those of the lean control mice (Figure 3.29). These findings support those of a previous study where a metabolite profiling of plasma from 20 weeks old ob/ob and db/db mice indicating reduced concentration of glycine compared to corresponding control mice (Giesbertz et al. 2015). Glycine is involved in multiple processes, such as collagen formation, the generation of purines, and glutathione production (Wang et al. 2013). The synthesis of glycine from glyoxylate is, at least in humans, a minor but relevant pathway, contributing to the endogenous production of glycine (Melendez-Hevia et al. 2009). The reduced hepatic glycine concentration in the livers of ob/ob mice, accompanied by the slightly increased oxalate excretion strengthens the assumption that the reduced expression of Agxt has physiological consequences in the ob/ob mouse model.