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In addition to the genetic mouse models mentioned above, liver tissues were collected and analysed from mice, which were fed with a Western diet, a type of high fat diet, for several weeks. After 6, 12, 18, 24, and 30 weeks on the Western diet, five mice per group were sacrificed and liver tissues were collected. On the 29th _{week of diet, four mice per group were}

placed in a single mouse metabolic cage to collect 24 h urines and to analyse the urinary oxalate concentrations in these mice. This diet study was conducted by Dr. Ahmed Ghallab who kindly allowed us to collect urine as well as liver tissue for RNA analysis, and liver sections for histopathological characterisation.

3.9.2.1 Western diet induces hepatic lipid accumulation and displacement of nuclei

As illustrated in Figure 3.39 A, mice on the Western diet exhibited a time dependent increase in body and liver weight. The liver to body weight ratio was elevated after eighteen weeks on the Western diet compared to those mice on the normal diet. In addition, H&E staining of paraffin embedded liver slides showed an accumulation of intracellular lipid droplets in hepatocytes of the pericentral and middle zone. Moreover, several hepatocytes had nuclei that were shifted towards the periphery of hepatocytes (exemplified by yellow arrows in Figure 3.39 B).

Figure 3.39: Western diet induces a time-dependent increase in body and liver weight as well as hepatic lipid accumulation. A) Body and liver weights of mice on the normal or the Western diet (n = 5); B) H&E stainings of

paraffin embedded liver sections of mice on the normal or the Western diet for several weeks. The yellow arrows indicate examples of hepatocytes with a shifted nucleus. Scale bars represent 50 µm. ND: normal diet; WD Western diet; CV: central vein, PV: portal vein. * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

3.9.2.2 Western diet causes a repression of Agxt

After liver tissue was collected at the assigned time points, RNA was isolated and transcribed into cDNA. Afterwards, quantitative real-time analysis was performed to investigate the expression of Agxt and further genes associated with the glyoxylate metabolism. Moreover, IHC of paraffin embedded liver sections was performed to investigate the expression and distribution of Agxt in the liver tissue of mice which were on the normal or the Western diet for 24 weeks and 30 weeks.

The RNA level of Agxt was significantly reduced in livers of mice on the sixth week (fold change: -1.97 ± 0.34) of the Western diet, and stayed reduced until the end of the diet (fold change: - 2.97 ± 0.96; Figure 3.40 A). The staining of Agxt indicated reduced protein expression in livers of mice after 30 weeks on the Western diet (Figure 3.40 B).

Figure 3.40: Western diet reduces the expression of Agxt. A) Quantitative real-time PCR revealed a down-

regulation of Agxt in livers of mice after 6, 18 and 30 weeks on the Western diet relative to mice on the normal diet. Gapdh was used as the endogenous control (n = 5); B) Representative IHC indicated a reduction of Agxt protein level after 24 weeks (data not shown) and 30 weeks on the Western diet. Scale bars represent 200 µm. * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

The expression of further glyoxylate metabolism-associated genes was not altered as strongly or as early on the diet as seen for Agxt. Grhpr and Hao1 RNA expressions were not significantly reduced with the Western diet; however, there was a trend towards lower expression of Hao1 in liver of mice that were on the Western diet for 30 weeks (Figure 3.41).

All in all, Agxt was similarly downregulated in the liver of mice on the Western diet model as well as the ob/ob mouse model. In addition, Hao1 RNA expression was slightly but significantly downregulated in ob/ob mouse livers; whereas, only a trend towards decreased expression was observed in the livers of mice after 30 weeks on the Western diet.

Figure 3.41: Mice on the Western diet tend to have a reduced Hao1 expression after 30 weeks of diet.

Quantitative real-time PCR showed no significant deregulation of Grhpr or Hao1 at the RNA level in livers of mice on the Western diet relative to those of mice on the normal diet at each time point. The expression of Hao1 showed a reduced trend at the RNA level after 30 weeks on the Western diet (p < 0.077). Gapdh was used as the endogenous control (n = 5).

3.9.2.3 Mice on the Western diet are not hyperoxaluric

After 29 weeks on the Western diet, four mice as well as four control mice were placed in a single mouse metabolic cage and 24 h urine was collected on two consecutive days. As illustrated in Figure 3.42, mice on the Western diet excreted less urine, which was accompanied by increased creatinine and oxalate concentrations. After normalization of the urinary oxalate concentration to the creatinine concentration, daily oxalate excretion was quite similar between the control group and the Western diet group. One explanation may be the unchanged expression of Grhpr, which might compensate for the lack in glyoxylate detoxification due to decreased Agxt expression.

Figure 3.42: Mice on the Western diet are not hyperoxaluric. A) Daily excreted urine volume; B) Urinary

creatinine concentration; C) Urinary oxalate concentration of mice on the normal or the Western diet (29 weeks on diet; n = 4; quantification by LC-MS/MS); D) Daily urinary oxalate excretion normalised to creatinine excretion (n = 4). ND: normal diet; WD: Western diet; ns: not significant; * indicates p < 0.05; ** indicates p < 0.01.