ANDREA RODRÍGUEZ. Secretaria de Comisión
5.- PLENARIO INTERJUNTAS DE COMISIONES DEL ÁREA METROPOLITANA
defects of the glomeruli and podocyte injury – study I
E1-DN mice express a truncated EGF-R in β-cells of pancreatic islets under the pancreatic duodenal homeobox-1 promoter (369). Due to impaired epidermal growth factor signalling, the postnatal growth of the β-cell mass is prevented, and the homozygous E1-DN mice have persistent hypoinsulinaemia (369). As a result, the homozygous mice present hyperglycaemia at the age of two weeks, which tends to decrease over time but remains higher than in their wildtype littermates (369). In (369), the authors focused on the description of the mouse line, the altered β-cell function and establishment of diabetes. Here, we evaluate the development of the diabetic kidney complication in this transgenic model of hyperglycaemia.5.1.1. E1-DN mice are hyperglycaemic and albuminuric
As the onset of DKD occurs after weeks of hyperglycaemia, we used mice from 6 to 57 weeks of age to study the loss of kidney function in homozygous males. First, we confirmed that the homozygous E1-DN mice used for our study were hyperglycaemic at all time points analysed. We observed a tendency of improvement of the blood glucose over time (study I, figure 1a). Also, as in (369), heterozygous males had elevated blood glucose at 6 weeks but the level was normalised by the age of 40 weeks. Specifically, the blood glucose of homozygous male mice ranged from 30 mM at 6–7 weeks to 18 mM at 40 weeks. The heterozygous mice had 18 mM blood glucose at 6–7 weeks and 11 mM at 40 weeks, whereas the wildtype mice had 14 mM at 6–7 weeks and 10 mM at 40 weeks (study I, figure 1a). This confirms that E1-DN mice develop early- onset diabetes.
Next, we analysed the 24 h urine volume and its albumin content as a marker of the overall kidney function. In homozygous mice, the excreted urine volume in 24 h was elevated at all time points analysed compared to wildtype mice (although the statistical significance was not reached at all time points, study I, figure 1b). In heterozygous mice, however, the 24 h urine volume was elevated at weeks 6–7 but normalised by week 40 (study I, figure 1b), possibly reflecting the glycaemia (study I, figure 1a). The albumin content of the urine samples was measured and the AER was calculated for 24 h at 10 and 20 weeks. Homozygous E1-DN mice exhibited increased AER at both 10 and 20 weeks compared to both wildtype and heterozygous littermates (study I, figure 1c). At 20 weeks, the albuminuria was extremely high, in the range of milligrams per day. Strikingly, a strong variation was observed in the homozygous group in both blood glucose and AER, and we found a significant correlation between the blood glucose levels and the AER (r = 0.71, p < 0.001, study I figure 1d). Therefore, the elevation of the albumin concentration in the urine of homozygous and hyperglycaemic E1-DN mice justified more in-depth examination of the kidney phenotype.
5.1.2. Histological and structural changes in the kidneys of E1-
DN mice
To characterise the morphological defects that could lead to the increased AER, we performed histological and proliferation analysis of the tubular compartment of the kidney, and evaluated the structural changes occurring in the glomeruli.
In the tubular compartment, haematoxylin-eosin staining revealed flattened epithelial cells and enlarged tubular lumens in homozygous mice with an AER superior to 1000 µg / 24h (study I, figure 1a, b). Using Ki-67 staining, we found that the proliferation of the tubular cells was increased from 0.18% to 0.70% in these mice, compared to wildtype littermates (study I, figure 3c, d). Such increase in proliferation is often considered as a sign of a repair process (385) and is in line with possible atrophy of tubular cells.
In the rest of this study, we focused on examining glomerular defects. In homozygous E1-DN mice, the mesangial area, defined as mesangial cells, extracellular matrix and the GBM, was found to be increased by 25% or 22% using PAS staining or evaluation by electron microscopy, respectively (study I, figure 2). In the two homozygous mice with the highest albuminuria, the accumulation of mesangial matrix was classified as focal, global nodular sclerosis (study I, figure 2d). Using electron microscopy, we also found that in homozygous E1-DN mice, the foot processes were wider (613 nm vs 423 nm, study I, figure 6e) and the GBM thicker (370 nm vs 258 nm, study I, figure 6d) than in their littermate controls. Occasional bulging of the GBM was also observed (study I, figure 6b). These morphological changes are reminiscent of the pathological findings of the human DKD.
5.1.3. E1-DN mice show increased podocyte apoptosis and
decreased nephrin expression
To characterise the glomerular defects of the homozygous E1-DN mice in more detail, we measured the apoptosis rate in the glomeruli. We stained kidney sections of 20 and 57 weeks old E1-DN mice for cleaved caspase-3. At both age groups, increased glomerular apoptosis was found in homozygous mice compared to heterozygous and wildtype mice. At 20 weeks, the number of apoptotic cells per 10 glomeruli increased from 1.0 in heterozygous and wildtype mice to 6.5 in homozygous mice. At 57 weeks, the number of apoptotic cells raised to 4.4 in wildtype and heterozygous mice, and to 9.8 in homozygous mice (study I, figure 4a–c). Using double immunofluorescence with nephrin, we found that the glomerular cells positive for cleaved caspase-3 were podocytes (study I, figure 4d).
As nephrin has been shown to be decreased or mislocalised in DKD (231-234), and is necessary for normal glomerular function (22, 23), we compared its level of expression and localisation in homozygous E1-DN mice and wildtype littermates. The overall pattern of nephrin staining remained unchanged in homozygous E1-DN mice. However, the staining intensity was decreased in homozygous mice with albuminuria (study I, figure 5). As opposed to nephrin, the expression level of neither podocin nor ZO-1 was altered (not shown). Altogether, we show that in this model of
hyperglycaemia that shows signs typical of DKD, podocyte physiology is disturbed, resulting in reduced expression of nephrin and podocyte death.
To conclude, we found that homozygous and hyperglycaemic E1-DN mice develop serious renal complications that is reminiscent of human DKD with albuminuria, alterations in the glomerular ultrastructure, increased rate of podocyte apoptosis, and a reduction in nephrin expression.