5 Increased dynamin expression precedes proteinuria in glomerular disease 91 Glomerular Dnm1 and Dnm2 mRNA—but not dynamin protein area expression—are increased in Dahl rats prior to the onset of proteinuria Next, we measured glomerular dynamin expression in the Dahl salt-sensitive spontaneously proteinuric and hypertensive rat model (referred to here as simply Dahl rats) and the spontaneously hypertensive rat (SHR). Dahl rats under a low dietary salt intake developed significant proteinuria beginning at 6 weeks of age; in contrast, SHR rats did not develop proteinuria, even at 10 weeks of age (Figure 1A). Consistent with the spontaneous hypertension phenotype, both strains developed hypertension to a similar extent beginning at 8 weeks of age (Figure 1B). As Dnm2 is the ubiquitous and prevalent form of dynamin, we measured both glomerular Dnm2 and Dnm1 mRNA levels (Figures 1C and 1D respectively). When analysing only the Dahl group, no significant differences in glomerular Dnm2 mRNA levels were seen between at weeks 2, 6, 8, and 10. However, these levels were higher at 4 weeks of age than in all other time points (p < 0.0001 compared to weeks 2, 6, 8, and 10, Figure 1C). In SHR rats, a significant difference in glomerular Dnm2 levels was found between weeks 2 compared to weeks 8 and 10 (p < 0.001 and p < 0.01 respectively) and between weeks 4 and 8 (p < 0.05, Figure 1 C). A similar analysis of glomerular Dnm1 mRNA levels in Dahl rats shows a gradual increase from weeks 2 to 6 after which the levels decrease. This difference is significant between weeks 6 and 10 (p < 0.05, Figure 1D). SHR rats show very low expression of glomerular Dnm1 mRNA at week 2 which increases slightly, but not significantly up until 6 weeks (Figure 1D). None of the differences in mRNA levels at the different time points were significant in this group. When comparing dynamin mRNA levels between Dahl and SHR rats, the level of glomerular Dnm2 mRNA was significantly increased in Dahl rats compared to SHR rats at 2 and 4 weeks of age (Figure 1C, p < 0.05 and p < 0.0001 respectively). Glomerular Dnm1 mRNA was significantly higher in Dahl rats than in SHR rats at 2, 4, and 6 weeks of age (Figure 1D). Interestingly, the levels of both Dnm1 and Dnm2 mRNA at 2 weeks of age were higher in Dahl rats than in SHR rats, which is prior to the onset of proteinuria (i.e., at 6 weeks of age). Also when comparing the different age groups, animals that will become proteinuric (i.e., the Dahl rats) have an increase in glomerular Dnm2 expression prior to onset of proteinuria. When comparing PCR Ct values, Dnm2 mRNA levels were found to be over ten times higher than Dnm1 levels. With respect to dynamin protein levels, immunohistochemistry revealed that the protein was present at the tubular brush border and in podocytes in both Dahl and SHR rats (Figure 2C and D) at similar levels at both 4 and 6 weeks of age (i.e., after Dahl rats develop proteinuria) (p = 0.6, Figure 2G).
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