Figure 1.
Male 16-wk LP (n = 10) offspring body and kidney weights compared to age-matched control (n = 10).
(a) Birth weight; (b) Adult (16-wk-old) offspring; (c) Right kidney weight in 16-wk-old LP compared to NP rats; and, (d) Left kidney weight in 16-wk-old animals.**p = 0.005, ***p<0.0001.
Figure 2.
Daily urinary protein excretion in 16-wk-old LP offspring (n = 10) compared to appropriated age-matched controls (n = 10).
Figure 3.
Effect of maternal low protein diet on offspring glomeruli (from 5 LP rats) ultrastructure A: Three-dimensional organization of the outer surface of podocytes (p) surrounding capillaries in a control rat (from 5 NP rats).
a: Detail of a podocyte showing primary (1) and secondary (2) processes and pedicels (arrow) among which filtration slits can be seen. B: LP rat glomerulus with an intensive cohesive arrangement and bulbous and crushed podocytes b: Note the irregular surface of cell body with enlarged processes, pseudo cists formation (*), width and club-shaped pedicels and reduced number of filtration slits.
Figure 4.
Transmission electron micrographs showing: A) the filtration barrier from NP glomeruli (from 5 rats) showing all the structural components of the filtration barrier, including the fenestrations of the glomerular endothelium (arrowheads), the GBM, the podocyte pedicels (pp) and the filtration slits crossed by tenuous diaphragm membranes (arrows).
B–F) the filtration barrier from LP glomeruli (from 5 rats) showing a drastic decrease in the number of pedicels and slit diaphragms. Irregular electron-dense masses occur in the podocyte cytosol. Note in B the increased thickness of GBM compared to what is observed in A–G) in LP glomeruli, we found PECs in the process of differentiating into podocytes.
Figure 5.
We found an intensive rise of the TGF-β1 expression in LP (B, from 5 rats) compared to NP glomeruli (A, from 5 rats).
By western blot from total renal tissue extract we also found enhancement in the expression of this protein in LP offspring (C).
Figure 6.
Immunohistochemistry and western blot showing reduction in the expression of glomerular epithelial markers.
We can observe an extensive immunoreactivity for nephrin along NP glomerulus (from 5 rats) (A) compared to weak immunostained glomerulus in LP (from 5 rats) (B). E-cadherin glomerular expression was also reduced in LP (D) compared with NP(C) glomeruli. By western Blot from total renal tissue extract we found reduced expression of podocin (E, 39%) and nephrin (F, 62%) in LP.
Figure 7.
Immunohistochemistry showing enhanced glomerular expression of mesenchymal markers.
We verified that fibronectin (B), type I collagen (D) and desmin (F) immunoreactivity are intensively enhanced in LP (n = 5) when compared to that observed in NP (n = 5) (A, C and E, respectively).
Figure 8.
Immunohistochemistry showing enhanced glomerular expression of ZEB 2, an EMT-related mediator.
In NP (n = 5), we verified little immunoreactivity for ZEB2 in podocytes and none in PECs (A). In LP (n = 5), both podocytes and PECs (arrows) in the vascular stalk present intensive reactivity (B).
Figure 9.
Expression of the miR-200 family and miR-192.
Expression levels of mir-192, mir-141, mir-200a, mir-200b, mir-200c and mir-429 estimated by TaqMan RT-qPCR in isolated glomeruli of 16-wk old LP rats. The expression of each microRNA was normalized for U6 and U87 genes. Data are expressed as fold change (mean ± SD, n = 5) relative to control group (n = 5). *Significantly different from control group (P≤0.05).
Figure 10.
Expression levels of col 1α1, col 1α2, desmin, E-cadherin, fibronectin, TGF-β1, ZEB1, ZEB2 and ZO-1 estimated by SyBR green RT-qPCR in glomeruli of LP rats. The expression of each mRNA was normalized for GAPDH, β-actin and TBP genes. Data are expressed as fold change (mean ± SD, n = 5) relative to control group (n = 5). *Significantly different from control group (P≤0.05).
Figure 11.
Schematic representation of proposed fetal programming consequences in cells of the renal corpuscles from adult rats.
Gestational low protein diet leads to a reduction of 27% in nephron number and adult hypertension. We suppose that glomeruli overload is an initial insult that starts a cascade of events including an early inflammatory phase followed by a fibrogenic response. The downregulation of the microRNA-200 family, in response to enhanced TGFβ 1 expression, results in the reduction of epithelial markers and enhancement in mesenchymal markers and in ZEB 2, an EMT mediator. Podocytes will undergo EMT to escape apoptosis, which results in podocyte simplification, which leads to an impaired glomerular filtration barrier ensuring the onset of proteinuria. Depending of the severity and duration of injury, there may be a podocyte loss exacerbating proteinuria. PECs will also undergo EMT for differentiation into new podocytes in an attempt to attenuate the glomerular function loss (modified from Liu, 2010) [5].
Table 1.
Gene sequence studied in isolated glomeruli.