To identify morphological and developmental changes in protein and RNA expression patterns during nephrogenesis in the rhesus monkey, immunohistochemistry and quantitative real-time PCR (qPCR) were utilized to analyze temporal and spatial expression of WT1, Pax2, Nestin, Synaptopodin, α-SMA, CD31, VEGF, and Gremlin.
Kidney development in rhesus monkeys follows a pattern similar to humans as the intermediate mesoderm differentiates to form the mesonephros and the definitive metanephric kidney in the first trimester () (Matsell and Tarantal, 2002
; Hiatt et al., 2010
). The nephrogenic zone comprises roughly half of the developing tissue in the late first trimester and gradually decreases as gestation advances in human and nonhuman primates. Mature glomeruli and proximal and distal tubules were noted as early as the late first trimester in monkey kidneys () similar to humans (Saxen, 1987
; Hiatt et al., 2010
). With the development of the medullary region in the mid-second trimester, glomeruli begin maturation in the cortical region (). Reciprocal inductive processes between the ureteric bud and the metanephric mesenchyme (blastema) triggers branching and elongation of the ureteric bud (future collecting system) and condensation and differentiation of the mesenchyme (future excretory component) (Saxen, 1987
). The condensed mesenchyme was noted to form renal vesicles that differentiated into comma or C- and S-shaped bodies. With migration of the vascular endothelial component into the cleft of the S-shaped bodies, glomerular tuft formation begins and is followed by further differentiation of the distal tail of the S-shaped body into parietal epithelium of the glomerular capsule. The proximal epithelium of the S-shaped body then differentiates into a columnar structural entity forming podocyte precursors in the visceral epithelium of the glomerulus. Similar to humans, the nephrogenic zone disappears by the late third trimester in rhesus monkeys (). In contrast to the mouse (Saxen, 1987
), postnatal kidneys do not show evidence of nephrogenesis in human or nonhuman primates (; ) (Matsell and Tarantal, 2002
Hematoxylin and Eosin (H&E) staining of sequential stages of kidney development
Time course of nephrogenesis and expression of renal developmental markers in human, monkey, and mouse
Early first trimester expression of WT1 was noted in the anterior-medial border of the neural crest, and in a region between the dorsal aorta and the mesonephros (Figs. , ). Diffuse WT1 expression was observed in the metanephric mesenchyme, but not the ureteric bud, in the mid-first trimester (). WT1 staining, while not apparent in the early renal vesicle, intensified as the renal vesicle differentiated into the C- and S-shaped bodies (Figs. , ). In the second trimester, strong WT1 expression was noted in the tall columnar visceral epithelium of the developing glomerular tuft and the simple squamous parietal epithelium of the capsule (Figs. , ) where it was co-expressed with Pax2 from the late-second trimester until Pax2 expression diminished in the mid-third trimester (-G). Co-expression of WT1 with Nestin on glomerular tuft endothelium was noted in the second trimester (). By the late third trimester and continuing into the postnatal period and adulthood, WT1 immunoreactivity was evident only in the podocytes of the glomerulus and in the arteriole endothelium (Fig. 3H-I) where it was co-expressed with Nestin ().
WT1 and Pax2 staining of sequential stages of kidney development in rhesus monkeys
WT1 and Nestin expression in sequential stages of kidney development in rhesus monkeys
Pax2 was expressed in some cells of the mesonephros of the early first trimester embryo (), and in the ureteric bud in mid-first trimester () where expression continued throughout the second trimester. Additional structures with Pax2 immunoreactivity in the late first trimester included the condensed mesenchyme surrounding the ureteric bud, the renal vesicle, and the developing C-shaped body (). By the late second trimester, strong Pax2 staining was noted in the visceral and parietal epithelium () followed by Pax2 expression in the visceral epithelial layer of the glomerular tuft as podocyte differentiation proceeded (-G). Postnatal expression of Pax2 was noted only in the parietal epithelial layer of the Bowman’s capsule near the vascular or urinary poles and in isolated groups of cells just beneath the cortex, and in some, but not all, cells of the collecting ducts (Fig. 3H-I).
Early first trimester expression of Nestin was noted in the neural crest (Figs. , ) and in some cells in the mesonephros. The diffuse pattern of Nestin expression noted in the metanephric mesenchyme in the mid-first trimester largely disappeared by the end of the first trimester as Nestin expression was noted only in the induced blastema surrounding the ureteric bud tips (Figs. , ). Although Nestin was not expressed in the renal vesicles, strong Nestin expression was noted in early glomerular tuft formation () and on the bordering surface of WT1-positive visceral epithelium of the glomerulus. Mature glomeruli located deep in the kidney cortex were noted with Nestin-positive cells, which were likely podocytes. With advancing gestational age, the location of Nestin-positive glomeruli expanded peripherally towards the outer cortex (Figs. , ). From the mid-third trimester through early postnatal life, Nestin expression was noted solely on the glomerular podocytes () and vasculature including arcuate arteries and afferent arterioles (). Podocyte co-expression of Nestin, WT1, and Synaptopodin was evident from the mid-third trimester to the adult (Figs. , ). The early visceral epithelial layer (developing podocytes) was noted with strong cytoplasmic expression of WT1 while Nestin expression was localized on the developing endothelium and/or the bordering surface of the visceral epithelium. As glomerular maturation progressed, Nestin expression was reduced in the glomerular endothelium but increased in the visceral epithelium of the glomerulus. By the late third trimester, Nestin was co-expressed with Synaptopodin in podocytes () although Synaptopodin immunoreactivity in maturing glomeruli was noted to be greater than Nestin immunoreactivity.
Synaptopodin (SYN) and Nestin expression in sequential stages of kidney development in rhesus monkeys
Some, but not all, of the tubule-shaped structures located below the metanephric zone of differentiation were immunoreactive for Synaptopodin in the late first trimester (). The location and morphology of these Synaptopodin-positive structures suggests association with the ureteric bud. Synaptopodin staining was also noted in the region surrounding the notochord. In the early second trimester, limited Synaptopodin expression was observed on the membrane of visceral epithelial cells in contact with the glomerular capillary tuft (). Co-expression with Nestin was noted at this stage of development. This pattern intensified with differentiation and, by the late third trimester, Synaptopodin expression was noted solely on podocytes ().
Markers of Glomerular Development
Gremlin and α-SMA were included as markers in first and second trimester fetal kidneys to visualize early glomerular tuft development (). In the late first trimester and into the second trimester Gremlin staining was observed on the luminal surface of the ureteric bud and developing glomerular tuft endothelium where it was co-expressed with CD31. High levels of Gremlin expression were noted in the early third trimester () in a pattern consistent with endothelial or mesangial cells, and was co-localized with CD31 on the luminal surface of developing glomerular endothelium. As expression of these markers was diminished in the third trimester, additional markers were studied including VEGF. Expression of α-SMA was observed in vascular smooth muscle () of arcuate arteries, afferent arterioles, and occasionally in developing glomerular endothelium although co-expression with VEGF was not noted ().
Markers of glomerular tuft development including CD31, Gremlin, alpha-smooth muscle actin (α-SMA), and vascular endothelial growth factor (VEGF) in sequential stages of nephrogenesis in rhesus monkeys
Transcriptional Expression of Renal Developmental Markers
The transcriptional activity of key developmental genes was studied by quantitative RT-PCR analysis of expression patterns across gestation (). In general, patterns of transcript expression were similar to protein expression noted in the immunohistochemical analysis. Expression of WT1, Pax2, and α-SMA transcripts did not show any significant differences across gestation. In accordance with protein immunoreactivity described above, Nestin transcript expression declined 38% from the first to second trimesters (p=0.03) before increasing with podocyte maturation near term (relative copy number 8.8 in the second trimester compared with 17.5 at term; p=0.01). Likewise, Synaptopodin transcript expression was low in the first and second trimesters (1.0 and 0.7 relative copy numbers, respectively) before increasing with podocyte maturation in the third trimester (relative expression 2.2; p<0.05) and at term (relative expression 4.5; p<0.001). Expression of Gremlin mRNA increased more than two-fold from the first and second trimesters (16.1 and 21.5 relative copy numbers, respectively) to the third trimester (42.9 relative copy number; p<0.05) before declining sharply at term (relative expression 13.1; p<0.01).
Quantitative RT-PCR analysis of expression patterns of α-SMA, Gremlin, Nestin, Pax2, Synaptopodin, and WT1 across gestation
Summary of Renal Markers by Structure and Gestational Age
A summary of expression of markers by structure and gestational age is provided in . Of the markers studied, Pax2 and Synaptopodin were expressed in the ureteric bud in the first trimester kidney with dim Gremlin expression noted at the branching tips. Pax2 was also expressed in the condensed mesenchyme surrounding the ureteric bud and in the early renal vesicle. WT1 and Nestin were diffusely expressed in the metanephric mesenchyme, but intensified as the Pax2-positive condensed mesenchyme differentiated into C- and S-shaped bodies. The inner cleft of the tail of the S-shaped body contained the podocyte progenitors (visceral epithelium) expressing Pax2, Nestin, and WT1 from the early second trimester. As kidney maturation progressed, Pax2 expression was lost in these structures and concurrent with an increase in Synaptopodin expression. Mature podocytes were observed from the mid-third trimester through adulthood, and expression of WT1, Nestin, and Synaptopodin was also shown. Early renal vasculature and glomerular endothelium were CD31, α-SMA, and Gremlin-positive. As the glomerular structures matured, Gremlin expression was restricted to mesangial cells and afferent arterioles in the mid-third trimester only. α-SMA immunoreactivity was noted in renal vascular smooth muscle postnatally and into adulthood (see ).
Summary of distribution of renal developmental markers in fetal rhesus monkeys