Roles of mitogen-activated protein kinases and angiotensin II in renal development

Experimental and clinical evidence suggests that angiotensin II (AII) participates in renal development. Renal AII content is several-fold higher in newborn rats and mice than in adult animals. AII receptors are also expressed in higher amounts in the kidneys of newborn rats. The kidneys of fetuses whose mother received a type 1 AII receptor (AT1) antagonist during gestation present several morphological alterations. Mutations in genes that encode components of the renin-angiotensin system are associated with autosomal recessive renal tubular dysgenesis. Morphological changes were detected in the kidneys of 3-weekold angiotensin-deficient mice. Mitogen-activated protein kinases (MAPKs) are important mediators that transduce extracellular stimuli to intracellular responses. The MAPK family comprises three major subgroups, namely extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinases (JNK), and p38 MAPK (p38). Important events in renal growth during nephrogenesis such as cellular proliferation and differentiation accompanied by apoptosis on a large scale can be mediated by MAPK pathways. A decrease in glomerulus number was observed in embryos cultured for 48 and 120 h with ERK or p38 inhibitors. Many effects of AII are mediated by MAPK pathways. Treatment with losartan during lactation provoked changes in renal function and structure associated with alterations in AT1 and type 2 AII (AT2) receptors and p-JNK and p-p38 expression in the kidney. Several studies have shown that AII and MAPKs play an important role in renal development. However, the relationship between the effects of AII and MAPK activation on renal development is still unclear.


Introduction
In humans, the branching ureteric duct reaches the outer cortex approximately 12 weeks before term.New nephron formation ceases between the 28th and 36th gestational week (1).The nephrogenesis of rats begins on embryonic day 12 and is completed between 10 and 15 days after birth (1,2).Four different stages of glomerular development have been defined: a) vesicle, b) S-shaped body, c) developing capillary loop, and d) maturing stages (2).One-and 7-day-old rats present glomeruli in different stages of differentiation, while 15-and 30-day-old rats present glomeruli in the final stage of development.
Increases in fibronectin, α-smooth muscle actin (α-SMactin), proliferating cell nuclear antigen (PCNA), phosphoextracellular signal-regulated protein kinase (p-ERK), and angiotensin II (AII) expression are observed in the renal cortex of 1-and 7-day-old rats, followed by a decrease during renal development (3).The activation of these cells may be due at least in part to an increase in renal AII and mitogen-activated protein kinase (MAPK) content in the renal cortex (Figure 1).Fibronectin is one of the most important components of the extracellular matrix (ECM).Formation of the ECM represents a key event in kidney cell differentiation (4,5).Fibronectin is a glycoprotein that can interact with some proteins from the cell or ECM, inducing changes in cellular migration and adhesion (5).AII stimulates mesangial cells to produce various ECM components in cell culture studies (6), and also promotes cell proliferation and differentiation (7).It has also been shown that human fetal mesangial cells express α-SM-actin (8,9) and that large-scale cell proliferation occurs during kidney development (10).Up-regulated α-SM-actin synthesis by mesangial cells is frequently associated with increased cell proliferation and ECM production (11).
ERK has been related to cell proliferation and differentiation and is also expressed at higher intensity in 1-dayold rats (12,13).Several effects of AII are mediated through MAPK pathways (14,15).Kubo et al. (14) reported that AII and endothelin provoke increased MAPK activity in cultures of vascular smooth muscle cells, and that this activity is inhibited by losartan and by an endothelin receptor antagonist.In addition, evidence shows that MAPK expression in the developing kidneys is modulated by angiotensin-converting enzyme, and that c-Jun N-terminal kinases (JNKs), as well as AII-related intracellular signaling pathways of renal apoptosis in the developing kidney can be mediated by the p38 MAPK pathway (15).Taken together, the results of all these studies show that AII plays an important role in renal development and that many effects of this peptide, such as cell proliferation and differentiation as well as apoptosis, can be mediated by the MAPK pathways.

Evidence for a role of AII in kidney development
Clinical and experimental evidence shows that the renin-angiotensin system participates in renal development (16)(17)(18)(19)(20)(21)(22).Greater angiotensinogen expression has been observed in the rat kidney during late gestation and the newborn period than in adult kidney.Renin mRNA was detected after embryonic day 17 and was higher in fetuses on embryonic day 20 and in newborns than in adult rats (17,18).Renal AII content is several-fold higher in newborn rats and mice than in their adult counterparts.AII receptors are also expressed to a greater extent in newborn rats (19).
The mRNA for the type 1 AII receptor (AT 1 ) has been detected in the renal glomeruli of newborn rats during cell proliferation and differentiation (20).Renal expression of the type 2 AII receptor (AT 2 ) increases during fetal life and decreases after birth (20), suggesting that AII modulates the growth of various cell types and tissues (21,22).Mutations in genes that encode components of the renin-angiotensin system are associated with autosomal recessive renal tubular dysgenesis (23).Kidneys from fetuses with tubular dysgenesis show absence of tubule differentiation and marked thickening of arterial walls.These fetuses also present anuria, oligohydramnios and skull ossification defects.The kidney of 3-weekold angiotensin-deficient mice showed pelvic dilation with an atrophic papilla, A B C D E F Figure 1. Figure 1. Figure 1. Figure 1.medial hyperplasia of the interlobular artery and afferent arterioles, glomeruli with axial mesangial expansion, reduced glomerular area, patchy fibrosis, mononuclear infiltrate in the interstitium, as well as dilated tubules in the outer medulla with interstitial fibrosis around the dilated tubules (24).In conclusion, several lines of clinical and experimental evidence indicate that AII has an important role in pre-and postnatal renal development.

Evidence for the role of MAPK in kidney development
Many effects of AII are mediated through MAPK pathways (14,15,25).AII converting enzyme modulates the expression of components of the MAPK family in kidney from neonatal rats (15).Kumar et al. (25) suggested that the activation of p38 and JNK MAPKs was linked to AII signaling and demonstrated that such activation is associated with acute ischemic-reperfusion injury and may be linked, in part, to AT 2 stimulation.The authors showed that the improvement in left ventricular function induced by AT 2 blockade was associated with a normalized expression of p-JNK-1 and a slight increase in p38 expression.
Immunoblot analyses of kidney homogenates, as well as immunohistochemical studies, have indicated that ERK and p38 MAPKs are predominantly expressed in the developing kidney, the highest levels being observed in the embryo (10).The role of JNK MAPK in embryogenesis has been investigated, and it has been shown that the loss of the MKK4 gene, a JNK activator, results in embryonic death (26).A decrease in glomerulus number was observed in 15-day-old embryos cultured for 48 and 120 h with ERK or p38 inhibitors (27).Choi et al. (15) reported that the treatment of newborn rats with enalapril for 7 days increased JNK-2 and p38 MAPK expression in the kidney, and that these increases were temporally associated with higher numbers of apoptotic cells.However, the authors did not find any alteration in renal p-ERK expression in those animals.Nevertheless, it has been previously shown that p-ERK is related to cell proliferation and differentiation, while p-JNK and p38 MAPKs are in-volved in apoptosis (28).Therefore, the activation of the MAPK pathways by AII can result in apoptosis and cell proliferation and differentiation that are important events in renal development (Figure 2).

Renal development in offspring of dams submitted to a high sodium diet during pregnancy
It has been reported that a prenatal high salt diet increases blood pressure and salt retention in spontaneously hypertensive rats (29).da Silva et al. (30) and Hazon et al. (31) observed that adult offspring submitted to perinatal salt overload presented higher blood pressure, which was associated with increased renal AII content and absence of plasma renin changes in response to higher salt consumption (30).We observed a decrease in fibronectin, α-SM-actin, PCNA, p-ERK MAPK, AT 1 , and AII expression in the renal cortex of 1-day-old pups from dams subjected to sodium intake compared to same-age control rats (3,13).Therefore, there is a temporal association between the     reduction of AII and p-ERK MAPK expression and the decrease of α-SM-actin, fibronectin and PCNA expression in the renal cortex.There was an increase in p-p38 and p-JNK MAPK expression in 30-day-old pups of dams that received sodium intake (13).
Thirty-day-old rats from dams that received saline also presented higher systolic blood pressure and a decrease in glomerular filtration rate (3).We also found increased glomerulosclerosis and tubulointerstitial lesions in the renal cortex of 120-day-old offspring from dams subjected to an increase in saline intake compared to the same age control rats (32).These data show that sodium intake during gestation and lactation in rats leads to changes in the renal expression of AII receptors and MAPK in the pups that result in higher blood pressure and structural changes in the kidney of adult offspring.

Renal development in offspring of mothers that received angiotensin II antagonists
Human fetal kidneys from mothers that received an AT 1 antagonist during gestation presented poorly developed tubules, increased mesenchymal tissue, reduced numbers of proximal tubules, abnormal arterial and arteriolar walls, poorly developed vasa recta, and hyperplastic juxtaglomerular apparatus (33).Spence et al. (34,35) have shown in experimental studies that maternal treatment with losartan during late gestation and lactation is associated with lower pup body weights and higher pup mortality rates during the preweaning/postweaning periods, as well as irreversible histopathologic renal abnormalities in the F1 generation.Morphological renal findings, such as dilation of the renal pelvis, edema of the renal papilla, medial hypertrophy of intracortical arterioles, chronic renal inflammation, and irregular scarring of the renal parenchyma, have been described in rats.Akil et al. (36), using losartan to block AT 1 in pregnant rats, observed that the fetuses presented growth inhibition, delayed nephron maturation, increased TGF-β immunoreactivity and glomerular basal membrane thickness associated with apoptosis in devel-oping tubular structures during the early period of nephrogenesis.It was also reported that the fibrogenic effects of AII were mediated by AT 2 receptors whose expression was increased in injured tissues and organs undergoing remodeling.
Neonatal rats treated with an angiotensin-converting enzyme inhibitor presented persistent and irreversible histopathological renal abnormalities in adult life (37).These abnormalities mainly consisted of cortical tubulointerstitial inflammation, various degrees of papillary atrophy and pelvic dilation.
Chen et al. (38) reported in rat pups treated for 2 days with losartan down-regulation of genes encoding cytoskeletal and ECM components, resulting in malformation of the ECM and dysfunction of cell-cell and cell-matrix interactions.Machado et al. (39) observed that rats that received losartan during lactation presented albuminuria and alterations in renal structure in adult life that were related to the presence of an inflammatory infiltrate in the renal cortex.We observed that the 30-day-old pups from dams treated with losartan during lactation presented increased AT 2 , p-JNK and p-p38 expression in the kidneys and higher numbers of TUNEL (apoptosis) and PCNA-positive cells in the renal cortex (40).Albuminuria, decreased glomerular filtration rate as well as tubulointerstitial alterations and decreased glomerular area were also found in 30-day-old offspring from losartan-treated dams.Taken together, these data show that treatment with losartan during lactation can provoke changes in renal function and structure in rats associated with alterations in AT 1 , AT 2 , p-JNK and p-p38 expression in the renal cortex.However, the effects of AII on MAPK activation in the kidney of pups that received losartan during lactation have not yet been clarified.

Figure 1 .
Figure 1.Figure 1.Figure 1.Figure 1.Figure 1. Immunolocalization of angiotensin II (A and B), phospho-extracellular signalregulated protein kinase (p-ERK; C and D), phospho-c-jun N-terminal kinase (p-JNK; E and F) (arrows) in the renal cortex of rats at 1 (A, C, and E) and 30 days (B, D, and F) of age (scale bars = 20 mm; original magnification 250X).Note that the reactions are more intense in A and C than in B and D.