Changes in renal WT-1 expression preceding hypertension development

Hypertension is one of the most complex diseases to understand due to its strong genetic component. Although the genetic basis of the disease is firmly established and there have been very important developments in the field of molecular biology in recent years, few studies deal with hypertension as a result of possible changes in renal development by decoupling organogenic key factors [1]. In this regard, nephrogenic impairment is often associated with low birth weight and has been recognized as a powerful risk factor for kidney disease with low glomerular filtration rate, which would condition a more rapid progression of kidney disease and hypertension [2]. Of special interest is the fact that one important consequence of the altered nephrogenic process is hypertension. Kidney damage is a significant event in the development of hypertension and, thus, maternal food restriction alters key fetal nephrogenesis gene expression, such as Wilms’ tumor transcription factor 1 (WT-1) and other factors; this appears to contribute to hypertension development [3, 4]. In addition, some genes involved in hypertension development also depend on WT-1. The fact that renin gene transcription is regulated by WT-1, and that inherited mutations in the WT-1 gene can lead to hypertension, may explain the findings of patients with high renin and hypertension [5]. Accordingly, unpublished results from our laboratory assays performed in adult spontaneously hypertensive rats (SHRs) suggested an opposite relationship between WT-1 renal expression and blood pressure values. Furthermore, recent hypertension studies show low levels of vitamin D associated with the exaltation of the renin-angiotensin-aldosterone system (RAAS) [6‐8]; and, in that respect, our laboratory has demonstrated, in the primary culture of proximal tubular cells of SHRs, that heat shock protein 70 (Hsp70) protects against hypertension induced by angiotensin II by exerting a cytoprotective effect [9]. We have also demonstrated in adult SHRs that the induction of vitamin D receptor (VDR) modulates an increase in Hsp70 levels, with a decrease in the angiotensin II receptor, type 1 (AT₁) expression, providing renal protection [10]. These contributions are relevant considering previous reports, where WT-1 and Hsp70 are physically associated (localized) in embryonic rat kidney cells, in primary Wilms’ tumor samples, and in cultured cells with inducible WT-1 expression [11]. It has also been described that Hsp70 is an important cofactor for the function of WT-1, and suggested a potential role for this chaperone during nephrogenesis. Furthermore, it has been reported that human VDR is regulated by WT-1 [12]. These findings strengthen our recent demonstrations in the adult SHR model. Here we established, in mitochondria, a significant and consistent anatomical-functional correlate with the expression of the markers/modulators mentioned above (AT₁, VDR and Hsp70) [10]. Moreover, cumulative evidence suggests that reactive oxidative stress (ROS) and inflammation, with particular attention to mitochondria, plays an important role in hypertension development [13, 14]. In this regard, our laboratory reviewed and proposed a complex new regulation in the inflammatory pathway that involves the possible modulation of Hsp70 and WT-1 on mitochondrial signaling and where the net effect would favor cell survival by WT-1 stabilizing Bcl₂, and would limit the potential for the release of cytochrome C from mitochondria [15].

Finally, SHRs were originally inbred from Wistar rats and their Wistar–Kyoto (WKY) inbred non-hypertensive controls. These rats develop hypertension at about 6 weeks of age without physiological, pharmacological or surgical intervention; however, environmental factors affect the development of hypertension, and the importance of this model has been attributed to the similarity of its pathophysiology with essential hypertension in humans [16].

Based on the arguments stated above, we finally proposed to evaluate the following key hypothesis: Changes in the expression pattern of the WT-1 transcription factor and molecular mediators related to nephrogenesis could contribute to anatomical and functional kidney disorders, as well as to a hemodynamics disorders typical of the SHR model. Such modifications could respond to a Hsp70-dependent conditioning, as well as a probable modulation of the vitamin D receptor. In addition, we discuss the possibility that mitochondrial injury and dysfunction linked to master nephrogenic factors, such as WT-1, could play a central role in the pathogenesis of the essential hypertension model.

Both the SHR group and the control group, formed by Wistar Kyoto rats (WKY), were cared in accordance with the Guiding Principles in the Care and Use of Animals of the United States National Institute of Health. All experimental procedures were previously approved by the Institutional Animal Care and Use Committee of the Medical Sciences College, National University of Cuyo (Protocol approval N° 46/2015).

This paper is the first report showing that, in the natural history of hypertension, ultrastructural mitochondrial damage occurs first, before hemodynamic clinical manifestations appear. Therefore, altered mitochondrial energy metabolism linked to master nephrogenic factors, such as WT-1, could play a central role in the essential hypertension model, and should be further investigated. The present study shows WT-1 expression in the late nephrogenic process associated with hypertension development as well as the corresponding anatomical and functional correlation. Also for the first time, previous to the increase in blood pressure, we demonstrated low expressions of WT-1, VDR and Hsp70 in kidneys from neonatal SHRs.

As widely known, hypertension is considered one of the most important public health problems in developed countries, affecting about one billion people worldwide (9.4 million deaths/year; World Health Organization 2013). Hypertension occurs as an asymptomatic disease, although, paradoxically, many changes that precede the elevation of blood pressure produce specific organic lesions, some clinically defined. In addition, the multitude of contributing factors (environment, sodium intake, renin, insulin resistance, sleep apnea and age, among others) makes hypertension complex to study mainly due to the genetic component associated with its appearance. Of particular interest to this study is the relationship between hypertension and kidney disease, which yet remains unclear and is a matter of considerable research interest, with evidence suggesting that hypertension is both a cause and a consequence of kidney disease.

The kidney is an extremely complex structure, consisting of functional units having about 10,000 cells divided into at least 14 different cell types. This implies that the renal morphogenesis must be perfectly orchestrated, and that the conversion of mesenchymal mesodermal cells into polarized epithelial cells [26] is a critical step of this process. There is evidence that WT-1 may be directly involved in the regulation of cell proliferation and differentiation. In situ hybridization studies have shown WT-1 to be selectively expressed in the metanephric blastema and glomerular epithelium during embryonic and fetal development [27]. The expression patterns of WT-1 indicate an important role of this gene not only during urogenital development but also during fetal and postnatal life. Furthermore, using laser microscopy techniques, WT-1 was sub-located in the nucleus cell, regardless of cell type and stage of development. Moreover, it has been concluded that the initial growth of the ureteric bud is dependent on a signal from the metanephric blastema and WT-1 would be necessary for this [28‐30]. Therefore, WT-1 is essential for normal kidney development [31].

In contrast, nephrogenic deficiency has been recognized as a powerful risk factor for kidney disease, conditioning a more rapid progression of the disease and the possible development of hypertension [2]. Our group has confirmed this in the present study when quantifying/qualifying the glomeruli of SHRs compared to those of WKYs, from birth to hypertension development. Specifically, a slight decrease in the number and diameter of glomeruli was found in 4-week-old rats, while a significant decrease was found in 8-week-old rats. Similarly, we established significant changes in renal function (urea and creatinine) only in 8-week-old rats. In parallel, we could establish a close relationship of these parameters and WT-1 expression, which was clearly lowered especially in 8-week-old rats. This is consistent with previous reports from our laboratory where a low expression of WT-1 in neonatal rats with kidney disease was associated with glomerular changes and reduced nephrogenic markers such as Snail, BMP-7 and E-cadherin [32]. However, the most striking finding of the present study was to establish that, first, there were changes in WT-1 expression linked to glomerular alterations (in 4-week-old rats), and then, these were followed by hemodynamic changes with elevation of blood pressure (in 6-week-old rats). To support this, genes typically involved in the process of hypertension development are also WT-1 dependent [5, 33]. Interestingly, inherited mutations in the WT-1 gene can lead to life-threatening hypertension [5]. Thus, the renin gene transcription is regulated by the WT-1 (−KTS) protein and this could explain the findings in patients with WT-1 mutations that occur with increased plasma renin and hypertension. Therefore, and of particular interest to our current discussion, one important consequence of the altered nephrogenic process is hypertension development, which further amplifies the risk of onset and progression of kidney disease [33]; this makes it controversial to analyze whether hypertension is a cause or a consequence of kidney disease.

To emphasize our findings, WT-1 plays a key role in the onset of kidney formation, the progression of kidney formation and the maintenance of normal kidney function. The WT-1 protein participates in diverse cellular processes such as proliferation, differentiation and apoptosis and, in accordance with these functions, the number of target genes and/or proteins it affects is still increasing [34]. Particularly, the cells of the glomerulus, the proximal tubules and the distal tubules all derive from the metanephric mesenchyme where WT-1 already plays an active role [35]. According to this, in addition to glomerular alteration, we also established a significant change in the morphometric evaluation of fibrosis and apoptosis in glomerulus and tubular cells. Contrary to our results, apoptosis was markedly decreased in the kidneys of neonatal SHRs compared with their normotensive controls [36]. Nevertheless, it should be noted that the cited study was conducted in newborn SHRs within the first week of life, a period when we did not find significant changes. Previously, we had demonstrated an increase on renal interstitial fibrosis and apoptosis in adult SHRs [10]. However, this is the first study using the essential hypertension model which established a lesser WT-1 expression level occurring parallel to renal morphometric changes during late renal development and its consolidation as an adult organ. This is a key finding, considering that nephrogenesis in rats continues many days after birth. Thus, renal development in the newborn rat is comparable to that of the midtrimester human fetus, while renal maturation in the 2-week-old rat is analogous to that of the human infant [37]. Interestingly, Dr. Chevalier demonstrated that renal growth is impaired and nephron number is reduced in 14- to 19-day-old rats with obstructive nephropathy [38]. Using the same model, we have reported a significantly low expression of WT-1 in neonatal rats linked to glomerular changes and reduced nephrogenic markers [32]. Hence, kidney impairment is a significant event in hypertension development and, thus, alteration of a key nephrogenic gene expression such as WT-1 appears to contribute to hypertension development.

On the other hand, recent studies in hypertension have shown low levels of vitamin D linked to RAAS exaltation [6, 39, 40]. Moreover, Hsp70 regulates signaling pathway responses to cellular oxidative stress; and, in that respect, our laboratory has shown that Hsp70 protects against angiotensin II-induced hypertension [9]. We examined the effect of losartan on the expression/localization of Hsp70 in primary cultures of proximal tubular cells of SHRs, and demonstrated that the membrane translocation of Hsp70 could exert a protective effect by reducing the activity and expression of NADPH oxidase. In line with these studies, we also demonstrated in adults SHRs that structural and functional changes were reversed by VDR induction and that the association with enalapril worked even better. Thus, our recent data suggest that a low AT₁ expression through VDR induction could be a consequence of Hsp70-mediated cell protection [10]. These concepts could be critical in understanding the complex interplay between VDR and RAAS for hypertension and related inflammatory disorders. Unprecedentedly, and occurring previous to the increase in blood pressure, we demonstrated low expressions of VDR and Hsp70 linked to a poor WT-1 level in the renal cortices from 4-week-old SHRs. Contrary to this, AT₁ staining was observed in the same SHR epithelial cells. These values were very significant in 8-week-old rats. These data are revealing in the light of previous contributions. WT-1 and Hsp70 are physically associated in rat kidney cells, in primary Wilms’ tumor samples and in cultured cells with inducible expression of WT-1. Hsp70 is recruited to the characteristic sub-nuclear clusters containing WT-1. In addition, WT-1 requires amino-terminal transactivation domain for binding to Hsp70, and the expression of that domain itself is sufficient to induce the expression of Hsp70. Replacing the Hsp70 binding domain is sufficient to restore the functional properties of WT-1. These observations indicate that Hsp70 is an important cofactor for the function of WT-1, and suggest an emergent role of Hsp70 during nephrogenesis [11]. VDR, a ligand-activated transcription factor, is also regulated by WT-1. In humans, multiple assays suggested that, while WT-1 can choose from three binding sites within the VDR promoter, the VDR gene activation seems to occur through a single site. This site differs from a site-sensitive WT-1 previously identified in the murine VDR promoter [12]. Accordingly, the evidence suggests that there could be a modulation between Hsp70, VDR and WT-1. Moreover, the decoupling between Hsp70 and VDR associated with a possible poor expression of WT-1 could be keys to hypertension development.

In the present study, we have demonstrated that there was ultrastructural damage at the mitochondrial level in the renal cortices of neonatal SHRs. In addition, a presence of high AT₁ and NADPH oxidase activity coupled with low WT-1, VDR and Hsp70 expressions were consistent with the histological and structural changes demonstrated. Importantly, hypertensive-induced renal injury is characterized by activation of several deleterious pathways, including oxidative stress, RAAS, renal remodeling, poor vitamin D levels and apoptosis, all of which may compromise mitochondrial integrity and function. Mitochondria are the main generators of reactive oxygen species (ROS) in cells, yet their pathophysiological role in hypertension and related inflammatory diseases remains to be fully clarified [13]. Mitochondrial dysfunction has been implicated in the etiology of many diseases, like hypertension, and, among the many factors involved, our laboratory has characterized in mitochondria the RAAS exaltation and deficiency of vitamin D receptor [6, 10, 39]. In this paper, we have demonstrated that proximal convoluted tubules display normal tubular cells with baseline projections within normal parameters. They had abundant mitochondria, mostly with normal ultrastructure, except for some beginning to show changes, such as wider spaces than expected between the mitochondrial cristae. Moreover, in 8-week-old SRHs, the architecture of the renal tissue was disorganized, and the cellular structure was also altered. These cells contained vacuoles, mitochondria with cristae separated by widened spaces between them –some of them containing vacuoles and dense bodies surrounded by membranes. In the last decade, it has been shown that ROS and inflammation, with special attention to mitochondria, play an important role in the development of hypertension [13, 41]. In this regard, our laboratory proposed a complex interplay between inflammatory pathway regulation and Hsp70 [42]. One mechanism that has recently been discussed by our work group and other laboratories is the possible involvement of Hsp70 and WT-1 on mitochondrial signaling pathway, where the net effect would favor cell survival by WT-1 stabilizing Bcl₂, and would limit the potential for release of cytochrome C from mitochondria [15]. Also, Hsp70 interacts with the VDR and plays a role in controlling VDR concentrations within cells [43]. Vitamin D has also been demonstrated to be a nontoxic inducer of Hsp70 in the rat kidney [44]. NADPH oxidase activity was reverted in mitochondrial fractions from vitamin D inducer-treated animals [19]. Furthermore, VDR-modulated Hsp70/AT₁ expression may protect the structure and function of SHR kidneys [10]. In addition, transcriptional activation of the VDR by WT-1 can mediate programmed death of renal embryonic cells in response to 1,25-(OH)(2)D(3) [45]. Thus, previous evidence and our present findings support a key role of the vitamin D endocrine system in renal cell growth and differentiation during hypertension development. Moreover and in accordance with our current findings, maternal vitamin D deficiency accompanies changes in the expression of important renal factors that can slow maturation of glomeruli, extending the period of nephrogenesis [46].

Collectively, we propose an original hypothesis holding mitochondrial dysfunction as a precursor of hypertension. The potential for interaction between Hsp70 and VDR in mitochondria could be a cause or consequence of WT-1 modulation (Fig. 8, proposed hypothesis graph).

The originality of the proposal presented in this paper is the idea that, in the natural history of hypertension, ultrastructural mitochondrial damage occurs first, before hemodynamic clinical manifestations appear. The altered mitochondrial energy metabolism linked to master nephrogenic factors, such as WT-1, could play a central role in the essential hypertension model, and should be further investigated. Unprecedentedly, we show opposing relationships between blood pressure levels in relation to the WT-1 nephrogenic factor and related genes. To our knowledge, this is the first study that finds that hypertension may be a result of possible changes in renal development by decoupling organogenic key factors. Finally, although the relationship between hypertension and kidney disease remains unclear and a matter of considerable interest for research, our current results provide new evidence suggesting that essential hypertension would result as a consequence of kidney disease with nephrogenic disturbances. Further studies should be designed to validate our findings and allow the implementation of novel therapeutic tools for hypertension.