Nephron number, hypertension, and CKD: physiological and genetic insight from humans and animal models

Abstract

The kidneys play a vital role in the excretion of waste products and the regulation of electrolytes, maintenance of acid–base balance, regulation of blood pressure, and production of several hormones. Any alteration in the structure of the nephron (basic functional unit of the kidney) can have a major impact on the kidney’s ability to work efficiently. Progressive decline in kidney function can lead to serious illness and ultimately death if not treated by dialysis or transplantation. While there have been numerous studies that implicate lower nephron numbers as being an important factor in influencing susceptibility to developing hypertension and chronic kidney disease, a direct association has been difficult to establish because of three main limitations: 1) the large variation in nephron number observed in the human population; 2) no established reliable noninvasive methods to determine nephron complement; and 3) to date, nephron measurements have been done after death, which doesn’t adequately account for potential loss of nephrons with age or disease. In this review, we will provide an overview of kidney structure/function, discuss the current literature for both humans and other species linking nephron deficiency and cardio-renal complications, as well as describe the major molecular signaling factors involved in nephrogenesis that modulate variation in nephron number. As more detailed knowledge about the molecular determinants of nephron development and the role of nephron endowment in the cardio-renal system is obtained, it will hopefully provide clinicians the ability to accurately identify people at risk to develop CKD/hypertension and lead to a shift in patient care from disease treatment to prevention.

Function of the Kidney and Nephron

the basic function of the kidneys is to filter blood, reabsorb what is needed, and excrete what is not. The kidneys play a vital role in regulation of electrolytes, maintenance of acid–base balance, regulation of blood pressure (salt and water balance), and production of several hormones including calcitriol (active metabolite of vitamin D), erythropoietin (involved in red blood cell production), and renin (blood pressure control) (61). The kidney is divided into two major structures, the renal cortex and renal medulla, both containing segments of the nephron, the main functional unit of the kidney (Fig. 1). Each nephron contains a tuft of glomerular capillaries (that compose the filtration barrier via three structures: fenestrated endothelium, basement membrane, and podocytes), through which large amounts of fluid are filtered from the blood, and a long tubule (composed of distinct tubule sections) where the filtered fluid is converted into urine on its way to the renal pelvis and subsequently to the bladder (77). Kidney function is measured by glomerular filtration rate (GFR), which is defined as the volume of fluid filtered through the glomerular capillaries per unit time. Traumatic injury to the kidney can compromise renal function and lead to acute kidney injury or more subtle damage to structures of the nephron over time can lead to chronic kidney disease (CKD) or other cardiovascular complications (e.g., hypertension, cardiac hypertrophy, etc.) (59).

Fig. 1.

Overview of kidney organogenesis and schematic of mature nephron. During mammalian embryonic development, there are 3 paired renal organs that develop: the pronephros, the mesonephros, and the metanephros. The pronephros develop in the cervical region of embryo (cranial) and develop toward the caudal end of the intermediate mesoderm and eventually join/form the pronephric duct. The pronephric duct proceeds in a cranial-to-caudal direction. As it elongates caudally, the pronephric duct, through interaction with intermediate mesoderm (IM) form mesonephric tubules, which subsequently become the mesonephric duct (ND). The remaining IM are composed of a mesenchymal cell population called the nephrogenic cord (NC). The ND develops an outgrowth, known as the ureteric bud (UB), which invades the metanephric mesenchyme (MM), extends, and undergoes a series of branching due to reciprocal signals (Fig. 3). Around the tips of the ureteric bud tree forms the cap mesenchyme (CM), resulting from condensation of the MM. As the MM aggregates at the edge of the UB, it undergoes a mesenchymal-to-epithelial transformation and subsequently forms the renal vesicles (RV), followed by formation of comma-shaped bodies (CSB), S-shaped bodies (SSB), and finally the immature nephron (IN). Three segments emerge from the S-shaped body: the proximal segment differentiates into the glomerulus (G) and glomerular epithelial cells (podocytes); the midsection forms the proximal tubule (PCT) and loop of Henle (LH); and the distal segment becomes the distal tubule (DT). Several DT merge to form the cortical collecting duct (CCD). E, embryonic day.

Overview of Kidney and Nephron Development

The kidney has served as a model organ for developmental studies for over 40 yr, partly because of the relative uniformity with which it develops (branching morphogenesis) as well as the fact that it can be studied in culture (137). The kidney originates from a reciprocal interaction between distinct cell types within the intermediate mesoderm (IM) of which arise three successive pairs of renal structures (pronephros, mesonephros, and metanephros). These structures develop in an anterior to posterior direction (Fig. 1) (138). The ventral IM remains as a mesenchymal cell population called the nephrogenic cord (NC). The pronephros are rudimentary/transient structures that are nonfunctional in humans (but do function in lower animals). The pronephros develop from a group of cells in the IM that undergo a mesenchymal to epithelial transition [gestation day (GD) 28–35]. The epithelial cells arrange themselves in a series of tube-like structures that fuse to become the pronephric duct. After the pronephros atrophy/regress, the mesonephric duct or Wolffian duct is what remains and continues to grow until it joins the cloaca (75). Mesonephros develop by the formation of mesonephric tubules from the IM. The mesonephros becomes the main excretory structures (GD28-63) until the development of the permanent kidney. Each mesonephric tubule receives a blood supply by surrounding a capillary tuft. These structures are similar to the glomerulus of the fully developed nephron (32, 75). As development progresses, the more caudal mesonephros tubules differentiate as more cranial ones regress. The fate of the mesonephros (as the permanent kidney develops) is to become part of the epididymis, vas deferens, and seminal vesicles in males. In females, the mesonephros atrophies, leaving behind only remnants in the adult that partly compose the suspensory ligament of the ovary (160).

The development of the metanephros or definitive kidney also originates in the IM. The metanephros begins to develop around GD35 [or embryonic day (E)9.5–11.5 in rodents] as the mesonephros begins to regress (39). The metanephros begin functioning around GD63 (around the 9th week). Between the nephric duct (ND) and a specialized region of the NC, an epithelial outgrowth develops called the ureteric bud (UB). The UB invades the adjacent metanephric mesenchyme (MM) and forms the metanephric kidney (Figs. 1, 2) (29). The MM contains progenitor cells that will later form the epithelia of the metanephric nephrons and produce the inductive signals that promote and position the outgrowth of the UB from the ND (21, 82). As the MM aggregates at the edge of the UB, it undergoes a mesenchymal-to-epithelial transformation (MET) and subsequently forms the renal vesicles, followed by formation of comma- and S-shaped bodies (Fig. 1) (37, 121). Three segments emerge from the S-shaped body: the proximal segment differentiates into glomerular epithelial cells (podocytes); the midsection forms the proximal tubule and loop of Henle; and the distal segment becomes the distal tubule (37, 121). In total, these regions compose the mature nephron (Fig. 1).

Fig. 2.

Macroview of normal and abnormal kidney development. Normal development of the kidneys involve bilateral extension of the pronephric/mesonephric duct (ND) proceeding in a cranial-to-caudal direction. Ultimately, the ureteric bud (UB) grows out from ND, invades the metanephric mesenchyme (MM), and undergoes a series of branching (as described in detail in Fig. 1). Slight changes in the timing, expression, and function of genes/proteins likely account for the observed natural variation in branching and nephron number. For conditions of abnormal development, such as failure of a kidney to develop and/or other congenital defects including ipsilateral urogenital tissues, such as vas deferens, seminal vesicle and epididymis, or uterine horn (Mullerian duct) result from a truncation of the ND. Likewise, more profound alterations in timing, expression, and function of kidney genes/proteins could lead to a variety of congenital kidney defects.

Normal Variation of Nephron Number in Humans and Animals

In humans, nephron development (nephrogenesis) occurs during the embryonic period, and thus total nephron endowment is set at birth (66). Several research groups have attempted to accurately determine the average number of nephrons that compose the human kidney. The first study to utilize an unbiased stereology method to estimate nephron numbers was Nyengaard and Bendtsen (110). In this study, a fourfold difference in nephron number (331,000–1,424,000) was observed. Subsequently, Keller et al. (79) analyzed 20 human kidneys at autopsy and observed that nephron number ranged from 531,140 to 1,959,914, also a ~4-fold difference. Variation in nephron number was confirmed by Bertram’s group (68), which found that nephron complement ranged from 210,332 to 2,026,541 (average of 884,064) or ~10-fold variation in nephron number. Unfortunately, a major limitation of these studies is that measurements were performed at autopsy and likely didn’t capture the impact of important clinical parameters (chronic hypertension, diabetes, etc.) that could explain low nephron numbers (i.e., no way to discern whether low nephron number was from birth or significant number of nephron was lost due to chronic disease). Currently, there are a number of laboratories working toward developing efficient noninvasive methods for quantitation of nephron numbers (8, 15, 117, 161). For example, Beeman et al. (15) used magnetic resonance imaging technique along with injection of cationic ferritin to measure nephron numbers. This method yielded similar results as stereology and acid maceration measurements. The major advantage is that it is nondestructive, with the ability to measure glomeruli throughout the entire kidney. However, a concern is the potential toxicity of cationic ferritin as well as the ability to label only functioning glomeruli. These types of studies, with continued improvement, could potentially provide more definite measure of nephron number across healthy individuals.

There are estimates of nephron numbers determined in several animal models, including mice, rats, primates, pigs, and sheep; however, the literature is relatively sparse. Nephron complement per kidney ranges from 9,000 to 18,000 in mice (19, 30, 150) and from 13,000 to 26,000 in rats (45, 169). Studies in baboons have shown that nephron number range from 138,078 to 427,471 (57, 58). In pigs, total nephron complement varies from 1,624,672 to 4,613,980 or a ~3-fold difference (91, 163). A fourfold difference in nephron complement is observed in sheep (200,000–800,000) (7, 49, 177). In summary, it appears that normal variation in nephron number is a common finding between species, while the degree of variation appears to be greatest in humans.

Impact of Low Nephron Number on Cardio-Renal Function: Living with a Single Kidney

The most striking example of low nephron numbers is living with a single kidney. There are several reasons why an individual may have a single kidney, including: 1) a kidney was donated to a person requiring a kidney transplant; 2) a kidney was removed due to an acute injury (e.g., physical trauma) or a disease, such as cancer; or 3) an individual developed with only one kidney (109). The health outcomes across these groups provide an opportunity to understand the potential consequences of reduced nephron endowment on health and disease. In general, the data discussed below suggest that health outcomes vary, depending upon whether the single kidney is acquired or congenital and susceptibility to other diseases.

Acquired solitary kidney: unilateral nephrectomy.

The first reported therapeutic unilateral nephrectomy (surgical removal of one kidney) was performed in 1869 (145), and the first living kidney transplant was performed in 1954 (97). The medical benefits of unilateral nephrectomy as a cure for trauma and cancer are unquestionable (90, 153). However, for some in the medical community, the long-term outcome for living kidney donors remains a topic of concern despite a number of international surveys that report extremely low mortality of living kidney donors (0.02–0.04%) (51, 60, 94, 100). In support of these findings, a number of clinical studies have confirmed the safety of living kidney donation in adults (46, 72, 123), with some studies even demonstrating a higher life expectancy for donors compared with the general population (47). This apparent benefit of nephrectomy in donors may be related to their positive selection for low risk to develop chronic diseases (e.g., hypertension and diabetes), overall high nephron complement (in the remaining kidney), as well as careful follow-up after surgery (2, 87). In contrast, some reports have described the development of proteinuria (measure of renal injury) and hypertension after donation (135, 175), but these patients usually do not progress to end-stage renal disease (Table 1) (33, 120, 124). A recent study by Grams et al. (53) estimated that the 15 yr observed risks after donation among kidney donors were 3.5–5.3 times as high as the projected risks in the absence of donation. Similar to nephrectomy in adults, some studies that have evaluated uninephrectomy in children (43, 143) have found no significant difference in long-term outcome, while other studies identified increased risk of developing proteinuria and impaired renal function (Table 1). In summary, based on current studies, any significant negative outcomes for living kidney donors appear limited, assuming that donors are healthy and not susceptible to chronic diseases.

Table 1.

Cardio-renal implications of living with single kidney

	Proteinuria	Hypertension	Renal Impairment
Congenital solitary kidney
Argueso et al. (5)	++	++	++
Oldrizzi et al. (112)	+++++	++++	++
Kolvek et al. (83)	++++	++	++
Vu et al. (164)	+	−	−
Nephrectomized in childhood
Robitaille et al. (124)	+	−	−
Wikstad et al. (175)	+	+	−
Argueso et al. (4)	+++	++	+++
Baudoin et al. (13)	+++	+++++	+
Hegde and Coulthard (64)	+	−	−
Dursun et al. (43)	N/A	++++	+
Kidney donors
Fehrman-Ekholm et al. (47)	++	++++	++
Fehrman-Ekholm et al. (46)	++	++++	−
Talseth et al. (155)	+++++	++	++
Boudville et al. (20)	N/A	+	N/A
Ramcharan and Matas (119)	−	−	+
Muzaale et al. (104)	−	−	+

Renal impairment is based on elevated serum creatinine, decreased creatinine clearance, or formal glomerular filtration rate (GFR) measurement. +, 5–10%; ++, 11–20%; +++, 21–30%; ++++, 31–40%; +++++, >40% patients exhibiting given phenotype; N/A, no measurements were performed; −, no change between control and experimental group.

Congenital solitary kidney: born with a single kidney.

On occasion, some individuals can develop with only one kidney. This condition is commonly referred to as unilateral renal agenesis or congenital solitary kidney (CSK). In more extreme cases, it is possible that both kidneys do not develop (bilateral renal agenesis), which ultimately proves fatal. A number of malformations of the kidney are also possible, including hypoplasia, dysplasia, and multicystic dysplasia (Fig. 2). Due to common developmental pathways between kidneys and other urogenital organs, defects in the ureter [e.g., grossly dilated (megaureter)], ureter obstructions (ureterovesical junction obstruction), and/or other genital defects (absence of components of sex organs) frequently coexist with kidney malformations (147, 148). In total, these types of developmental defects are commonly referred to as congenital anomalies of the kidney and urinary tract (CAKUT) and collectively occur in 1:500 births (125).

Of particular interest are CSK individuals, because in the long term they exhibit a poorer prognosis compared with more benign forms of the CAKUT (80, 133, 178). Argueso et al. (4) reported the incidence of CSK at ~1:1,000 births, with similar incidence between males and females. This estimate is supported by several large studies that observed an incidence of CSK of ~0.15% (9, 98, 174, 182). In the short term, most CSK children usually demonstrate no clinical symptoms unless they have accompanying urinary tract malformation that require intervention. However, there are some inconsistent findings when these individuals are followed with age. Some authors report that CSK is more or less a harmless congenital malformation (164, 176), whereas other studies demonstrate that ∼30% of CSK adults require dialysis by the age of 40 yr (133). In total, 25–30% of people exhibiting CSK develop proteinuria and hypertension, and 13% experience renal insufficiency (Table 1) (1, 65).

A comparison between CSK, childhood uninephrectomy, and kidney donation individuals appears to show variation between the onset and/or progression of hypertension and impaired renal function. This variation may be due, in part, to inadequate long-term follow-up, limitation in population size, and/or the consideration of the impact of confounding factors (“second hit”), including genetic susceptibility and environmental influences. However, it does seem clear from the clinical data that there may be an important difference between developing with a single kidney and being born with two kidneys and undergoing a nephrectomy (i.e., loss/removal of kidney), as early in utero compensation or alterations in kidney development in the CSK individuals may lead to increased risk for renal impairment later in life.

Impact of Low Nephron Number on Cardio-Renal Function: Two-Kidney Individuals

Nephron number and predisposition to hypertension.

The large natural variation in nephron numbers between typical two-kidney individuals leads to an important question, “how many nephrons are needed to maintain normal kidney function?” Or conversely, “is there a minimum number or threshold at which low nephron numbers would cause a predisposition to disease?” Unfortunately, there are no definitive answers to these questions, mainly due to the inability to measure nephron numbers accurately (especially by minimally invasive techniques) as well as to account for nephron loss with age or through confounding factors, such as hypertension and CKD. The first study to examine the impact between nephron number and blood pressure was proposed almost three decades ago. In 1988, Brenner and colleagues (22) identified an association between nephron number and blood pressure in humans and hypothesized that any reduction in nephron number would be accompanied by glomerular hyperfiltration, glomerular enlargement, and culminate in systemic hypertension. This hypothesis has been well documented in different experimental animal models and humans by investigating loss of nephrons (via renal ablation and/or nephrectomy) on renal hemodynamics, blood pressure, and/or renal function measures (56, 89, 169).

More recently, another study demonstrated an inverse relationship between nephron number and blood pressure based on a German population. For normotensive individuals, the mean nephron number was ~1,429,200, whereas in hypertensive individuals, the mean nephron number was half at ~702,379 (17). This relationship has not been confirmed in African Americans (71). For Hispanic Americans and Caucasians, limited studies suggest that nephron number in hypertensive people is on average 250,000 less than in those without hypertension (69). Interestingly, a recent study in mice using blood pressure high 2 (BPH2) and their normotensive (BPN3) or low blood pressure control (BPL1) identified a significant correlation (R² = 0.779; P < 0.0001) between nephron number and systolic blood pressure (34).

Nephron numbers and predisposition to CKD.

In contrast to hypertension, there have been relatively few direct studies that have examined the relationship between nephron number and renal pathology in humans. Similar to the association to blood pressure, an inverse association has been observed between nephron number and percent of injured glomeruli (glomerulosclerosis), although the relationship was not significant (140). In another study of 140 US individuals, a significant inverse association between nephron number and glomerulosclerosis and intimal thickening in interlobular arteries was observed (36). A direct correlation between nephron number and birth weight in both white and African Americans has also been identified (70). Using birth weight as a surrogate measure of nephron number, some studies have found that low birth weight or prematurity is associated with various measures of CKD (62) including microalbuminuria (173), reduced GFR (78), and end-stage renal disease (85).

Nephron number and cardio-renal disease: insights from animal studies.

In general, animal models have provided more direct insight into the association between nephron number and cardio-renal disease. For example, a number of mice models have been used to investigate the consequences of low nephron number. There is at least one study using a premature mouse model to investigate the association between nephron number and hypertension. The study found that mice born 2 days premature exhibited 24% fewer nephrons and subsequently developed hypertension, albuminuria, and decreased GFR compared with full-term mice (150). A number of genetically modified knockout mice models have been identified with altered nephron numbers. For example, knockout of fibroblast growth factor receptor 2 (fgfr2) leads to a 24% decrease in nephron number. By 1 yr of age, mutant mice exhibit a significant increase in systolic blood pressure and more glomerular/tubular injury vs. controls (116). The loss of one GDNF allele results in a 30% reduction in nephron number. Aged GDNF heterozygous mice present with elevated arterial pressure, glomerular hypertrophy, and hyperfiltration compared with wild-type mice (30). In contrast, transforming growth factor-β2 heterozygous [Tgfb2(+/−)] mice exhibit 30% more nephrons compared with wild-type control. Heterozygous mice exhibit lower blood pressure and mean glomerular volume compared with wild-type animals (166).

Rat models have also been utilized to investigate the association between nephron number and hypertension and renal function impairment. The Munich Wistar Frömter (MWF) rat demonstrates less nephron endowment compared with the spontaneously hypertensive rat (SHR). The transfer of region of SHR genome on rat chromosome 6 to MWF background (congenic strain) led to increased nephron number and reduced albuminuria (142). This study also provided evidence that there are genetic factors involved in nephrogenesis on RNO6, as well as a link between nephron number and renal injury. The unilateral urogenital anomalies (UUA) rat is a model of CSK that was derived from a Wistar stock. UUA animals born with CSK demonstrate 1.2- fold more nephrons in the remaining kidney, but an overall reduction of nephron numbers due to the loss of one kidney. At 50 wk of age, CSK rats have significantly higher serum creatinine and albuminuria compared with two-kidney littermates (3). Recently, we developed a new model of CSK, the HSRA (heterogeneous stock-derived model of unilateral renal agenesis) rat, which exhibits nephron deficiency (~20% fewer nephrons) in the remaining kidney, and progressive kidney injury and decline in renal function with age. The extent of injury and decline in renal function in the HSRA-S (born with single kidney) is significantly more than observed in uninephrectomized two-kidney (HSRA-UNX) and two-kidney control (HSRA-C) littermates (169).

Aside from rodent models used to study nephron number and cardio-renal disease, sheep models have also been employed. Fetal nephrectomy in the sheep resulted in the remaining kidney exhibiting a 45% increase in nephron endowment, but overall a 30% reduction compared with a sham-operated fetus with two kidneys (35). At 6 and 12 mo of age, fetal uninephrectomized sheep develop hypertension and decreased GFR vs. sham-operated animals (86, 102). Additionally, pregnant sheep exposed to betamethasone (corticosteroid/anti-inflammatory) exhibit a 26% reduction in nephron number in offspring. Later in life, these sheep develop higher blood pressure and lower GFR compared with unexposed sheep (18, 183).

Low nephron numbers and impact of “second hit.”

There are a number of animal studies that have investigated the impact of additional stressors (i.e., second hit) that induce hypertension, such as angiotensin II (ANG II), N^G-nitro-l-arginine methyl ester (L-NAME), deoxycorticosterone acetate (DOCA), and salt-loading, in the context of reduced nephron numbers (136). In general, these studies demonstrate that renal injury and/or cardiovascular dysfunction is more severe than nephrectomy alone. ANG II used in combination with nephrectomy hastened glomerulosclerosis, proteinuria, and/or kidney function decline compared with sham animals (88). Interestingly, Tsukamoto et al. (158) showed ANG II infusion in uninephrectomized mice plus salt loading has a significant impact apart from renal injury as mice demonstrate hypertensive heart disease that leads to a heart failure phenotype.

L-NAME, a nitric oxide synthase inhibitor, combined with subtotal nephrectomy with and without salt-loading (48, 162) generated marked glomerulosclerosis, ischemic injury, interstitial expansion, and elevated creatinine compared with controls. DOCA-salt is widely utilized to induce hypertension, mainly through volume expansion. Animal studies combining DOCA-salt treatment with nephrectomy resulted in more proteinuria, kidney injury, and myocardial remodeling (6, 74). Our work with nephron-deficient HSRA-S animals demonstrated that the model is highly susceptible to develop hypertension-induced (DOCA + 1% NaCl) renal injury, which was significantly greater than injury observed in HSRA-UNX animals (168). In contrast, the induction of hypertension in two-kidney control (HSRA-C) had little to no impact on kidney injury or kidney function. The impact of salt-loading alone, along with nephrectomy at young age was studied by Carlström et al. (25). The study demonstrated that nephrectomy performed at a young age (3 wk of age) or salt-loading separately resulted in salt-sensitive hypertension. However, the combination of young nephrectomy and long-term high-salt treatment resulted in more pronounced hypertension and salt sensitivity than either alone (25).

Age itself can also be viewed as secondary stressor. A long-term study by Rodríguez-Gómez et al. (126) demonstrated that both male and female uninephrectomized rats exhibited proteinuria and increased blood pressure after 18 mo, compared with sham animals. Male uninephrectomized rats tended to have earlier and more severe lesions than female uninephrectomized rats. In total, there is strong experimental evidence to support the idea that uninephrectomy/nephron deficiency alone can cause a predisposition to progressive kidney injury, but when combined with a secondary stressor this predisposition can be significantly accelerated.

Kidney Development and Molecular Basis of Nephrogenesis

Major molecular factors involved in nephrogenesis.

There are several key developmental events and signaling cascades in renal development that have been elucidated, mostly through studies in mice (31). However, there are likely still genes and genetic factors yet to be identified as well as a lack of understanding of the interactions and coordination of these factors to generate the complex structure of a kidney (75). In general, the development of the metanephric kidney and lower urinary tract is coordinated by complex interactions among numerous transcription/growth factors and intracellular signaling molecules (29, 37). These genes can be coexpressed in the MM, stroma, angioblasts, UB, and cloaca (95, 121). However, cells at the UB tip express many genes that are not expressed by cells in the tubular portions and vice versa (139). The discussion below will focus on providing a review of important genes and networks that function at crucial stages of renal development and are also associated with CAKUT (Fig. 3, Table 2).

Fig. 3.

Molecular signaling involved in nephrogenesis. The development of the kidney is coordinated by the complex interactions among numerous transcription/growth factors and intracellular signaling molecules. The most important signaling pathway is GDNF-RET, which controls metanephric mesenchyme (MM) development and ureteric bud (UB) outgrowth and branching. GDNF is secreted by the MM and binds a receptor complex that includes RET and a membrane-tethered GFRA1 co-receptor located on the nephric duct (ND). GDNF signals through the RET complex to promote UB invasion of the MM. GDNF binding to the RET receptor leads to activation of signaling pathways promoting proliferation and differentiation. There are several key transcriptional regulators including Odd1, Eya1, and Six1/Six2 identified as being necessary for interaction between UB and MM. Loss of function in any of a number of genes including Pax2, Eya1, Six1, Wt1, and Hoxa11/Hoxd1 (and others) leads to a loss of Gdnf expression and either renal agenesis or renal hypoplasia. Likewise, mice knockouts of Ret or Gfra1 result in failure of the UB to form and can also result in renal agenesis. In this context, any alterations in the timing, expression, and/or function of these genes/proteins have the ability to influence nephron composition on a continuum from low to high.

Table 2.

Key genes involved in kidney development and associated with congenital abnormalities of kidney and urinary tract

Gene	Function	Abnormalities	Reference
Gdnf, GFRα1, c-Ret	promote UB induction/branching	renal agenesis	Pichel et al., 1996; Sanchez et al., 1996 (114, 132)
Eya1, Six1, Six4, Six5	induce expression and secretion of Gdnf	renal agenesis	Hoskins et al., 2007; Rodriguez-Soriano et al., 2001; Xu et al., 2003 (67, 127, 180)
Odd1, HoxA-11, HoxC-11, HoxD-11	regulate Gdnf expression	renal agenesis	James et al., 2006; Patterson et al., 2001; Stricker et al., 2006 (76, 113, 151)
Six2	maintain MM cells, allow continued branching	renal hypoplasia	Weber et al., 2008 (171)
Pax2, Sall1	interact among UB, MM, or stroma; mesoderm differentiation	renal hypoplasia	Negrisolo et al., 2011; Weber et al., 2006 (107, 170)
Bmp4, Bmp7	inhibit Wnt expression, inhibit UB branching	renal hypoplasia	Luo et al., 1995; Tabatabaeifar et al., 2009 (93, 154)
Fras1, Frem1	control differentiation of stromal and mesenchymal cells	renal dysplasia	Saisawat et al., 2012 (131)
FoxC1, FoxC2	DNA promoter binding to Eya1, ectopic UB budding	duplex ureters	Nakano et al., 2003 (105)
Robo2, Slit2	similar to FoxC2 regulation UB budding	duplex ureters	Zu et al., 2009 (185)
Rara, Rar2b	upregulate c-Ret	ectopic ureter	Batourina et al., 2001 (12)
Spry1	downregulate c-Ret	ectopic ureter	Rozen et al., 2009; Yosypiv et al., 2008 (129, 181)
WT1, Lmx1β, Sema3a	stimulate podocyte differentiation and angioblasts.	nephrotic syndrome	Chen et al., 1998; Gao et al., 2005 (26, 50)
REN, AGT, ACE AGTR1	stimulate renal tubular growth	renal tubular dysgenesis	Beck et al., 2011; Mounier et al., 1987 (14, 103)
Wnt4	mesenchymal signal for epithelial transformation	low nephrons	Iglesias et al., 2007 (73)
Hnf1β	develop renal capsule	horseshoe kidney	Nakayama et al., 2010 (106)
Pkd1, Pkd2	pattern of tubular and collecting duct	polycystic kidneys	Rossetti and Harris, 2007 (128)

The induction of the UB into the MM is directly controlled by glial-derived neurotrophic factor (Gdnf) and the tyrosine kinase receptor c-Ret, along with the co-receptor GFRα1 (27, 141). The levels and spatial expression of Gdnf play a central role in initiating budding and are regulated by multiple transcription and growth factors. Mice with inactivated Gdnf die from renal agenesis soon after birth (101). The expression of Gdnf itself is determined by a complex signaling cascade involving eyes absent homolog 1 (Eya1), and SIX homeobox (Six)1, 4, and 5 (23). Eya1 knockout mice show loss of Gdnf in the ND (179). However, Eya1 appears not to stimulate transcription directly, and the effect on Gdnf is due to the loss of expression of Six1, Six4, and Six5 in Eya1 mutant animals (111). Eya1 mutant animals also exhibit renal agenesis. Transcription factors odd skipped-related 1 (Odd1) is one of the earliest acting genes involved in metanephros formation and likely acts upstream of Eya1 in the NC to promote MM development (167). In addition to Odd1, loss of homeobox 11 (Hox11) family genes, including Hoxa-11, Hoxc-11, and Hoxd-11, leads to an arrest of MM differentiation and loss of Six and Gdnf expression (172).

Six2 is a homeodomain transcription factor expressed in the MM, which maintains MM cells in an undifferentiated state, thereby allowing continued UB branching and nephron formation to proceed (82). Paired box 2 (Pax2) is a homeobox transcription factor related to the activation of Gdnf (24). End-stage renal disease occurs in almost 100% of individuals with Pax2-associated renal hypoplasia (134). Polymorphisms in Pax2 are also associated with reduced kidney size in neonates (118). Sal-like 1 (Sall1) is expressed in the mesenchyme and recently implicated in renal hypoplasia. In Sall1-mutant animals, Gdnf and Eya1 remain expressed, indicating that Sall1 lies downstream of these signals or possibly in a separate pathway (108). Bone morphogenetic protein 4 (Bmp4) is a potent antagonist of Gdnf and plays a crucial role in inhibiting ectopic budding of the ureter (99). Homozygous Bmp4 knockout mice die early in the antenatal period but present with ectopic ureteric budding (171). Bmp7 is required for suppressing tubulogenesis and for the survival of mesenchymal cells (41). In Bmp7 knockout mice, kidneys initially form normally with appropriate UB branching, comma- and S-shaped body formation, but nephrogenesis is inhibited from E14.5 onward. Bmp7 becomes depleted in Six2 mutant kidneys (42), suggesting that Six2 lies upstream of Bmp7. Recessive mutations in human FRAS1 and FREM2 were recently detected in patients with nonsyndromic renal dysplasia. These two genes are related to extracellular matrix protein 1, which controls the differentiation of stromal and mesenchymal cells (131). In Fras1 null mutant mice, decreased FRAS1/FREM2 complex reduces the expression of transcription factors Hoxd11/Six2 and results in defective interactions between the UB and mesenchyme (115).

Additionally, studies in mice have demonstrated that genes such as forkhead box protein C (Foxc), Slit homolog 2 (Slit2), and its receptor, Roundabout homolog 2 (Robo2), confine Gdnf expression to the caudal part of the NC (84). Mutations in genes encoding these proteins lead to increased Gdnf expression to the rostral part of the embryo and can promote the outgrowth of multiple ureters (55). Mutations in Robo2 have been identified in patients with vesicoureteral junction defects and vesicoureteral reflux (16, 92). Vitamin A has also been shown to be involved in kidney signaling (52). The retinoic acid receptor-α (Rara) is expressed at low levels throughout the embryonic kidney, whereas retinoic acid receptor-β2 (Rarb) expression is restricted to stromal cells. The deletion of either Rara or Rarb does not lead to kidney defects; however, in double-null Rara and Rarb mutants, UB growth is reduced and c-Ret expression is downregulated (96). The inhibition of c-Ret expression and signaling activity is also modulated through sprouty homolog 1 (Spry1). Sprouty proteins are required for limiting branching during kidney growth (11). Mutations in Spry1 result in ectopic ureters by rendering the duct more sensitive to Gdnf levels (10).

Podocyte differentiation is regulated by transcription factors Wilms tumor 1 (WT1), LIM homeobox transcription factor 1β (Lmx1β), and semaphorin3a (Sema3a) (122, 152). Lmx1b and FoxC work together to modulate podocyte development (63). WT1 and Sema3a expression is restricted to the podocyte layer during nephron maturation as well as acting as a PAX2 repressor (130). This repression seems to be crucial since ectopic activation of PAX2 in podocytes of transgenic mice leads to severe glomerular disease (40, 165). Wingless-type integration site family 4 (Wnt4) induces MM cells to undergo MET and differentiate into nephron epithelia (149). Activation of Wnt4 seems to be controlled by a complex network of genes, including WT1 and Pax2 (144, 156). Wnt4 knockout mice almost completely lack nephrons (81).

Other important factors involved in nephrogenesis.

Hepatocyte nuclear factor 1β (Hnf1β) is involved in renal capsule formation as defects in Hnf1β lead to kidneys fused at inferior lobes and located lower than usual (commonly referred to as horseshoe kidneys) (44). Mutations in Hnf1β are detected in 33% of children with nonsyndromic multicystic dysplastic kidney or renal hypodysplasia (159). Polycystic kidney disease (PKD) is also a congenital kidney disorder, caused by cysts that arise from tubules. Pkd1 and Pkd2 represent an example of patterning defect (146). The increased tubular cell proliferation together with decreased integration of cells into the plane of tubular epithelium or loss of oriented cell division may account for cyst formation (157). The developing mammalian metanephros expresses all components of the renin–angiotensin system (29). In humans, their expression in the embryonic kidney is as early as the fifth week of gestation when metanephric organogenesis is initiated (28). Mutations in the genes encoding for AGT (angiotensinogen), REN (renin), ACE (angiotensin-converting enzyme), or AGTR1 (AT1 receptor) lead to autosomal recessive (homozygous mutation) renal tubular dysgenesis (54, 184).

Perspective: Relevance of Understanding Molecular Determinants of Nephron Development, Endowment, and Role in Cardio-Renal Disease

The kidneys play a vital physiological role by efficiently removing waste and regulating blood pressure, but its ability to do so depends heavily on events that occur during kidney organogenesis. It is clear that kidney development requires the coordinated spatiotemporal expression of genes and numerous signaling pathways that lead to the interactive induction between UB and MM, resulting in branching morphogenesis. Thus, any alterations in the timing, expression, and/or function of genes involved in this process have the ability to influence nephron composition on a continuum from low to high (Fig. 3). Through the use of animal models (particularly mice), a significant understanding of the specific gene/proteins that play a role in this process has been achieved. However, there is still a critical need to understand how epigenetics, gene expression, and proteomic networks work together (systems biology) to result in nephrogenesis/kidney development.

There is no doubt that animal models provide a more integrative view of kidney development; however, a major limitation (regardless of species) is the ability to evaluate nephron endowment noninvasively. The ability to determine nephron number accurately early in life before the impact of disease (or environmental factors) would provide the ability to establish the exact association between nephron number and cardiovascular disease. The current data certainly suggest that there is a significant inverse relationship between nephron number and cardiovascular disease (CVD) risk (Fig. 4). Individuals that exhibit high nephron endowment and low genetic susceptibility to CVD (e.g., hypertension) are less likely to develop any impairment in renal function. These individuals would also likely not exhibit any adverse effects from either losing a kidney due to injury or serving as a kidney donor. Conversely, individuals that exhibit low nephron endowment and high genetic susceptibility to CVD are most at risk of developing significant loss of renal function with age, which would likely be exacerbated with the loss or donation of a kidney (Fig. 4). In the context of CSK, an individual could still exhibit a relatively high nephron endowment, despite only having one kidney (e.g., a CSK individual could have more nephrons than a two-kidney individual), with no adverse impact on the occurrence or progression of CKD. Thus, it is not simply a matter of having one kidney (either CSK or loss due to injury or donation) or two, but rather a measure of total nephron composition and susceptibility to confounding disease factors (“second hit”) that can lead to cardio-renal disease later in life.

Fig. 4.

Impact of nephron number and genetic susceptibility on cardio-renal disease. Both nephron number and genetic susceptibility to cardiovascular disease (CVD) exist on a continuum from low to high. Individuals that exhibit high nephron endowment and low genetic susceptibility to CVD (e.g., hypertension) are less likely to develop any impairment in renal function, whereas individuals that exhibit low nephron endowment and high genetic susceptibility to CVD are most at risk to develop significant loss of renal function with age. There likely exists a point (threshold) at which a certain minimum number of nephrons can lead to and/or exacerbate pathological conditions, such as chronic kidney disease (CKD).

It is relatively easy to recognize the impact of low or high nephron number on CVD, but a major challenge is to identify whether there is a point or “threshold” at which normal renal function is maintained (or below a point that leads to increased risk of developing CKD and hypertension). The ability to make such determinations as well as to integrate genetic with a more extensive systems-biology understanding of the determinants of nephron development will ultimately provide clinicians the chance to identify accurately people at risk for CKD and hypertension. This, in turn, will provide physicians a powerful tool for transforming patient care, from disease treatment to preventive care.

GRANTS

M. R. Garrett is supported by National Institutes of Health (NIH) Grant HL-094446 and the Robert M. Hearin Foundation. Support was also provided through funds from the NIH, including Mississippi INBRE (P20GM-103476); Center for Psychiatric Neuroscience (CPN)-COBRE (P30GM-103328); and Obesity, Cardiorenal and Metabolic Diseases-COBRE (P20GM-104357).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

X.W. and M.R.G. prepared figures; X.W. and M.R.G. drafted manuscript; X.W. and M.R.G. edited and revised manuscript; X.W. and M.R.G. approved final version of manuscript; M.R.G. interpreted results of experiments.