Response to Nephron Loss in Early Development

Introduction

Glomerular endowment is highly variable in humans. Although each kidney contains approximately 1 million nephrons, glomerular number ranges from 210,000 to 2,700,000 per kidney representing a 10-fold difference. ^, Both environmental and genetic factors influence nephrogenesis, a complex developmental program including renal cell fate determination, stem and progenitor renewal and differentiation, nephron patterning and lineage maturation, and the coordinated development of the kidney and urinary tract. Human nephrogenesis is not complete until approximately 36 weeks of gestation, and infants born preterm have decreased nephron number at birth. Some studies suggest that nephrogenesis ends at birth in both term and preterm humans; however, conflicting data suggests that nephrogenesis continues for a limited time postnatally. Importantly, studies suggesting that postnatal nephrogenesis occurs have shown that this process is abnormal and may result in nonfunctional glomeruli.

Congenital anomalies of the kidney and urinary tract, which are usually associated with gene mutations, may also result in decreased nephron formation at birth and accelerated decline in nephron number over time. Furthermore, with age and insults such as sepsis and hypotension leading to acute kidney injury (AKI), there could be a further reduction in the number of functional nephron throughout life. When faced with a nephron deficit, the kidney undergoes adaptive responses that have both short- and long-term effects. This chapter reviews the causes and consequences of low nephron number, emphasizing the unique features of the vulnerable developing kidney.

Key Determinants of Nephron Endowment

While considerable variation exists with regard to the number of nephrons in humans, a relationship between glomerular number and birth weight has been reported. It has been estimated , from autopsy studies , that each kilogram in birth weight confers on average 260,000 more nephrons. A dditionally, 60% of nephrons are formed during the third trimester, making preterm birth a risk factor for low nephron endowment. In an elegant human autopsy study, Sutherland and colleagues showed that during the first 40 days of life nephrogenesis continues. Yet the glomeruli formed were abnormal appearing, suggesting that nephrons formed after birth may not function normally. This suggests that preterm birth may result in a functional nephron deficit. Since low nephron number can lead to chronic kidney disease (CKD) preterm birth and low-birth-weight infant s are expected to have an increased risk of CKD. Indeed, recent epidemiologic studies have identified preterm birth and low birth weight as risk factors for CKD.

A broader understanding of normal nephrogenesis has identified genetic and environmental factors that play a key role in determining glomerular number. The kidneys develop through branching morphogenesis, whereby an outgrowth of the wolffian duct, the ureteric bud (UB), undergoes iterative branching events that are dependent on signaling with the surrounding metanephric mesenchyme (MM). The epithelial-mesenchymal interactions at the tips of the branching UB are essential for nephron formation. Ret , a receptor tyrosine kinase, is expressed in the UB, and its ligand, glial-cell derived neurotrophic growth factor, glial cell line-derived neurotrophic factor (GDNF), is secreted by surrounding mesenchyme. The interaction between Ret and GNDF results in UB branching that induces cells in the MM to condense around UB tips, transition to renal progenitor cells, and ultimately form the nephron. Disruptions in genes and pathways that regulate the progenitor cell or mesenchymal population and those involved in the branching morphogenesis result in deficits in nephron number. Fig. 104.1 highlights the major factors that contribute to the number of nephrons in the metanephric kidney.

Genetic Factors

Advanced DNA sequencing and analysis has led to the discovery of hundreds of single genes that, if mutated, are responsible for isolated or syndromic kidney and urogenital malformations that can alter nephron endowment. Six2 is expressed in the cap mesenchyme surrounding the UB, and the progenitor cell population is reduced when it is inactivated. Perturbations in the UB or its ability to branch also affect final nephron number. For example, infants with a common Ret variant (the RET[1476A] allele) have a kidney volume that is 10% smaller than controls. The association between low kidney volume and low nephron number in Ret variants is supported by animal studies; homozygous Ret knockout mice do not develop kidneys, and heterozygotes have reduced nephron number.

Although most mutations known to affect nephron number result in fewer nephrons, variants that may increase nephron number also exists. El Kares and associates found that newborns with a variant in ALDH1A had a 22% greater kidney volume. ALDH1A encodes an enzyme, RALDH2, which converts nutritional vitamin A to active retinoic acid; retinoic acid is critical for UB branching. Manipulation of mechanistic target of rapamycin (Mtor), a regulator of cellular growth, metabolism, proliferation, and survival, can alter nephron number. Disinhibition of Mtor signaling through deletion of Hamartin, a negative regulator of Mtor, results in a 25% increase in nephron number. Altered heterochronic genes may also impact glomerular number by prolonging the duration of nephrogenesis. Yermalovich and colleagues discovered that the heterochronic genes Lin28 and Let7 , which code for highly conserved RNA binding proteins, have an important role in the cessation of nephrogenesis in mice. Lin28b is expressed in MM during nephrogenesis and when present inhibits Let7 expression. Over time Lin28 expression decreases allowing for increased Let7 activity, which includes inhibition of Lin28. Overexpression of Lin28b in the developing MM results in ectopic, nephron containing renal mass. Alternately, suppressing Let7 miRNA during nephrogenesis results in prolonged nephron formation in the outer nephrogenic zone, effectively increasing nephron number. While manipulating heterochronic gene expression poses significant risk for oncogenesis, understanding what signals terminate nephrogenesis in humans could lead to therapeutic interventions to potentially increase nephron endowment.

Although genetic factors are important, other regulators such as epigenetic factors and microRNAs are likely to play a significant role in the determination of nephron number in humans. Indeed, Nakagawa and colleagues showed that deleting an essential microRNA-activating enzyme, Dicer1, in developing renal stromal cells resulted in hypoplastic kidneys with abnormal tubules and vasculature. Other groups have gone on to show that in mice, microRNAs regulate kidney development through inhibiting gene expression, particularly pro-apoptotic transcripts in nephron progenitors. ^,

Environmental Factors

Infants with intrauterine growth restriction (IUGR) are born with low birth weight and fewer nephrons than those whose birth weight is appropriate for gestational age. Data from experimental models of IUGR suggest that the timing of growth restriction determines nephron reduction, with perturbations that occur during periods of robust nephrogenesis associated with lower nephron endowment and higher blood pressure later in life. Fetal rats of mothers whose uterine artery has been ligated, or rats with spontaneous IUGR, have reduced nephron number. ^, Reduced maternal calorie or protein intake leads to reduced nephron number in experimental animals and may program the individual for salt-sensitive adult hypertension. ^{, , ,} Intrauterine malnutrition in Aborigine humans is associated with low birth weight and impaired renal function in adulthood, suggesting that optimizing fetal growth may reduce renal disease and hypertension. In a provocative study, mild vitamin A deficiency was shown to lead to inborn nephron deficits in the rat, with a linear relationship between maternal plasma retinol levels and the number of glomeruli in the pups. This study and many others suggest that small variations in maternal nutritional status may have significant implications for the ultimate complement of nephrons.

The connection between in utero nutritional deprivation and altered nephrogenesis is not fully understood. Animal data has implicated an adverse effect of increased renin-angiotensin signaling during nephrogenesis. Studies have also shown altered DNA methylation patterns in human offspring exposed to in utero nutrient deprivation. Recently Wanner and colleagues showed that deletion of a specific DNA methyltransferase in mice leads to impaired nephrogenesis that resembles models of growth restriction-induced nephron deficits, supporting the role of epigenetic modification in nephron formation. In some cases nutrient deprivation can increase nephron number. In rats, vitamin D deficiency during pregnancy increased nephron number by roughly 20%, suggesting that vitamin D deficiency may stimulate nephrogenesis.

Beyond nutrition, there are a wide range of fetal and early neonatal exposures that have been linked to altered nephron endowment. Fetal exposure to toxins, such as maternal smoking or alcohol consumption, can impair kidney development. Exposure of the fetus to maternal angiotensin-converting enzyme (ACE) inhibitors or to prostaglandin synthesis inhibitors impairs nephrogenesis and renal maturation. ^, Maternal exposure to glucocorticoids, aminoglycosides, β-lactam antibiotics, or cyclosporine may also impair nephrogenesis and contribute to hypertension.

Infants of diabetic mothers have an increased incidence of congenital malformations, and maternal hyperglycemia in rats leads to nephron deficits. Early postnatal hyperglycemia may also alter nephrogenesis. In a study of preterm lambs, hyperglycemia was associated with accelerated nephron maturation. This altered nephrogenesis was thought to be due to increased reactive oxygen species found in hyperglycemic kidneys. Perinatal factors such as asphyxia and severe circulatory disturbances can also cause irreversible nephron loss.

Early human fetal urinary tract obstruction can result in abnormal kidney development and reduced number of nephrons, and the number of glomeruli can be related to the time of developmental arrest. Similarly, animal models of ureteral obstruction also result in reduced glomerular number, and ureteral obstruction impairs nephrogenesis in direct proportion to the duration of obstruction. These findings further support the interdependence of UB and MM for proper kidney development.

Preterm Birth

Experimental data focused on the effect of prematurity on glomerular endowment is limited in part due to a lack of animal models. Baboons delivered prior to the completion of gestation are suitable models for studying the effects of prematurity on renal development as they have a long gestation and can survive in a primate intensive care unit after premature birth. When baboons are delivered at a gestational age equivalent to a 27-week human gestation, there is no difference in the number of nephrons at 3 weeks of life compared to gestational age matched controls, suggesting that baboons born preterm undergo postnatal nephrogenesis. However, nephrogenesis after early delivery does not appear to recapitulate normal kidney development in utero, as there are histologic abnormalities within the glomeruli. Mice are commonly used laboratory animals to address many research questions. Unlike humans and primates, they complete nephrogenesis about 4 days after birth. The delivery of pups 1 to 2 days prior to term gestation results in low nephron endowment and evidence of CKD at 5 weeks with albuminuria, hypertension, and lower glomerular filtration rate (GFR).

There are few human studies that examine the effect of a premature birth on the development of the kidney. Autopsy studies have provided insight into the postnatal adaptations that occur in infants born during a rapid phase of nephrogenesis. In a study of premature infants where the investigators matched to controls based on gestational age ( n = 32), the kidneys were examined by stereologic and histologic methods to count and measure the glomeruli. The investigators found an accelerated termination of kidney development with a decreased nephrogenic zone and more abnormal glomeruli with a larger surface area. In a similar study, 66 infant kidneys were examined from premature infants weighing less than 1000 g who were grouped based on gestational and postnatal age, in addition to exposure to AKI. The investigators provided evidence that glomerulogenesis ceased 40 days after birth. Additionally, there were smaller radial glomerular counts in the premature infants, particularly if there was exposure to AKI, implying fewer nephrons. The kidneys from the premature infants also had increased mesangial tufts and capsular areas when they lived for more than 40 days, which may predispose them to vasomotor or nephrotoxic injury in the postnatal period or in later life. Taken together, these studies suggest that there may be a limited period of postnatal nephrogenesis but that if so, ex-utero nephrogenesis is likely abnormal.

The association between preterm birth and low nephron number in non-autopsy samples has been studied mostly through imaging modalities that use kidney size as surrogate for nephron endowment. A study following preterm children through 18 months found that children born prematurely had smaller kidneys than term children. In a Dutch cohort of premature infants (less than 32 weeks) renal ultrasound performed at 20 years of age revealed smaller renal volumes compared with term controls; the average reduction was 47 mL. These studies suggest that these children born preterm have lower nephron number; however, as explored in subsequent parts of this chapter, kidney size is not a precise surrogate for nephron number.