**2. The effects of preterm birth on nephrogenesis**

The human kidney develops from a ridge of mesodermal tissue (known as the nephrogenic cord) which is found along the posterior wall of the abdominal cavity on either side of the primitive aorta (Blackburn, 2003). Development of the permanent kidney involves the formation of the pronephros and mesonephros (transitory organs) and the metanephros (the permanent kidney) (Saxen, 1987; Clark and Bertram, 1999; Sweeney and Avner, 2004; Moritz

Effects of Preterm Birth on the Kidney 63

neonates have provided insight into how preterm birth affects the structure of the kidney and the number of glomerular generations formed within the kidney. As well, carefully controlled experimental studies in the nonhuman primate provide valuable insight into the effects of preterm birth on nephrogenesis and on the total number of nephrons formed.

Table 1 summarizes the main *in vivo* clinical studies that have investigated the effects of preterm birth on kidney length and volume. Overall, the findings in relation to the effects of preterm birth on kidney size are conflicting; however, it is difficult to compare between studies due to the varying time points of assessment and the differences in the control

In order to establish the normal expected renal growth in preterm neonates, in a recent study van Venrooji *et al* (2010) examined kidney lengths and volumes of 30 very preterm neonates (gestational age ranging from 23.6 weeks to 30.6 weeks) at 1, 4 and 8 weeks after birth through ultrasound measurements. Significant correlations were found between average renal size (volume and length) with both body weight and age. The study also found no significant difference in growth rates between the extremely low birth weight group (<1.0 kg; GA 23.6 – 27.1 weeks) and the very low birth weight group (1.0 – 1.5 kg; GA 26.1 – 30.6 weeks). In another study, Huang and colleagues (2007) compared postnatal kidney growth in preterm neonates (<34 weeks gestation) to kidney growth *in utero* in a control group (28-40 weeks of gestation). Kidney volumes were measured in 56 preterm neonates at postnatal time points ranging from 14 to 96 days after birth. In the control group, kidney volumes were measured within 48 hours of birth. Kidney volumes of preterm neonates with a postconceptional age (defined as the sum of gestational age and postnatal age) equivalent to less than 31 weeks of gestation were significantly larger compared to controls, whereas the preterm neonates with a postconceptional age greater than 31 weeks of gestation had a significantly smaller kidney volume compared to controls. In the preterm infants where kidney volume increased, it is likely that the increase in kidney size is indicative of the response of the neonatal kidney to increased functional demands. However, if this is the case, it is unclear why the renal response was different in the preterm neonates greater than 31 weeks of postconceptional age where kidney volume was significantly less than controls. Another study by Kent *et al* (2009) compared MRI measurements of kidney volume and kidney volume relative to body weight in extremely preterm neonates (25-28 weeks gestation) to term-born controls. Kidney volumes in the preterm neonates were measured once they reached term-corrected age (37-40 weeks gestation) and within the first 4 weeks of life in the term controls. Interestingly in that study,

Findings from older preterm infants, children and adults have been more consistent across studies and generally demonstrate a decrease in kidney size (relative to body size) compared to term controls. Firstly, Schmidt *et al* (2005) reported significantly smaller relative kidney volumes in preterm infants (born at less than 37 weeks of gestation) at 3 months of term-corrected age compared to 3-month-old term infants. Furthermore, preterm children at 18 months of term-equivalent age had slimmer shaped kidneys compared to term-born infants perhaps suggestive of decreased glomerular generations. In the most comprehensive study of renal growth in preterm infants to date, (involving 466 infants from 3 months through to two years of age), Drougia *et al* (2009) showed that kidney length was

**2.1 Clinical** *in vivo* **studies** 

groups used in each study.

no differences were found between groups.

et al., 2008). Development of the metanephros, begins at approximately week 5 of gestation with the outgrowth of the ureteric bud from the Wolffian duct (Saxen, 1987). Subsequent events include invasion of the mass of metanephric mesenchyme by the ureteric bud, followed by reciprocal inductive interactions between the ureteric bud and metanephric mesenchyme that lead to both dichotomous branching of the ureteric bud and the formation of nephrons at the ureteric bud tips (Moritz et al., 2008). Formation of the functional units of the kidney, the nephron, commences at approximately week 9 of gestation (Figure 1)(Blackburn, 2003).

As shown in the timeline in Figure 1, nephrogenesis in the human kidney is not complete until ~34-36 weeks of gestation with the majority of nephrons formed during the third trimester (from ~20 weeks of gestation onwards) (Hinchliffe et al., 1991). In very preterm and extremely preterm neonates, nephrogenesis is still on-going at the time of birth and continues in the *ex-utero* environment. Hence it is imperative to get a good understanding of how preterm birth affects the developing kidney and in particular the effects on nephrogenesis.

Fig. 1. A timeline of human nephrogenesis during gestation. Nephrogenesis is rapidly ongoing at the time when most preterm neonates are delivered.

To date, there have been few studies examining the effects of preterm birth on nephrogenesis. *In vivo,* clinical studies have utilised renal ultrasound and magnetic resonance imaging (MRI) to estimate kidney size as a proxy measure of nephron endowment. However, such extrapolations should be treated with caution. Although kidney size is generally a good predictor of nephron number, this may not be the case in the preterm infant, with kidney size likely to be influenced by glomerular and tubular hypertrophy and increased interstitial mass due to the increased postnatal functional demands. Hence, it is often difficult to make predictions based on parameters such as kidney size (Lodrup et al., 2008). In this regard, autopsy studies in deceased preterm

et al., 2008). Development of the metanephros, begins at approximately week 5 of gestation with the outgrowth of the ureteric bud from the Wolffian duct (Saxen, 1987). Subsequent events include invasion of the mass of metanephric mesenchyme by the ureteric bud, followed by reciprocal inductive interactions between the ureteric bud and metanephric mesenchyme that lead to both dichotomous branching of the ureteric bud and the formation of nephrons at the ureteric bud tips (Moritz et al., 2008). Formation of the functional units of the kidney, the nephron, commences at approximately week 9 of gestation (Figure

As shown in the timeline in Figure 1, nephrogenesis in the human kidney is not complete until ~34-36 weeks of gestation with the majority of nephrons formed during the third trimester (from ~20 weeks of gestation onwards) (Hinchliffe et al., 1991). In very preterm and extremely preterm neonates, nephrogenesis is still on-going at the time of birth and continues in the *ex-utero* environment. Hence it is imperative to get a good understanding of how preterm birth affects the developing kidney and in particular the effects on

Fig. 1. A timeline of human nephrogenesis during gestation. Nephrogenesis is rapidly on-

To date, there have been few studies examining the effects of preterm birth on nephrogenesis. *In vivo,* clinical studies have utilised renal ultrasound and magnetic resonance imaging (MRI) to estimate kidney size as a proxy measure of nephron endowment. However, such extrapolations should be treated with caution. Although kidney size is generally a good predictor of nephron number, this may not be the case in the preterm infant, with kidney size likely to be influenced by glomerular and tubular hypertrophy and increased interstitial mass due to the increased postnatal functional demands. Hence, it is often difficult to make predictions based on parameters such as kidney size (Lodrup et al., 2008). In this regard, autopsy studies in deceased preterm

going at the time when most preterm neonates are delivered.

1)(Blackburn, 2003).

nephrogenesis.

neonates have provided insight into how preterm birth affects the structure of the kidney and the number of glomerular generations formed within the kidney. As well, carefully controlled experimental studies in the nonhuman primate provide valuable insight into the effects of preterm birth on nephrogenesis and on the total number of nephrons formed.

#### **2.1 Clinical** *in vivo* **studies**

Table 1 summarizes the main *in vivo* clinical studies that have investigated the effects of preterm birth on kidney length and volume. Overall, the findings in relation to the effects of preterm birth on kidney size are conflicting; however, it is difficult to compare between studies due to the varying time points of assessment and the differences in the control groups used in each study.

In order to establish the normal expected renal growth in preterm neonates, in a recent study van Venrooji *et al* (2010) examined kidney lengths and volumes of 30 very preterm neonates (gestational age ranging from 23.6 weeks to 30.6 weeks) at 1, 4 and 8 weeks after birth through ultrasound measurements. Significant correlations were found between average renal size (volume and length) with both body weight and age. The study also found no significant difference in growth rates between the extremely low birth weight group (<1.0 kg; GA 23.6 – 27.1 weeks) and the very low birth weight group (1.0 – 1.5 kg; GA 26.1 – 30.6 weeks). In another study, Huang and colleagues (2007) compared postnatal kidney growth in preterm neonates (<34 weeks gestation) to kidney growth *in utero* in a control group (28-40 weeks of gestation). Kidney volumes were measured in 56 preterm neonates at postnatal time points ranging from 14 to 96 days after birth. In the control group, kidney volumes were measured within 48 hours of birth. Kidney volumes of preterm neonates with a postconceptional age (defined as the sum of gestational age and postnatal age) equivalent to less than 31 weeks of gestation were significantly larger compared to controls, whereas the preterm neonates with a postconceptional age greater than 31 weeks of gestation had a significantly smaller kidney volume compared to controls. In the preterm infants where kidney volume increased, it is likely that the increase in kidney size is indicative of the response of the neonatal kidney to increased functional demands. However, if this is the case, it is unclear why the renal response was different in the preterm neonates greater than 31 weeks of postconceptional age where kidney volume was significantly less than controls. Another study by Kent *et al* (2009) compared MRI measurements of kidney volume and kidney volume relative to body weight in extremely preterm neonates (25-28 weeks gestation) to term-born controls. Kidney volumes in the preterm neonates were measured once they reached term-corrected age (37-40 weeks gestation) and within the first 4 weeks of life in the term controls. Interestingly in that study, no differences were found between groups.

Findings from older preterm infants, children and adults have been more consistent across studies and generally demonstrate a decrease in kidney size (relative to body size) compared to term controls. Firstly, Schmidt *et al* (2005) reported significantly smaller relative kidney volumes in preterm infants (born at less than 37 weeks of gestation) at 3 months of term-corrected age compared to 3-month-old term infants. Furthermore, preterm children at 18 months of term-equivalent age had slimmer shaped kidneys compared to term-born infants perhaps suggestive of decreased glomerular generations. In the most comprehensive study of renal growth in preterm infants to date, (involving 466 infants from 3 months through to two years of age), Drougia *et al* (2009) showed that kidney length was

Effects of Preterm Birth on the Kidney 65

significantly decreased in small-for-gestational age preterm infants (born 28-34 weeks of gestation) compared to those born at term. In addition, Kwinta *et al* (2011) recently reported in 6 to 7 year old children, reduced absolute and relative kidney volumes in those born extremely low birth weight (26 – 29 weeks of gestation) compared to children born full-term. Furthermore, extremely low birth weight (birth weight <1.0kg; 26.3 - 27.7 weeks of gestation) children in a similar age group (5-6 year olds), had significantly reduced right and left kidney volumes and lengths compared to very low birth weight children (birth weight 1.0-1.5 kg; 29.9 - 31.3 weeks of gestation) (Zaffanello et al., 2010). This is the only study to date demonstrating significant differences in kidney size due to severity of prematurity. To our knowledge, there has only been one study to date examining kidney size in preterm individuals in adulthood. In that study, 20-year-old adults born preterm (less than 32 weeks of gestation) had significantly smaller absolute and relative left kidney lengths and volumes compared to 20 year-old term controls; the difference was only significant in females

There have been three published human autopsy studies (apart from case studies) that have examined the effect of preterm birth on postnatal nephrogenesis (Rodriguez et al., 2004; Faa et al., 2010; Sutherland et al., 2011b). Since non-uniform portions of the kidney are usually collected at autopsy, stereological methods cannot be accurately employed. Under these circumstances, the medullary ray glomerular generation counting method (Hinchliffe et al., 1992b) (also referred to as radial glomerular count or glomerular generation count) is a useful technique to provide insight into renal maturity and potentially nephron endowment. The method involves counting all developed glomeruli along one side of clearly distinguishable medullary rays in histological renal sections. Glomeruli are counted from the inner to outer renal cortex. Importantly, in our studies we have found a strong correlation between glomerular generation number and nephron number, which supports

In one of the first autopsy studies conducted, the number of radial glomerular counts in kidneys from extremely preterm neonates (56 neonates) was compared to 10 full-term infants (Rodriguez et al., 2004). Radial glomerular counts were found to be significantly reduced in preterm infants; however, since many of the preterm infants were also intrauterine growth restricted (IUGR) it is difficult to determine the effects of preterm birth *per se* from this study. In a smaller study, Faa *et al* (2010) have reported significantly reduced radial glomerular counts and marked inter-individual variability in the number of glomerular generations among the kidneys from preterm neonates compared to term newborns. In that study, 8 human fetuses, 12 preterm neonates and 3 full-term neonates

As follow on to these studies, we have recently undertaken a study examining kidneys obtained at autopsy from 28 preterm infants and 32 still-born gestational controls (Sutherland et al., 2011b); the preterm group included 6 infants that were also IUGR. Importantly, analyses comparing growth restricted and non-growth restricted kidneys demonstrated no significant differences, although the findings are limited by the small sample of growth restricted neonates. In contrast to the studies described above, we found accelerated nephrogenesis in the preterm group demonstrated by an increase in the number of glomerular generations, a decreased nephrogenic zone width (suggesting

were examined; it is unknown whether any of the neonates were also IUGR.

(Keijzer-Veen et al., 2010a).

**2.2 Human autopsy studies** 

the validity of the technique (Sutherland et al., 2011a).


Table 1. The main studies that have examined renal size (volume and length) in preterm neonates, children and adults (PCA=post-conceptional age, ELBW=extremely low birth weight, VLBW=very low birth weight, SGA=small for gestational age, AGA=appropriately grown for gestational age)

**Study Groups** 

Preterm (n=56) Gestational controls (n=44)

VLBW (n=16)

Preterm (n=17)

Preterm 28-34 weeks (SGA: n=100 AGA: n=54) Preterm 34-36 weeks (SGA: n=80 AGA: n=61) Term (AGA: n=90 SGA: n=81)

Preterm (n=59) Term (n=801)

VLBW (n=43)

Term (n=37)

Preterm SGA (n=22) Preterm AGA (n=29) Term AGA (n=30)

**and sample size Outcomes on renal size** 

Term (n=13) No differences in volume

Larger relative volume in preterm <31 weeks PCA Smaller relative volume in preterm >31 weeks PCA

No differences in volume or length

Smaller right kidney length in preterm SGA 28-34 week group compared to AGA group at all postnatal time points

Smaller relative volumes at 3 and 18 months

ELBW have smaller volumes (Right, Left, Total) ELBW have smaller length (Right and Left)

Smaller absolute volume; not significant when adjusted for body surface area

> Length and volume appeared to be in normal range; no comparison group

Smaller length and volume in preterm (SGA and AGA) female group compared to term

Term (n=38) ELBW have smaller volume

**Age at assessment** 

37-40 weeks (term equivalent)

36 and 40 weeks (term equivalent) 3, 6, 12 and 24 months

> 40 weeks 3 and 18 months

Zaffanello (2010) 26-31 5-6 years ELBW (n=36)

Kwinta (2011) 26-29 6-7 years ELBW (n=78)

Rakow (2008) <32 (mean=27) 9-12 years Preterm (n=33)

Soriano (2005) 23-35 6-12 years Preterm (n=27)

Table 1. The main studies that have examined renal size (volume and length) in preterm neonates, children and adults (PCA=post-conceptional age, ELBW=extremely low birth weight, VLBW=very low birth weight, SGA=small for gestational age, AGA=appropriately

(2010) <32 (mean=31) 20 years

(2010) 23-30 1, 4, 8 weeks ELBW (n=14)

**Author & Year** 

Van Venrooij

Kent (2009) 25-28

Drougia (2008) 28-41

Schmidt (2005) <37

Rodriguez-

Keijzer-Veen

grown for gestational age)

**Gestational age at birth (weeks), range** 

Huang (2007) 24-36 14-96 days

significantly decreased in small-for-gestational age preterm infants (born 28-34 weeks of gestation) compared to those born at term. In addition, Kwinta *et al* (2011) recently reported in 6 to 7 year old children, reduced absolute and relative kidney volumes in those born extremely low birth weight (26 – 29 weeks of gestation) compared to children born full-term. Furthermore, extremely low birth weight (birth weight <1.0kg; 26.3 - 27.7 weeks of gestation) children in a similar age group (5-6 year olds), had significantly reduced right and left kidney volumes and lengths compared to very low birth weight children (birth weight 1.0-1.5 kg; 29.9 - 31.3 weeks of gestation) (Zaffanello et al., 2010). This is the only study to date demonstrating significant differences in kidney size due to severity of prematurity. To our knowledge, there has only been one study to date examining kidney size in preterm individuals in adulthood. In that study, 20-year-old adults born preterm (less than 32 weeks of gestation) had significantly smaller absolute and relative left kidney lengths and volumes compared to 20 year-old term controls; the difference was only significant in females
