**3.4 Proteinuria**

70 Basic Nephrology and Acute Kidney Injury

Acute kidney injury (AKI; previously referred to as acute renal failure) is reported to occur in 8% to 24% of preterm neonates admitted to neonatal intensive care units (Stapleton et al., 1987; Hentschel et al., 1996). The current diagnosis of AKI is primarily based on the RIFLE system, which categorises the stages of increasing AKI severity: Risk, Injury, Failure, Loss and End-stage kidney disease (ESKD) (Bellomo et al., 2004). This system was further modified following recommendations from the acute kidney injury network (AKIN) (Mehta et al., 2007). The initial clinical indication of AKI risk includes a 50% increase in serum creatinine (or ≥ 0.3 mg/dl within a 48 hour period), and/or a urine output less than 0.5 mg/kg/hr for a period of six hours (Bellomo et al., 2004; Mehta et al., 2007), which are changes indicative of a significantly reduced GFR. Classifications for the definition of AKI

The causes of AKI in the preterm neonate are primarily pre-renal in origin, arising from conditions which affect renal perfusion such as hypotension, hypoxia and sepsis (Stapleton et al., 1987; Cataldi et al., 2005). These in turn lead to apoptotic, necrotic and inflammatory processes within the kidney (Ueda and Shah, 2000; Bonventre, 2007). Importantly, AKI in the preterm neonate may subsequently lead to long-term chronic renal disease (Abitbol et

In a study involving 172 preterm neonates by Cataldi *et al.* 2005, the risk factors for AKI were found to be maternal and neonatal drug administration (non-steroidal antiinflammatory drugs (NSAIDs) and antibiotics, especially ceftazidime), a low Apgar score, and a patent ductus arteriosus. Interestingly, gestational age did not affect risk of AKI, however, the majority of AKI cases (79%) weighed < 1.5 kg at birth (Cataldi et al., 2005). In a larger study by Cuzzolin *et al.* (2006), involving 281 preterm neonates, a number of risk factors for AKI were also identified. These included maternal NSAID administration, low Apgar score, respiratory distress syndrome, neonatal drug administration (antibiotics and NSAIDs), and a number of clinical interventions (intubation at birth, catheterization,

Given the importance of the early diagnosis and treatment of AKI, there has been much recent focus on the discovery of novel urinary biomarkers. The expectation of a new biomarker is to enable the diagnosis of cellular injury before a decline in renal function occurs. For example, serum creatinine is not elevated until 48-72 hours after an acute injury has occurred (Moran and Myers, 1985); such a prolonged delay before diagnosis and treatment likely results in further renal injury. As Rosner (2009) describes, it would be optimal if a biomarker could be developed to: 1) assess the response to, and any adverse effects of therapeutic interventions 2) indicate the severity of renal injury 3) inform on the etiology of the injury and 4) identify the location of injured cells. In a systematic review of the current literature, Parikh *et al.* (2010) determined that the molecules with the most promise for the diagnosis of established AKI include interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1), N-acetyl-beta-D-glucosaminidase (NAG) and neutrophil gelatinaseassociated lipocalin (NGAL). NGAL, IL-18, fatty acid binding protein (FABP), and cystatin-C are the most encouraging biomarkers for the early diagnosis of AKI, given that the upregulation of these molecules following injury onset precedes the rise in serum creatinine

In the preterm neonate, a small number of studies have been conducted for the assessment of urinary NGAL levels, with mixed results. These studies have shown that the highest NGAL levels are evident in the neonates that are critically ill, with and without evidence of

in a neonatal specific population, however, have not been developed.

phototherapy, and mechanical ventilation).

by many hours (Parikh et al., 2010).

**3.3 Acute kidney injury**

al., 2003).

Proteinuria, the presence of high levels of protein in the urine, may be of glomerular and/or tubular origin. The number of different proteins that have been identified in the adult urinary proteome is 1,543, and these are primarily of membrane, extracellular and lysosomal origin (Adachi et al., 2006). Despite this large number, unless renal function is impaired, proteins are normally only present at very low levels in urine, due to the function of the glomerular filtration barrier and tubular reabsorption capabilities.

Presence of high molecular weight (HMW) proteins in the urine, such as albumin traditionally indicates a disruption in the integrity of the glomerular filtration barrier. Recent debate, however, has suggested that the contribution of tubular reabsorption of albumin from the filtrate may be greater than previously considered (Comper et al., 2008). In general, albuminuria is a strong marker for renal and cardiovascular disease, and a risk factor for mortality (Matsushita et al., 2010; Methven et al., 2011). Normally, adults excrete less than 30 mg of albumin per 24 hours (Mathieson, 2004). Urinary albumin levels between 30 – 300 mg in 24 hours is considered microalbuminuria, with levels greater than 300 mg classified as macroalbuminuria (Mathieson, 2004). Traditionally, 24 hour urine samples were required for reliable estimates of urinary protein. However, single random spot samples with protein levels corrected for urine creatinine, have been shown to be significantly correlated with results from 24 hour collections, and are equally effective in the prediction of outcomes (Ralston et al., 1988; Methven et al., 2011). In neonates, 24 hour urine collection is difficult, therefore analysis of urinary protein levels are undertaken using spot urine samples obtained using urine collection bags.

Low molecular weight (LMW) proteins, such as α1-microglobulin, β2-microglobulin and retinol binding protein pass freely through the glomerular filter and undergo reuptake via proximal tubule cells (Tomlinson, 1992). Megalin and cubulin have been identified as important receptors involved in tubular protein uptake, with mutations in the receptors resulting in proteinuria (Christensen and Birn, 2001). To date, LMW protein levels in the urine are not routinely measured in the clinical setting. Importantly, however, amongst the LMW proteins there may be potential novel biomarkers of tubular cell injury and this requires further research (Rosner, 2009; Parikh et al., 2010).

In the preterm neonate, few studies have been conducted to examine urine protein excretion. In general, there is a high variability in urine albumin levels between individual neonates (Clark et al., 1989; Fell et al., 1997), with the highest levels exhibited by those with a low gestational age at birth and those that are clinically unstable (Galaske, 1986; Clark et al., 1989; Tsukahara et al., 1994; Fell et al., 1997; Awad et al., 2002b). The majority of studies have only been conducted during the first week of life following preterm birth. However, in a study by Tsukahara *et al.* (1994) urine albumin levels were assessed in preterm and term

Effects of Preterm Birth on the Kidney 73

(foundation group of cells) surrounded by a layer of podocytes with scant, if any, capillarisation. Our findings thus strongly suggest that it is the very immature glomeruli (possibly those formed in the extrauterine environment) that are particularly vulnerable to preterm birth. Given the gross abnormalities observed in these glomeruli, it is unlikely that these glomeruli will ever be functional and thus, it is expected that they will be subsequently resorbed into the surrounding tissue. In the short-term, such abnormalities will likely lead to marked impairment of renal function in the neonate if a high proportion of the nephrons are affected, or to minor impairment if only a small proportion are abnormal. When the kidney is severely affected this will adversely impact on the number of functional nephrons at the beginning of life and thus reduce the long-term functional reserve of the kidney, rendering it

The cause(s) of the glomerular abnormalities in the preterm infant is currently unknown. Importantly in this regard, we have shown that there is a wide variation in the proportion of abnormal glomeruli within the kidneys of preterm infants, with the kidneys of some preterm infants appearing morphologically normal whereas in others a large proportion of the glomeruli appear abnormal (Sutherland et al., 2011b). Given the wide variation in the proportion of abnormal glomeruli within the kidneys of preterm infants, this suggests that it is not preterm birth *per se* that leads to the glomerular abnormalities; instead they are likely due to factors often associated with preterm birth as shown in Figure 3. It is likely that these deleterious effects may relate to: 1) adverse factors in the *in utero* environment that have led to premature delivery, 2) factors in the neonatal care of the preterm infant and 3) pharmacological interventions/therapies administered to mothers prior to birth and/or the infant after birth. There are many factors which apply to each of these categories; below we

Fig. 3. Depiction of the potential factors that may contribute to impaired nephrogenesis in

the preterm kidney and consequently lead to a reduced nephron endowment and

vulnerability to renal disease in adulthood

vulnerable to hypertension and secondary life style insults.

have selected some that we consider are important for future research.

neonates over the first 28 days of life. Urine albumin levels were found to remain relatively stable postnatally over the one month period in the term neonates, whereas in the preterm neonates, urine albumin was seen to decrease with increasing postnatal age. These findings suggest that the glomerular filtration barrier following preterm birth is structurally immature, until beyond one month of age.

Urinary β2-microglobulin levels have also been shown to be significantly greater in the preterm infant compared to term-born infants throughout the first month of life (Aperia et al., 1981; Tsukahara et al., 1990; Tsukahara et al., 1994), and are decreased with increasing gestational and postnatal age (Takieddine et al., 1983). Similarly, levels of 1-microglobulin and RBP are higher in preterm neonates than neonates born at term (Clark et al., 1989; Fell et al., 1997; Awad et al., 2002a). To date, however, it remains unclear whether the increased urinary high- and low- molecular weight protein levels reported in preterm neonates are associated with renal immaturity and/or injury. The high variability in urinary protein levels may also reflect differences in the postnatal clinical course in preterm neonates; further studies are necessary to verify whether this is the case.

#### **3.5 Long-term effects of preterm birth on renal function**

Renal function in preterm-born children and adults, has to date only been investigated in a small number of studies, with inconclusive results. In school-aged children, Rakow *et al.* (2008) found no difference in GFR or urinary levels of both HMW and LMW proteins between children born less than 32 weeks gestational age, and those that were born at term. Similarly, Vanpee *et al.* (1992) determined no difference in renal function in preterm and term-born children at 8 years of age, despite lower GFR and higher urine albumin levels being evident in the preterm group at 9 months of age. In contrast, however, a study by Rodriguez-Soriano and colleagues (2005) reported that GFR was significantly reduced in preterm-born children compared to term controls, with impairments in electrolyte excretion also evident. Furthermore, in children examined at 6-8 years of age, Iacobelli et al. (2007) demonstrated microalbuminuria in 8.3% of the preterm neonates, which was associated with postnatal factors such as neonatal hypotension and increased catch-up growth. Increased risk of renal demise was also evident in individuals born preterm who were obese during childhood (Abitbol et al., 2009).

Two studies have also been conducted to examine renal function in young adults (20-30 years of age), with both Keijzer-Veen *et al.* (2007) and Kistner *et al.* (Kistner et al., 2000) finding no effect of preterm birth on GFR or albuminuria. To the contrary, in a cohort of 19 year old young adults, those who were born preterm as well as IUGR, there was a significant reduction in GFR (Keijzer-Veen et al., 2005). Given these results in preterm-born children and adults, there is some suggestion that preterm birth adversely affects the growth and functional capacity of the kidney and may result in progressive renal failure later in life. Importantly, adverse consequences appear to be more likely to occur in combination with other insults. Therefore, future research must be directed towards identifying these insults and their effects on the structure and function of the kidney.

### **4. Preterm birth leads to glomerular abnormalities – Areas of future research**

One of the most important findings we have shown thus far, is the presence of abnormal glomeruli in both the human and nonhuman primate (baboon) preterm kidney. These abnormal glomeruli are located in the outer renal cortex and are in the most immature stage of development (stage 1); they are composed of an undifferentiated glomerular anlage of cells

neonates over the first 28 days of life. Urine albumin levels were found to remain relatively stable postnatally over the one month period in the term neonates, whereas in the preterm neonates, urine albumin was seen to decrease with increasing postnatal age. These findings suggest that the glomerular filtration barrier following preterm birth is structurally

Urinary β2-microglobulin levels have also been shown to be significantly greater in the preterm infant compared to term-born infants throughout the first month of life (Aperia et al., 1981; Tsukahara et al., 1990; Tsukahara et al., 1994), and are decreased with increasing gestational and postnatal age (Takieddine et al., 1983). Similarly, levels of 1-microglobulin and RBP are higher in preterm neonates than neonates born at term (Clark et al., 1989; Fell et al., 1997; Awad et al., 2002a). To date, however, it remains unclear whether the increased urinary high- and low- molecular weight protein levels reported in preterm neonates are associated with renal immaturity and/or injury. The high variability in urinary protein levels may also reflect differences in the postnatal clinical course in preterm neonates;

Renal function in preterm-born children and adults, has to date only been investigated in a small number of studies, with inconclusive results. In school-aged children, Rakow *et al.* (2008) found no difference in GFR or urinary levels of both HMW and LMW proteins between children born less than 32 weeks gestational age, and those that were born at term. Similarly, Vanpee *et al.* (1992) determined no difference in renal function in preterm and term-born children at 8 years of age, despite lower GFR and higher urine albumin levels being evident in the preterm group at 9 months of age. In contrast, however, a study by Rodriguez-Soriano and colleagues (2005) reported that GFR was significantly reduced in preterm-born children compared to term controls, with impairments in electrolyte excretion also evident. Furthermore, in children examined at 6-8 years of age, Iacobelli et al. (2007) demonstrated microalbuminuria in 8.3% of the preterm neonates, which was associated with postnatal factors such as neonatal hypotension and increased catch-up growth. Increased risk of renal demise was also evident in individuals born preterm who were obese

Two studies have also been conducted to examine renal function in young adults (20-30 years of age), with both Keijzer-Veen *et al.* (2007) and Kistner *et al.* (Kistner et al., 2000) finding no effect of preterm birth on GFR or albuminuria. To the contrary, in a cohort of 19 year old young adults, those who were born preterm as well as IUGR, there was a significant reduction in GFR (Keijzer-Veen et al., 2005). Given these results in preterm-born children and adults, there is some suggestion that preterm birth adversely affects the growth and functional capacity of the kidney and may result in progressive renal failure later in life. Importantly, adverse consequences appear to be more likely to occur in combination with other insults. Therefore, future research must be directed towards identifying these insults

**4. Preterm birth leads to glomerular abnormalities – Areas of future research**  One of the most important findings we have shown thus far, is the presence of abnormal glomeruli in both the human and nonhuman primate (baboon) preterm kidney. These abnormal glomeruli are located in the outer renal cortex and are in the most immature stage of development (stage 1); they are composed of an undifferentiated glomerular anlage of cells

immature, until beyond one month of age.

during childhood (Abitbol et al., 2009).

further studies are necessary to verify whether this is the case.

**3.5 Long-term effects of preterm birth on renal function** 

and their effects on the structure and function of the kidney.

(foundation group of cells) surrounded by a layer of podocytes with scant, if any, capillarisation. Our findings thus strongly suggest that it is the very immature glomeruli (possibly those formed in the extrauterine environment) that are particularly vulnerable to preterm birth. Given the gross abnormalities observed in these glomeruli, it is unlikely that these glomeruli will ever be functional and thus, it is expected that they will be subsequently resorbed into the surrounding tissue. In the short-term, such abnormalities will likely lead to marked impairment of renal function in the neonate if a high proportion of the nephrons are affected, or to minor impairment if only a small proportion are abnormal. When the kidney is severely affected this will adversely impact on the number of functional nephrons at the beginning of life and thus reduce the long-term functional reserve of the kidney, rendering it vulnerable to hypertension and secondary life style insults.

The cause(s) of the glomerular abnormalities in the preterm infant is currently unknown. Importantly in this regard, we have shown that there is a wide variation in the proportion of abnormal glomeruli within the kidneys of preterm infants, with the kidneys of some preterm infants appearing morphologically normal whereas in others a large proportion of the glomeruli appear abnormal (Sutherland et al., 2011b). Given the wide variation in the proportion of abnormal glomeruli within the kidneys of preterm infants, this suggests that it is not preterm birth *per se* that leads to the glomerular abnormalities; instead they are likely due to factors often associated with preterm birth as shown in Figure 3. It is likely that these deleterious effects may relate to: 1) adverse factors in the *in utero* environment that have led to premature delivery, 2) factors in the neonatal care of the preterm infant and 3) pharmacological interventions/therapies administered to mothers prior to birth and/or the infant after birth. There are many factors which apply to each of these categories; below we have selected some that we consider are important for future research.

Fig. 3. Depiction of the potential factors that may contribute to impaired nephrogenesis in the preterm kidney and consequently lead to a reduced nephron endowment and vulnerability to renal disease in adulthood

Effects of Preterm Birth on the Kidney 75

chorioamnionitis, is likely to exacerbate postnatal renal dysfunction and further studies are required to determine whether renal inflammation at the time of birth is associated with the

The administration of supplemental oxygen to preterm infants experiencing respiratory distress is standard therapy, however, the effects of high levels of oxygen in the bloodstream on the development of organs is not well understood. Although the levels of supplemental oxygen administered to the preterm infant have been substantially reduced in recent years, the levels of oxygen in the bloodstream remain elevated above normal; hence it is important that research is conducted into the effects of hyperoxia on nephrogenesis in the preterm infant. Certainly, experimental studies in the lung have shown that hyperoxia can lead to the generation of oxygen free radicals, infiltration of inflammatory cells, collagen deposition, cellular apoptosis and subsequent tissue injury (McGrath-Morrow and Stahl, 2001; Dieperink et al., 2006; Alejandre-Alcazar et al., 2007; Chen et al., 2007; Chetty et al., 2008).

The mode of ventilation of the preterm infant also has the potential to impact on the developing kidney, since alterations in airway pressure are reported to lead to significant cardiopulmonary haemodynamic changes (Polglase et al., 2009) which may subsequently affect renal perfusion. For instance, mechanical ventilation has been shown to alter renal hemodynamics by leading to an increase in intrathoracic pressure, therefore decreasing cardiac output, leading to renal vasoconstriction and decreased GFR (Arant, 1987). It is likely that changes in renal blood flow will directly influence the growth of the kidney. Hence, it is important in future research to determine the effects of altered renal hemodynamics on growth of the developing kidney and how this is influenced by different

In general, postnatal growth of the preterm infant is markedly attenuated when compared to the term infant and when compared to the normal rate of growth *in utero* for the same post-conceptional age (Ehrenkranz, 2000; Clark et al., 2003). Extrauterine growth restriction in premature neonates (defined as growth below the 10th percentile of intrauterine growth expectation) is likely to directly influence nephrogenesis. In support of this concept, Bacchetta et al (2009) reported lower GFRs (albeit in the normal range) in 7 year old children who had been born very preterm (< 30 weeks gestation) and who were either IUGR or extrauterine growth restricted. However, it is important to note that in some preterm infants there can be a disproportional increase in kidney size (relative to body weight) after birth (Huang et al., 2007; Sutherland et al., 2011b) most probably due to the increased functional demands on the kidney. Importantly we have shown in our preterm baboon studies that under these circumstances, there remains a significant correlation between kidney size and

Taken together, these studies highlight the importance that neonatal nutrition can potentially have on kidney development in the preterm infant. Since nephrogenesis is

formation of abnormal glomeruli in the neonatal period.

Whether this is also the case in other tissues has not been examined.

**4.2 Factors in the neonatal care of the infant** 

**4.2.1 Hyperoxia** 

**4.2.2 Ventilation** 

modes of ventilation.

**4.2.3 Extrauterine growth / nutrition** 

nephron number (Gubhaju et al., 2009).
