**3. Results**

#### *3.1. Natural Course of sCr*

For the time course of sCr, the initial increase in sCr peaked at postnatal day (P) 10 and postnatal week 2 in EPT at 25–26 and 23–24 weeks of gestation, respectively (Figure 1A). The peak sCr showed a higher tendency without statistical significance in EPT at gestation of 23–24 weeks than in those at 25–26 weeks. After this, sCr gradually declined until postnatal week 9 in both subgroups and reached sCr at birth at postnatal week 6 and 7 in EPT at 25–26 and 23–24 weeks' gestation, respectively. In EPT of 25–26 weeks' gestation without HS PDA, sCr showed a similar time course without statistical significance with the EPT of 25–26 weeks' gestation with HS PDA (Supplementary Figure S1).

**Figure 1.** Time course of the mean serum creatinine levels within different gestational groups and prevalence of acute kidney injury (AKI) by stage in a week interval. (**A**) Serum creatinine profile for the 6 weeks of life in accordance to different gestational age groups; (**B**) Prevalence of AKI by stage in a week interval. \* *p* < 0.05 in comparison between infants at 23–24 and those at 25–26 weeks of gestation.

#### *3.2. AKI Prevalence*

Table 1 demonstrates the cumulative AKI stage within the first six postnatal weeks according to neonatal KDIGO classification stratified by gestational age group. While only 6% and 8% were at AKI stage 0 in EPT at 25–26 and 23–24 weeks of gestation, respectively, the prevalence of AKI stage 3 tended to be higher (72%) in EPT at gestation of 23–24 weeks than in EPT of 25–26 weeks (57%) without statistical significance.

For the time course of the prevalence of AKI by stage in a week interval, postnatal increase in the prevalence of AKI stage 3 peaked at postnatal week 2 in both groups, and afterwards prevalence gradually declined till postnatal week 6 (Figure 1B). The prevalence of AKI stage 3 at postnatal week 2 and 6 in EPT at gestation of 23–24 weeks was higher significantly than that in EPT at gestation of 25–26 weeks.

#### *3.3. Clinical Characteristics According to AKI Stage*

Demographic and clinical characteristics in each study group in accordance with AKI stages are described in Table 2. In EPT with AKI stage 3, total GA was significantly lower, and male gender in EPT at gestation of 25–26 weeks had higher GA than in EPT with AKI 0–2. While total oliguria and oliguria in EPT at 25–26 weeks of gestation with AKI stage 3 were higher compared with those with AKI stage 0–2 significantly, no differences were observed in other clinical variables between AKI stage 3 and stage 0–2 groups.

#### *3.4. Adverse Outcomes According to AKI Stage*

While sepsis in EPT at 25–26 weeks of gestation with AKI stage 3 was slightly higher than infants with AKI stage 0–2, no significant differences were found in other adverse outcomes, including mortality and BPD, between the AKI stage 3 and stage 0–2 groups (Table 3).


**Table 2.** Demographics and clinical characteristics of EPT in period II: Stage 0–2 AKI vs. stage 3 AKI.

\* *p* < 0.05 compared with Stage 0–2 AKI. † *p* < 0.05 compared with infants at 23–24 weeks of gestation.

**Table 3.** Adverse outcomes of infants in period II: stage 0–2 AKI vs. stage 3 AKI.


*p* < 0.05 compared with Stage 0–2 AKI.

#### *3.5. Adjusted ORs for Risk of Adverse Outcomes by AKI Stage 3*

The adjusted ORs for the risk of unfavorable outcomes were not increased in AKI stage 3 in multivariate analyses (Table 4). In addition, the adjusted ORs for outcomes were not elevated by prolonged duration (per week) of AKI stage 3 (Table 5).

**Table 4.** Adjusted ORs \* for risk of adverse outcomes by presence of Stage 3 AKI.


OR, odds ratio; AKI, acute kidney injury; CI, confidence interval; BPD, bronchopulmonary dysplasia; IVH, intraventricular hemorrhage; PVL, periventricular. leukomalacia; ROP, retinopathy of prematurity; NEC, necrotizing enterocolitis. \* adjusted for birth weight, gestational age, small for gestational age, antenatal steroid use, 1-min and 5-min Apgar scores, hypertension in pregnancy, chorioamnionitis.


**Table 5.** Adjusted ORs \* for risk of adverse outcomes by duration (per week) of stage 3 AKI.

OR, odds ratio; AKI, acute kidney injury; CI, confidence interval; BPD, bronchopulmonary dysplasia; IVH, intraventricular hemorrhage; PVL, periventricular. leukomalacia; ROP, retinopathy of prematurity; NEC, necrotizing enterocolitis. \* adjusted for birth weight, gestational age, small for gestational age, antenatal steroid use, 1-min and 5-min Apgar scores, hypertension in pregnancy, chorioamnionitis.

## **4. Discussion**

This present study is the first human study demonstrating the natural postnatal evolution of sCr and the prevalence of AKI in the peri-viable EPT at gestation of 23–26 weeks with HS PDA who received exclusive conservative management. In this present study, while sCr at birth was about the same between the study groups, representing maternal levels [8,9], the peak sCr and the peak prevalence of AKI stage 3 during the first two postnatal weeks were higher in EPT at gestation of 23–24 weeks than in EPT of 25–26 weeks. These findings sugges<sup>t</sup> that very low initial GFR and tubular immaturity, and its slow improvement, might be inversely related to GA in these peri-viable EPT [7,11,14,27,41]. Furthermore, as HS PDA closed averagely at postnatal day 41 and 53 in EPT of 25–26 and 23–24 weeks' gestation, respectively [31,32], our data suggests that renal immaturity inversely related to GA, rather than HS PDA induced renal hypo-perfusion [17,27–29], are primarily responsible for the initial postnatal rise in sCr and the peak prevalence of AKI stage 3 during the first two postnatal weeks [42].

sCr after peak declined rapidly, and reached a birth sCr at postnatal week 6 and 7, compatible with corrected GA of 31–32 weeks in EPT at 25–26 and 23–24 weeks of gestation, respectively, and after then, sCr approached a stable plateau in both GA subgroups, indicating a steady state between endogenous Cr production and excretion [13,27,41]. In the preterm infant, the GFR is lower until the full nephrogenesis is finished by 34–35 weeks of gestation [43–46]. Overall, our data sugges<sup>t</sup> that despite its inverse relationship with GA, the postnatal renal maturation and the ensuing logarithmic increase in GFR are accelerated by 2–3 weeks in these peri-viable EPT [47].

Although AKI in premature infants has been known to be related to raised mortality [11,17–21] and morbidity rates, including BPD [2,22,23] and IVH [24], evidence supporting their direct causal relationships are lacking. In contrast, while sepsis rate was more elevated in AKI stage 3 than in stage 0–2, only in EPT at gestation of 25–26 but not of 23–24 weeks, neither the presence nor the prolonged duration of AKI stage 3 was associated with elevated mortality or any morbidities rates including BPD and IVH. The reasons for our results, which are contradictory to other studies showing increased mortality and/or morbidities [2,11,17–24], are di fficult to explain. Few data are available for the peri-viable EPT of 23–24 week's gestation, and actively treated with pharmacologic agents for HS PDA could be cofounders in other studies. A further controlled study with a homogeneous patient population and same clinical managemen<sup>t</sup> policy might be necessary to clarify these contradictory findings.

Fluid therapy and drug dosing in premature infants need to be adjusted according to renal function, i.e., GFR [3,4,45,46]. Considering our results, which showed greater and delayed peak of sCr and very high prevalence of AKI stage 3 at the first two postnatal weeks in these peri-viable EPT with HS PDA, judicious fluid restriction might be prerequisite for the success of non-interventional treatment for HS PDA [2,48,49]. In our prior studies [31,32], fluid volume of 67 mL/kg/day at day of birth, and raising up to ≤115 mL/kg/day for the first month was accomplished without restricting caloric support or elevating the risk of renal dysfunctions and electrolyte imbalance. Acute fluid

overload in the newborn infants was associated with adverse outcomes, including mortality [50] and morbidities [2,48]. Furthermore, the extent of volume overload in critical adult patients also correlated with worse clinical course [51–53]. In contrast, fluid restriction was associated with reduced mortality [54] and morbidities, such as PDA and NEC [55]. Overall, these findings sugges<sup>t</sup> that judicious fluid restriction might be essential for the success of the non-interventional conservative treatment of HS PDA in EPT [45].

Heterogeneous time intervals and variable number of follow-up sCr measurements for review could be limitations of this retrospective uncontrolled observational single center study. The absence of long-term outcome assessments including growth and neurodevelopment might be another limitation of this study. However, a relatively large sample size (*n* = 50) of the peri-viable EPT at gestation of 23–24 weeks with HS PDA exclusively managed with a conservative treatment, as well as less variation in clinical managemen<sup>t</sup> policies, might be a strength of this single-center study.
