Kidney volume, kidney function, and ambulatory blood pressure in children born extremely preterm with and without nephrocalcinosis

The study was approved 5by the Ethical Committee at Karolinska University Hospital. Written and oral consent was obtained from all parents and children.

We identified 213 infants born before 28 weeks gestational age (GA) between 2008 and 2011 at the Karolinska University Hospital, Stockholm, Sweden (Fig. 1). Neonatal renal ultrasound was performed in 105 infants, but only 68 had traceable results and images. All neonatal investigations were performed by pediatric radiologists. NC was defined as hyper-echogenic reflections in cortex and or medulla visualized in longitudinal and transverse projections. Of the investigated 68 infants, 34 were diagnosed with NC (NC+) during their late neonatal period and 34 infants showed no signs of NC (NC−). There was no history of hyperoxaluria, cystinuria, or any type of renal tubular acidosis or a history of antenatal or postnatal diagnosis for urogenital malformation in any patient. Twenty-three families refused to participate and 4 children died after discharge from the neonatal unit. Twenty children with NC and 21 without NC during their neonatal period consented to participate in the study. The 172 non-participants (children without ultrasound investigation and children with ultrasound investigation but lost to follow-up or declined to participate or those with incomplete images for review) were not different from the participants with regard to perinatal characteristics. A total of 19 healthy children born at term with appropriate birthweight, without any congenital abnormalities, and with no history of kidney diseases selected from delivery room records were recruited as controls. All children were in good health at the time of the visits.

Clinical data was collected from the neonatal charts with particular attention to factors that could influence renal function, such as nephrotoxic substances: aminoglycosides, vancomycin, loop diuretics, thiazide diuretics, and antenatal and postnatal steroids. GA, birth weight, Apgar scores, intrauterine growth retardation, respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD) as defined by NIH, acute kidney injury (AKI) defined and staged by the KDIGO guidelines, patent ductus arteriosus (PDA) requiring treatment, sepsis episodes (clinical and/or culture verified), necrotizing enterocolitis (NEC) Bell stage II or more, surgical interventions for NEC, retinopathy of the premature (ROP) grade III or higher (and or any plus disease), and intraventricular hemorrhage (IVH) or parenchymal hemorrhage as defined by Papile were documented. Small for gestational age (SGA) was defined as a birth weight < − 2 standard deviations (SD) according to Swedish reference data for normal fetal growth [18].

At the visit, patient and parental medical histories, as well as maternal and paternal height and weight, were registered. The same research nurse performed all anthropometric measurements for weight, height, head circumference, and waist circumference on the children, who were wearing light indoor clothing. Height was measured using a wall-mounted stadiometer. Waist circumference was measured midway between the lower rib margin and the iliac crest using a normal measuring tape. Body mass index (kg/m²), body surface area (BSA = 0.007184 × Height^0.725 × Weight^0.425) [19] and waist-to-height ratio were calculated.

Office blood pressure was measured in all patients using an automated oscillometric device (GE Healthcare Dinamap Carescape V100). After a 30-min rest, three consecutive measurements were taken on the child’s non-dominant arm with an appropriate cuff size (bladder width that was at least 40% of the arm circumference [20]). We used a SPACELABS 90217A (SpaceLabs Medical Inc., Redmond, Washington, USA) device for ABPM, using the same cuff size as for the office measurements. The device was programmed to register blood pressure every 20 min between 07:00 AM and 21:00 PM and every 60 min during the night (21:00 PM–07:00 AM). Due to compliance difficulties in 21 children (12/20 NC+, 9/20 NC−) with neuropsychiatric disorder (ADHD, autism, and autism spectrum), we accepted > 50% of successful readings instead of the standard > 75% for statistical analysis [21]. The rest periods were described by the patient’s parents, and day and night measurements were adjusted accordingly. The non-dipper pattern was defined as nocturnal BP_Systolic or BP_Diastolic < 10% relative to the diurnal mean value [22]. Hypertension was defined as > 95th percentile according to gender and length. BP > 90th but < 95th percentile was defined as high normal BP [22].

Morning urine samples were collected from all study participants and analyzed for sodium, potassium, creatinine, albumin, calcium, phosphate, magnesium and protein HC (α-1-microglobulin) and immunoglobulin G. Sodium and potassium were measured using potentiometry with ion-selective electrodes (Cobas 8000, Cobas C ISE2, Roche, Basel Switzerland). Urine phosphate, calcium, and magnesium were measured using photometric technique (Cobas 8000 Cobas CC 701 Roche, Basel Switzerland), and urine albumin was measured using immunochemical and turbodimetric method (Cobas 8000 Cobas CC 701 Roche, Basel Switzerland). Protein HC and immunoglobulin G were measured by immunochemical and nephelometric method (BN Pro Spec, Siemens Healthcare, Erlangen, Germany).

Blood samples were taken in 55 patients (NC+,17; NC−, 21; control, 17) after placing a local application of a topical anesthetic cream containing 2.5% lidocaine and 2.5% prilocaine (EMLA; Astra Zeneca, Sodertalje, Sweden). Blood samples were investigated for plasma sodium and potassium using potentiometry with ion-selective electrodes (Cobas 8000, Cobas C ISE2, Roche, Basel Switzerland); calcium, phosphate, alkaline phosphatase, creatinine, and urea using photometric technique; and cystatin C using immunochemical and turbidometric method (Cobas 8000 Cobas CC 701 Roche, Basel Switzerland). For the estimation of GFR, we used the simplified Schwartz formula (with k = 36) as well as the cystatin C–based CAPA formula [23, 24].

Ultrasound of the kidneys during the neonatal period (2008–2011) was performed by pediatric radiologists using a Siemens S 2000 with a 6C2 curved transducer (Siemens, Erlangen, Germany) at an average age of 36 weeks postmenstrual age following local guidelines. All images from the neonatal period for the 41 included children were reviewed by a senior pediatric radiologist in 2018. Diagnosis of NC was confirmed in 39 of the 41 cases. In each group (NC+/NC−), one patient was misdiagnosed and moved to the opposite group. Kidney volume for the neonatal period was calculated in 36 out of the 41 patients using the equation for an ellipsoid described elsewhere [25] and expressed as a ratio to BSA (KV/BSA) [26]. In the remaining 5 patients, the reviewed images were incomplete and volume calculation therefore was not possible.

Ultrasound of the kidneys at school age visits was performed by a single experienced user. All investigations were performed with a Philips EPIQ 7G with SW1.5.2 software (Philips Ultrasound, Inc. 22100 Bothell Everett Hwy Bothell, WA 98021-8431, USA) using a C9-2 curved transducer. Multiple (at least two) measurements of kidney length, width, and depth were performed. An average of these measurements was entered in the equation for an ellipsoid, and KV was calculated [25]. Results from volumetric kidney measurements were adjusted for BSA by using linear regression analysis but also by using the ratio of kidney volume (KV) to BSA [26]. Predicted KV was calculated for each individual using the equation described by Dinkel et al. [25].

For descriptive statistics, continuous variables were presented with mean and standard deviation (SD). Continuous variables, approximately normally distributed, were analyzed with respect to the three defined groups, using analysis of variance (ANOVA). In order to adjust for continuous prognostic variables, covariance analysis was used (ANCOVA). Stepwise regression analysis was used to examine the impact of a set of prognostic variables. The coefficient of determination, R² was used to compare the precision of different models. Non-normal continuous variables were analyzed with Kruskal-Wallis test. Dichotomous variables were analyzed with cross tables and Pearson’s chi-square test. As additional analyses, the kidney volume was analyzed with a mixed effects model including right and left volume in the same analysis. A hierarchical model with the child as the main unit was set up, taking into consideration the correlation between right and left side, with the covariance structure unstructured (UN). In all statistical analyses, the relevant assumptions were checked. The significance level alpha was set to 0.05.

The neonatal characteristics and morbidities did not differ significantly between the NC+ and NC− groups; however, the NC+ group tended to be younger, smaller, and with more vancomycin, less prenatal steroid use, and more frequent IVH for all grades (Table 1). Children in the control group at visit were insignificantly older but significantly heavier, taller, and had larger head circumference, BSA, and LBM, but lower waist-to-height ratio. Between NC+ and NC−, only SDS for height and waist-to-height ratio were different at visit (Table 2). None of the kidney ultrasound investigations showed signs of persistent NC. One child from the control group was referred for follow-up ultrasound because of mild unilateral pelvic dilatation (excluded).

Unadjusted total kidney volumes (KV) were significantly lower for both preterm groups in comparison with controls (NC+ = 90.1 ml, NC− = 93.8 ml, control = 118.4 ml, p = 0.0004, ANOVA, Fig. 2). After adjusting KV for BSA, the analysis was no longer significant between NC+ and controls (p = 0.056) (Table 3). The mixed effects model analysis where BSA, gender, and each kidney side were included showed a significantly lower right and left KV for girls in the NC+ group compared with girls in the control group (p = 0.016). The effects of the following factors on total KV by using stepwise linear regression were also tested: age at visit, AKI, PDA, NEC, BPD, sepsis, and treatment with furosemide (days) and antenatal steroids. None of the tested confounding factors seemed to have the potential to explain a difference in total KV between the two preterm groups. Total KV calculated as the ratio of KV and BSA (KV/BSA) [26] was significantly lower for the NC+ group of preterm-born children compared with controls (p = 0.016), but not reaching significance for the NC− group (p = 0.08) (Table 4, Fig. 2). Both preterm groups taken together had significantly lower kidney volume in comparison with controls (Table 4). There was no significant difference between NC+ and NC− groups. Regardless of the method of the statistical analysis, there were differences in kidney volume between boys and girls (Fig. 3) as well as laterality of the kidney shown in Tables 3 and 4. Total KV calculated as a ratio to BSA (KV/BSA) for the neonatal period was not significantly different between infants with or without NC (NC+: 131.4 ml (SD 21.1); NC− 109.9 ml (SD 29.7), p = 0.07 using non-parametric Kruskal-Wallis test). The KV/BSA ratio from the neonatal period compared with the measurements at school age showed that children who had suffered from NC (NC+) during the neonatal period had significantly lower KV/BSA ratios than those without NC (NC−) (NC+ 81.05, NC− 103.4; p = 0.0036). Among NC+ children, only 2 of 18 (11%) had a rise in KV/BSA ratio from neonatal to school age, while 9 of 18 (50%) in the NC− group showed a rise in KV/BSA ratio (p = 0.01).

All groups had normal estimated glomerular filtration rate (eGFR) calculated by the cystatin C–based CAPA formula but showed lower values for the NC− group compared with controls as well as for both preterm groups taken together (NC+ 120, NC− 113, control 126.3 ml/min/1.73m², p = 0.012, 0.035 respectively). The difference between the NC+ and NC− groups was not significant (p = 0.11).

There was no difference between the groups when using the simplified Schwartz formula (eGFRcreatinine with k = 36) (NC+ 114.4, NC− 112.5, control 104.6 ml/min/1.73m², p = 0.3). Although entirely normal, plasma creatinine was significantly higher in controls compared with both groups of preterm-born children (NC+ 38.5 (5.9); NC− 40.1 (7.9); control 46.2 μmol/l (8.6), p = 0.011). Urinary proteins and electrolytes were not significantly different between the three groups (data on request).

ABPM readings were successful in 34 of the 41 EPT-born children. Neurodevelopmental disorders of autism spectrum disorders and/or ADHD were present in 16 children born EPT (12/20 NC+ (60%); 4 of 21 NC− (19%)). In seven of these children, ABPM measurements were not possible due to compliance problems or had to be discarded because of low quality. However, none of the investigated children with ADHD or autism spectrum diseases was on medical treatment at the time of investigation. The results of ABPM in the 34 performed were in the normal range for all preterm-born children. ABPM could verify the high office blood pressure measurements in only one of the five patients mentioned above. The majority of the children had systolic values below or at the 50th percentile (Table 5). There was no difference between NC+ and NC− with regard to the distribution among the percentiles. Seventeen of 34 preterm-born children (50%) (NC+ 9; NC− 8) had a day-to-night decline (night dipping) of < 10% (Table 5).