Glomerular developmental delay and proteinuria in the preterm neonatal rabbit.

Recent advances in neonatal care have improved the survival rate of those born premature. But prenatal conditions, premature birth and clinical interventions can lead to transient and permanent problems in these fragile patients. Premature birth (<36 gestational weeks) occurs during critical renal development and maturation. Some consequences have been observed but the exact pathophysiology is still not entirely known. This experimental animal study aims to investigate the effect of premature birth on postnatal nephrogenesis in premature neonatal rabbits compared to term rabbits of the same corrected age. We analyzed renal morphology, glomerular maturity and functional parameters (proteinuria and protein/creatinine ratio) in three cohorts of rabbit pups: preterm (G28), preterm at day 7 of life (G28+7) and term at day 4 of life (G31+4). We found no significant differences in kidney volume and weight, and relative kidney volume between the cohorts. Nephrogenic zone width increased significantly over time when comparing G31 + 4 to G28. The renal corpuscle surface area, in the inner cortex and outer cortex, tended to decrease significantly after birth in both preterm and term groups. With regard to glomerular maturity, we found that the kidneys in the preterm cohorts were still in an immature state (presence of vesicles and capillary loop stage). Importantly, significant differences in proteinuria and protein/creatinine ratio were found. G28 + 7 showed increased proteinuria (p = 0.019) and an increased protein/creatinine ratio (p = 0.023) in comparison to G31 +4. In conclusion, these results suggest that the preterm rabbit kidney tends to linger in the immature glomerular stages and shows signs of a reduced renal functionality compared to the kidney born at term, which could in time lead to short- and long-term health consequences.

Introduction

Recent advances in neonatal care have allowed the survival of extremely premature infants. At present more than 95% of infants born preterm survive into adulthood in most industrialized nations [1]. The prenatal conditions that cause preterm labor, the insult of preterm birth itself and the interventions during the clinical management of these fragile patients can all lead to transient and permanent effects on organ structure and function. This is often more evident for the pulmonary and nervous system compared to other organs, such as the kidneys.

Premature birth (<36 gestational weeks) affects the normal development and maturation of the renal system during a critical period. Nephrogenesis in humans is completed in week 34–36 of gestation, with a rapid formation of new nephrons from week 20 of gestation [2]. Preterm birth arrests this process and can result in a lower nephron number which does not recover later in life. Preterm birth itself is associated with neonatal acute kidney injury due to possible insults like decreased kidney perfusion (patent ductus, resuscitation, sepsis) or nephrotoxic medications. The resulting kidney damage is associated with renal pathology later in life such as hypertension, proteinuria and a decreased glomerular filtration rate [3-10].

Animal models are needed to study the exact pathogenesis or underlying mechanisms of kidney injury and disease caused by premature birth. Of the available models, mice and rats are most often used to study nephropathy, usually induced by surgical operation, drugs or toxins, or genetic modification. For neonatal pathology the rat seems an excellent model as active nephrogenesis persists until day 10 after birth. However, the rabbit model holds a position between the small and larger animal models and could thus provide a unique position for the translation of research to the clinic [11].

In this study we investigated the effect of preterm birth on nephrogenesis in preterm neonatal rabbits compared to term rabbits of the same corrected age.

Materials and methods

Ethical approval

The experiment was approved by the Ethics committee for Animal Experimentation of the Faculty of Medicine (KU Leuven; p060/2016) and performed according to current animal welfare guidelines and recommendations. Time-mated pregnant does (New Zealand White and Dendermonde hybrid rabbits) were provided by the animalium of the KU Leuven and housed in separate cages until caesarian section at a gestational age of 28 days (preterm) or 31 days (term). The cages were set at a 12 hour light-dark cycle, room temperature and free access to water and pellet feed.

Experimental procedure

Prior to caesarian section, does were sedated with intramuscular ketamine 35 mg/kg bodyweight (Nimatek®; Eurovet Animal Health BV, Bladel, The Netherlands) and xylazin 6 mg/kg bodyweight (XYL-M®; VMD, Arendonk, Belgium). Does were placed in a supine position after adequate sedation and euthanized by administrating an intravenous injection of a mixture of 200 mg embutramide, 50 mg mebezonium, and 5 mg tetracain hydrochloride (T61®; Intervet International BV, Boxmeer, The Netherlands). Then, the uterus was rapidly exposed by creating an abdominal incision and all pups were delivered through hysterotomy.

From each mother allocated to preterm delivery (G28), the first 3 fetuses were randomly selected for fetal harvest. These fetal controls were prevented from breathing by not removing the fetal membranes. The remaining pups were dried, stimulated and put in an incubator at 32°C, 75% humidity, and 95% O2. Term pups, all delivered through hysterotomy, were also put in an incubator at 32°C, 75% humidity, and 95% O2 during the first hour after birth.

One-hour survivors were then weighed and labeled. All pups, term and preterm, were housed in an incubator at 32°C, 75% humidity, and 21% O2. From then onwards, all pups were fed twice daily through a 3.5 Fr orogastric tube with milk replacer (Day One®, Protein 30%, Fat 50%; FoxValley; IL, USA) prepared according to manufacturer’s guidelines. The quantity of feeding increased per postnatal day (PN) from 40 (PN day 0), to 50 (PN day 1), to 75 (PN day 2), and finally 100 mg/kg bodyweight on PN day 3 until harvest. Immunoglobins (Col-o-Cat®, SanoBest; Hertogenbosch, The Netherlands) were added during the first 2 PN days and probiotics, electrolytes and vitamins (Bio-Lapis®; Probiotics International Ltd.; Somerser, UK) were added during the first 5 PN days. A single intramuscular injection of vitamin K1 was administered on PN day 2 (0.002 mg/kg BW, Konakion pediatrique®; Roche, Basel, Switzerland). From PN day 2 onwards both term and preterm pups were given daily intramuscular injections of benzylpenicillin (20,000 I.U./kg BW Penicilline®; Kela, Sint-Niklaas, Belgium) and amikacin (20 mg/kg BW day, Amukin®; Bristol-Myers-Squibb, Brussels, Belgium).

Tissue collecting and processing

Pups were deeply sedated with ketamine 35 mg/kg bodyweight (Nimatek®; Eurovet Animal Health BV, Bladel, The Netherlands) and xylazin 6 mg/kg bodyweight (XYL-M®; VMD, Arendonk, Belgium). After adequate sedation, the abdomen was opened through laparotomy and pups were euthanized by exsanguination. Blood from G28 + 7 and G31 + 4 pups was aspirated from the right ventricle, heparinized and centrifuged to retrieve plasma. Urine from G28 + 7 and G31 + 4 pups was aspirated directly from the bladder. Then, left kidneys were harvested and the perinephric fat was removed. Kidneys were weighed and fixated in PFA 4% for 24 hours. Then, kidney volume was measured by water displacement and the fixated kidneys were transferred to ethanol 70% for storage. Relative kidney volume and weight were calculated by dividing the kidney volume and weight by bodyweight at harvest.

One complete paraffin section at a thickness of 5 μm through the mid-hilar region on the longitudinal axis was collected for each kidney and stained with hematoxylin and eosin (H&E). Sections were digitalized using a Carl Zeiss Axio Scan.Z1 scanner at 40x magnification (0,220 μm2 per pixel) and assessed blinded, by coding of the file names. Measurements were done by a single researcher using QuPath for Windows, Version 0.1.2 [12]. Unblinding was done after finishing measurements in all histology slides.

Histological assessment

Kidney slides were digitally divided in equal poles (based on surface area) of which one pole was randomly selected (for more details see S1 Fig).

The distribution of maturity was quantified by counting and scoring all identifiable glomerular cross-sections in the selected half. Glomerular cross-sections unidentifiable for their maturity stage were excluded. Glomerular maturity stages were scored according to parameters described in Fig 1. Each stage was then averaged and calculated as the percentage of the total glomerular cross-sections counted.

Fig 1

Glomerular maturity stages found in the neonatal rabbit kidney.

Stages are based on criteria previously defined [13–15]. Glomeruli were divided in either immature stages (A-C) or mature stages (D-F). (A) Vesicle (V): a cluster of mesenchymal cells, exclusively found in the nephrogenic zone, bordering a ureteric bud (UB). (B) Comma- or S-shaped body: formed by the elongation and twisting of the vesicle. (C) Capillary loop: The lower limb of the S-shaped body formed an immature glomerulus, characterized by a crescent shape and few capillaries. (D) Stage I: no lobulation and at least half of the circumference is lined with dark-staining podocytes. (E) Stage II: little lobulation and less than half of the circumference is covered with dark-staining podocytes. Some open capillaries may be seen. (F) Stage III: fully matured glomerulus showing full lobulation and open capillaries. No podocytes lining the glomerular tuft. (Bar = 50 μm).

Glomerular density—the number of glomerular cross-sections per surface area—was calculated by dividing the total glomerular cross-sections by the renal cortex surface area in mm2. The renal cortex surface area was defined as the area between the medulla and the fibrous renal capsule and was measured by tracing these borders.

Nephrogenic zone width [μm] was defined as the area in the outer renal cortex just below the fibrous renal capsule to the last appearance of comma- and S-shaped bodies (S1 Fig) [16]. Histology slides were divided in eight equal sectors in which one measurement per sector, following the major organ surface area, was performed. Finally, the average nephrogenic zone width was calculated for each slide.

Renal corpuscle cross-sectional surface area [μm2] was measured by tracing the inner lining of the Bowman’s capsule. All glomerular cross-sections (stages I-III for the inner cortex, and capillary loops for the outer cortex) were numbered. For both the inner and outer cortex 30 glomerular cross-sections or the maximum amount possible, were randomly selected using a random number generator. Then, the average renal corpuscle cross-sectional surface area for the inner and outer cortex was calculated.

Functional parameters

Urinary creatinine [mg/dL] was determined through Roche enzymatic method, and urinary protein [g/L] with the benzethonium chloride method [17]. From this the protein/creatinine ratio was determined [g/g creatinine]. Plasma creatinine concentration [mg/dL] was determined by colorimetric assay (modified Jaffe reaction) [18]. Functional parameter data is presented as mean [CI-95%].

Statistical analyses

Statistical analyses were performed using GraphPad Prism version 7.04 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com). A p value < 0.05 was considered statistically significant. All variables were assessed for normality using the Shapiro-Wilk test. Bodyweight, absolute and relative kidney volume and kidney weight, glomerular density, nephrogenic zone width, renal corpuscle cross-sectional surface area and the distribution of maturity were tested with a one-way analysis of variance (ANOVA) and a post hoc Tukey’s multiple comparisons test. Functional data was tested with an independent samples T-test including Levene’s test for equality of variances. Correlations on continuous variables were tested using the Spearman’s Rank-Order correlation. Best-fit linear lines were plotted using linear regression analysis.

Results

Animal survival and biometry

A total of 19 pups were used in this experiment (S1 Table). Eight G28 pups were harvested directly after birth and six G28 pups were raised for harvesting on PN day 7 (G28 + 7). Six term pups were raised and harvested on PN day 4 (G31 + 4). Preterm or term pups that died before reaching their postnatal endpoint were excluded from the study (one G28 + 7 pup). This resulted in 8 G28, 5 G28 + 7 and 6 G31 + 4 pups. Birthweights of G28 and G28 + 7 were comparable. Even though harvest weight yielded no significant difference between G28 + 7 and G31 + 4, bodyweight tended to be higher in the term group (p = 0.245).

Kidney morphology

Relative kidney weight was found to be higher in G28 + 7 compared to G31 + 4 (p = 0.023) (Fig 2). We found no significant differences in absolute kidney volume and weight, and relative kidney volume between G28 + 7 and G31 + 4, but in both term and preterm it tended to increase compared to fetal controls. Moreover, we found that the average nephrogenic zone width increased overtime when comparing G31 + 4 to G28 (p < 0.001). But, this effect was not found when comparing G28 + 7 to G28. Nevertheless, the nephrogenic zone width was not significantly different between G28 + 7 and G31 + 4. The total glomerular cross-sectional count (Fig 3), renal cortex surface area (S1 Table) and glomerular density (Fig 2) tended to increase compared to fetal controls, but were comparable between G28 + 7 and G31 + 4. Post hoc Tukey’s revealed no significant differences in renal corpuscle surface area between G28 + 7 and G31 + 4 (S1 Table). The renal corpuscle surface area, in the inner cortex and outer cortex, tended to decrease significantly (p < 0.001) after birth in both preterm and term groups. In addition, we found a negative correlation between the renal corpuscle surface area–for both the inner and outer cortex–and the glomerular cross-sectional count (Fig 3). Moreover, we found a positive correlation between the glomerular cross-sectional count and bodyweight (r = 0.7931), kidney volume (r = 0.8811) and kidney weight (r = 0.8957), indicating that the larger kidney cross-sections have more glomerular cross-sections.

Fig 2

(A): Visual illustration of the animal model. Time points highlighted in blue are harvesting time points. (B): A graphic overview of the total immature and mature stage glomeruli across G28, G28 + 7 and G31 + 4, in absolute [mean] and relative counts [mean / %].

Fig 3

(A) Glomerular cross-sections counted. (** = p < 0.001. Error bars: mean ± SD) (B-F) Correlations plotted using Spearman’s Rank-Order correlation on glomerular cross-sections and the following variables: bodyweight, kidney volume, kidney weight, and renal corpuscle surface area for the inner and outer cortex. Best-fit slope (solid line) is accompanied by a 95%-CI (dotted line).

Glomerular maturity

Immature stages, taken overall, were found to be dominant in each group (Fig 2B and Table 1), specifically, vesicles in G28 (27.3%) and capillary loops in G28 + 7 (27.6%). Even though immature stages were dominant in G31 +4, we mostly found stage I glomeruli (31.0%). In comparison to fetal controls both G28 + 7 and G31 + 4 tended to have an increased absolute count of immature stages and stage I and II glomeruli. We found that G28 + 7 tended to have a higher relative count of overall immature glomerular stages than G31 + 4 (p = 0.136), and that G31 + 4 tended to have more mature glomeruli than G28 + 7. Furthermore, post hoc Tukey’s yielded no significant differences between G28 + 7 and G31 + 4 in vesicles (p = 0.064) and stage I glomeruli (p = 0.057). No significant differences were found in the relative counts of comma or S-shaped bodies, capillary loops, stage II or stage III glomeruli.

Table 1

Distribution of maturity stages.

Stages presented by the absolute mean, 95% confidence intervals and as the percentage of the total glomerular cross-sections counted.

Stage		G28			G28 + 7			G31 + 4
		Absolute	95%-CI	Relative	Absolute	95%-CI	Relative	Absolute	95%-CI	Relative
Immature Stages		122.9	[96.9, 148.9]	65.5%	241.0**	[198.0, 284.0]	66.5%	208.5**	[170.9, 246.1]	57.7%
	Vesicle	52.8	[41.8, 63.8]	27.3%	79.2*	[64.5, 93.9]	21.9%*	60.0	[41.1, 78.9]	16.6%**
	Comma or S-shaped Body	33.5	[29.0, 38.0]	17.6%	61.8**	[52.6, 71.0]	17.1%	69.3**	[59.6, 79.0]	19.2%
	Capillary Loop	40.1	[28.1, 52.1]	20.6%	100.0**	[69.8, 130.2]	27.6%	79.2*	[57.8, 100.6]	21.9%
Mature Stages		70.0	[62.0, 78.0]	34.5%	121.2*	[97.1, 145.3]	33.5%	152.7**	[104.2, 201.2]	42.3%
	Stage I	45.8	[38.3, 53.3]	24.0%	88.6*	[68.2, 109.0]	24.4%	114.2**	[81.0, 147.4]	31.0%*
	Stage II	16.4	[12.6, 20.2]	8.8%	29.4	[26.5, 32.3]	8.1%	36.5*	[16.6, 56.4]	10.0%
	Stage III	3.1	[1.3, 5.0]	1.7%	3.2	[0.5, 5.9]	0.9%	2.0	[0.4, 3.6]	0.6%

* p < 0.05 compared to G28.

** p < 0.001 compared to G28.

We found a significant difference in urinary protein (p = 0.019) and protein/creatinine ratio (p = 0.023) between G28 + 7 and G31 +4 (Fig 4). No differences were found in urinary creatinine nor in plasma creatinine.

Fig 4

Functional parameters.

Visual representation of the functional parameters measured: urinary creatinine, protein and the protein/creatinine ratio. Comparison between G28 + 7 and G31 + 4 reveals significant differences in proteinuria and in the protein/creatinine ratio. (* = p < 0.05. Error bars: mean ± SD).

Discussion

In humans preterm birth exacts a toll on many organ systems, especially the lung, brain and kidney. This results in an increased morbidity later in life with obstructive lung pathology, impaired neurocognitive outcomes and chronic renal disease with hypertension [3-10], but the pathophysiological background is still not entirely known. This is in part due to the lack of data from human subjects, because of ethical concerns and a large developmental variability [19], which forces researchers to rely on animal studies. In recent years we have developed a rabbit model to study the changes in pulmonary development that are induced by prematurity and hyperoxia. In the case of lung development, the rabbit mimics the human neonatal situation quite well as rabbits also reach the alveolar stage before birth. Also for the central nervous system we found that the rabbit was a good proxy for humans as they develop a form of encephalopathy of prematurity [20]. In order to further explore the translational value of the preterm rabbit model we set out to study the effect on renal development.

We evaluated the effect of preterm birth on nephrogenesis in preterm neonatal rabbits compared to term rabbits of the same corrected age. For this we established three cohorts: G28, G28 + 7 and G31 + 4. This allowed us to study the effect of 3 additional intrauterine days by comparing G28 + 7 (28 intrauterine—and 7 extrauterine days) and gestational corrected G31 + 4 (31 intrauterine–and 4 extrauterine days) to the ‘fetal preterm stage’ at a gestation of 28 days. To our knowledge this is the first study to study the effects of prematurity on glomerular maturation and functionality.

When we analyzed the major structural aspects of the renal structures, we found that the kidney-to-bodyweight ratio was significantly higher in preterm rabbit pups (G28), which is in line with studies performed in human and non-human primate neonates [15,21,22]. But the difference of three days of intrauterine life did not result in major structural discrepancies between the two cohorts. The kidney-volume-to-bodyweight ratio and glomerular density did not differ between the G28 + 7 and G31 + 4 cohorts.

Yet the glomerular density as such may not be a sensitive enough marker to quantify differences between these two conditions. We performed a detailed assessment of the different stages of glomerular development and found out that the preterm rabbit kidney at a postnatal age of 7 days (G28 + 7) contains more immature glomerular structures (vesicles) compared with ‘term’ kidneys at a similar age (G31 + 4), which contain more stage I glomeruli. This suggests a significant disruption of normal nephrogenesis induced by prematurity. One can only speculate on the exact pathophysiological processes that would result in an altered nephrogenesis after birth in our model (e.g. water- and electrolyte disturbances, toxic effects of medication…).

In contrast to our findings, Sutherland et al. [15] found an accelerated maturation, with a decreased relative amount of vesicles and a smaller nephrogenic zone width in autopsied preterm human neonates whose post-natal survival ranged from 2 to 68 days, compared to still-born gestational controls. However, it remains unclear whether this difference was found because of the effect of prematurity or through the administration of antenatal steroids. Additionally, a study comparing preterm and term baboons found an interruption of normal kidney development, objectified through a decrease in glomerular generations and glomerular relative area with an increase of the renal corpuscle area [23].

It has been reported that nephrogenesis in the rabbit is completed in the 2nd or 3rd postnatal week [24]. In this respect the rabbit differs significantly from humans where the formation of new glomeruli end in the final weeks of gestation and no new structures are formed postnatally. But when neonates are born premature their kidneys are not yet fully developed and in that respect their ongoing nephrogenesis is similar to the preterm rabbit pups. In our experiments we confirm that nephrogenesis is indeed still ongoing after birth in the both the preterm and term cohorts by the presence of a nephrogenic zone and changes in total glomerular count, kidney volume and weight (compared to the fetal kidneys at G28). At present we cannot perform long term experiments to study further kidney maturation in preterm rabbits as the pups fail to survive beyond 10 days when fed artificially. So we cannot determine if this delayed nephrogenesis after preterm birth catches up and results in structurally and functionally normal mature kidneys. To our knowledge, the current literature provides no reference to what gestational age in humans corresponds to a gestational age of 28 days in the rabbit. We could argue that the term rabbit could be used as a ‘preterm’ model as we have proven that nephrogenesis continues after birth in the term rabbit. However, if term rabbits are used, the insult of premature birth itself and its effects would be minimized.

Next to the structural changes, we studied the functional differences between the two cohorts. It became clear that the kidneys of the preterm cohort suggest have a reduced functionality, shown by an increased urinary protein/creatinine ratio and higher levels of urinary protein. Different hypotheses can be put forward to explain an increased protein/creatinine ratio in preterm rabbits. A study by Stelloh et al. has shown that preterm birth in mice correlates with a decrease in nephron endowment and thus induces stress on the remaining glomeruli, leading to proteinuria and hyperfiltration [25]. In our data, however, there is no compensatory increase in glomerular size, which is typically seen along with hyperfiltration, arguing against this theory. An alternative explanation are potential structural differences within the glomerulus. Wharram et al. have previously shown in a transgenic rat strain–in which podocytes express the human diphtheria toxin receptor—that a <40% podocyte reduction resulted in transient proteinuria and a reversible reduction of renal function. In contrast, a >40% podocyte reduction was responsible for segmental sclerosis, sustained proteinuria and a reduction in renal function [26]. In addition, Tsukahara et al. have described increasing and sustained high levels of albuminuria and urinary beta-2 microglobulin with increasing degrees of prematurity in human preterm neonates, whilst in term human neonates the protein levels gradually decreased during the first 28 days of life. Thus, demonstrating an increase in glomerular permeability and a decrease in tubular resorption with increasing degrees of prematurity [27]. However, Guignard JP. and Drukker A. found that in the preterm infant creatinine backflows along the tubule as a consequence of a ‘leaky’ immature tubular and vascular structures [28]. From this, we hypothesize that the increase in proteinuria in the absence of glomerular hypertrophy, found in preterm rabbits, may be caused by changes in the glomerular structure, such as podocyte immaturity or an “absolute” podocyte depletion (a decrease in the total number of healthy podocytes per glomerulus [29]) or through premature tubular processes such as tubular backflow of creatinine [28] and reduced tubular resorption [27].

This study is not without limitations. Firstly, an estimate of the total glomerular count (stereology based), was not obtained. We did, however, measure the glomerular density as an estimate for the amount of glomeruli per area. This measurement is thought to be susceptible to confounding factors such as section thickness and glomerular hypertrophy [29]. Also, the small number of animals in the experimental arms are likely to result in type II statistical errors. This argues a lack of significant differences where there in fact may be a biological effect and thus accepting a possibly false null hypothesis. To minimize the effects of these possible confounders we have consistently used the same section thickness and measured the cross-sectional renal corpuscle surface area in all cohorts. Also, in the current setting we were not able to perform the experiment using cationic ferritin enhanced-MRI, a novel and promising tool to quantify perfused glomeruli. This method, however, would not provide insight into differences in the developmental stages of the glomeruli that were found [30,31]. Secondarily, kidneys were embedded in paraffin: a method known to cause 40–50% shrinkage of soft tissues such as the kidney [32]. Furthermore, the prophylactic use of amikacin antibiotics in both preterm and term groups could have altered our results through nephrotoxicity [33]. However, Mingeot-Leclercq et al. have described amikacin to be nephrotoxic, after glomerular filtration, in the epithelial cells of the proximal tubules. Also, the use of benzylpenicillin and amikacin is a commonly used combination of antibiotics in clinical settings and provides us with a reflection. And, in previous experiments the rabbits were prone to develop dermal infections further substantiating the use of the current antibiotic regiment. Also, all experimental groups received these antibiotics in a dosage adjusted for bodyweight.

In conclusion, we have combined histology and functional parameters providing new insight in postnatal nephrogenesis in the preterm rabbit. We have shown that the preterm rabbit tends to linger in the immature glomerular stages compared to term. Importantly, preterm rabbits also show signs of proteinuria and a higher protein-to-creatinine ratio, suggestive of hyperfiltration which could in time lead to short- and long-term health consequences. Future research could focus on the processes that are involved in these changes and in the use of the preterm rabbit for pharmacokinetic studies.

Illustrative overview of the histology methods performed.

(A): Representative image of the nephrogenic zone as found in the outer renal cortex. The lower border of the nephrogenic zone is highlighted (white line). (B): Example histology slides for G28, G28 + 7 and G31 + 4. Provides a quick overview of the relative size. (C): Illustrative example of the analysis methodology. The kidney was divided in 8 sectors using grid lines. The upper or lower pole was randomly selected and all glomerular cross-sections were counted and staged. The renal cortex surface was measured by tracing just underneath the outer renal capsule and along the boundary between medulla and cortex. The nephrogenic zone width was measured in 8 sectors; the renal corpuscle surface area was measured in the 4 sectors in the randomly selected pole.

(TIF)

Click here for additional data file.

Baseline characteristics of units of analysis.

G28, G28 + 7, and G31 + 4, presented as mean and 95% confidence intervals.

(PDF)

Click here for additional data file.

24 Aug 2020

PONE-D-20-11602

Glomerular developmental delay and proteinuria in the preterm neonatal rabbit.

PLOS ONE

Dear Dr. de Winter,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please carefully address all the reviewers' remarks in your revised version of the manuscript.

Please submit your revised manuscript by Oct 08 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Umberto Simeoni

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2.At this time, we request that you  please report additional details in your Methods section regarding animal care, as per our editorial guidelines:

(1) Please provide additional details regarding the care of the rabbits prior to caesarian section.

(2) Please describe the care received by the rabbits after delivery, including the frequency of monitoring and the criteria used to assess animal health and well-being.

Thank you for your attention to these requests.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: no comments as i have a major conflict of interest with all the authors involved

no comments as i have a major conflict of interest with all the authors involved

no comments as i have a major conflict of interest with all the authors involved

no comments as i have a major conflict of interest with all the authors involved

Reviewer #2: The aim of this paper is to determine the effect of preterm birth on nephrogenesis of the rabbit, a proposed model for understanding the documented low nephron number in preterm human infants. In the Introduction, it is stated that “more than 95% of infants born preterm survive to adulthood” (line 48), but no reference is provided. Surely, this differs among populations.

The authors have based their study on 19 rabbits, 8 studied at preterm delivery, 6 born term and studied 7 days after birth, and 5 born preterm and studied 4 days after birth. The group G28+7 was raised in 95% oxygen and treated with amikacin. A sagittal section of the kidney was used for morphometric determination of relative glomerular number, glomerular size and maturation, and urine protein and creatinine concentration.

The results show that there was no difference between groups in kidney weight, kidney volume, or renal corpuscle surface area. Size of the nephrogenic zone was greater and proportion of immature glomeruli was lower in G31+4 vs. G28, but not different between G31+4 and G28+7. Urine protein concentration was increased (but with great variation) in G28+7 vs. G31+4 (with little variation). Table I is truncated, missing data for G31+4. An inverse relation was demonstrated between relative glomerular number and glomerular size.

The discussion admits that morphometric techniques utilized in the study may not be sensitive enough to detect differences between groups G31+4 and G28+7. This may explain the lack of increase in glomerular size in the G28+7 group demonstrated in the preterm mouse model cited in reference 24. P values between 0.05 and 0.15 suggest that type 2 error resulting from the small number of animals in each group (and high variation demonstrated in urine protein concentration in the G28+7 group) would support this. Stereologic approaches, including MRI image analysis, could circumvent this (Charlton JR et al. Pediatric Nephrology 2020 online publication https://doi.org/10.1007/s00467-020-04534-2). The MRI technique to study nephron number and structure has been employed in a rabbit model of neonatal acute kidney injury (Pediatric Research (2020) 87:1185–1192; https://doi.org/10.1038/s41390-019-0684-1).

Later study time points would also be highly desirable, but the authors indicate that these are not feasible in the model, because the preterm animals do not survive longer than a week with tube feeding. Additional concerns that limit the utility of the model are the exposure of the preterm neonatal rabbit to a nephrotoxin (amikacin) and a hyperoxic environment.

Despite the lack of documented glomerular hypertrophy, the authors account for increased urine protein concentration on the basis of hyperfiltration, citing the paper by Tsukahara (reference 26). However, albumin and beta-2-microglobulin were measured by Tsukahara et al., and changes in B2M were more pronounced than those in albumin, suggesting that tubular immaturity is a greater determinant than glomerular injury in preterm neonates. An additional factor to be considered is the backflow of creatinine across the immature renal tubule that artificially increases the urine protein/creatinine concentration in preterm urine (Guignard JP & Drukker A. Pediatrics, 1999 http://www.pediatrics.org/cgi/content/full/103/4/e49).

Reviewer #3: As you point out, human nephrogenesis is completed by 34-36 wk EGA. What gestational age in humans corresponds to d28 in the rabbit? Is this a model for previable human fetuses, or 28 wk ones?

Also, post-natal growth restriction is common in small premature infants. This corresponds with the fact that your 28+7 group weighed less than your 31+4 group. Since absolute kidney size was not different significantly, could the kidney-to-bodyweight ratio just reflect poor growth of the rest of the body, and not say anything about the kidney per se? I suspect providing adequate nutrition to these premie bunnies was very difficult, and like human ELBW, their somatic growth rate would not mimic that seen in utero.

Of note, on my copy, Table 1 was too wide for the page, and much of it was missing and could not be read.

Also, consider an additional reference with relevance to your discussion: Li J, et al. Nephrology 25 (2020) 116–124. doi: 10.1111/nep.13623 . They report that even though premies were small & lighter at follow up, kidney volume & length were not different from mature controls. How do your data fit with these human findings?

Keeping preterm rabbits alive if much trickier than term rabbits. Could term rabbits be used as a model for preterm human kidneys? Human nephrogenesis stops by 36 wks, but rabbits apparently continues postterm. Maybe term rabbit kidneys are a good model for premie human kidneys. Please comment on that.

I've made additional minor comments on the attached PDF Mark-up.

Reviewer #4: The authors of this manuscript investigated the effect of premature birth on nephrogenesis in preterm neonatal rabbits compared with term rabbits of similar corrected age.

This is an important neonatal topic in general, as emerging research focusing on the effects of prematurity on the kidney and its consequence for long-term renal outcome is being increasingly explored.

The manuscript was well-written and the study was thoughtfully designed. Nevertheless, there are some concerns with this manuscript.

Comments:

1. Animal models of kidney disease are reasonable to use to understand the pathogenesis of renal disease associated with the developmentally regulated changes in nephrogenesis and will aid in developing strategies to mitigate preterm kidney injury, however there are many inherent difficulties in bridging the gap from bench to bedside. The preterm rabbit model is an acceptable model as it the smallest model that includes a factor of prematurity and these animals are most likely to survive following surgical manipulation.

2. Translational research involving animals require careful experimental design, therefore the authors should explain why amikacin, a nephrotoxic agent, was used in the study design which aimed to describe normal development of the kidney with regards to glomerular development and renal function. Amikacin has been shown to increase creatinine level and the effect can persist for several days after cessation of therapy.

3. The authors should explain the respiratory state of those pups delivered at G28 and maintained for 7 days prior to being euthanized. It would seem intuitive that if there is a component of respiratory insufficiency, then hypoxia (a pathologic state) would be a contributory factor to the findings reported in these animals and do not adequately represent the real-life scenario where respiratory support and oxygen therapy may mitigate some of the changes described.

4. Authors should state whether the does are genetically identical – if not, how does that affect the model?

5. Authors should clarify how the pups from each mother was assigned to each arm of the study (that is equal distribution of pups from different mothers, especially between G28 and G28+7 arms of the study), because a similar in-utero environment may contribute to the ultimate developmental changes seen and may not represent the average effect seen across mothers and pups.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Janine Y Khan

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-20-11602 reviewer comments.pdf

Click here for additional data file.

11 Sep 2020

Reviewer #1 provided no comments due to a major conflict of interest with all of the authors involved.

Reviewer #2:

1. “The aim of this paper is to determine the effect of preterm birth on nephrogenesis of the rabbit, a proposed model for understanding the documented low nephron number in preterm human infants. In the Introduction, it is stated that “more than 95% of infants born preterm survive to adulthood” (line 48), but no reference is provided. Surely, this differs among populations.”

• We have provided a reference for the statement that more than 95% of infants born preterm survive into adulthood in most industrialized nations in line 48 of the Introduction. (Raju TNK, Pemberton VL, Saigal S, et al. Long-Term Healthcare Outcomes of Preterm Birth: An Executive Summary of a Conference Sponsored by the National Institutes of Health. J Pediatr. 2017;181:309-318.e1)

2. “The authors have based their study on 19 rabbits, 8 studied at preterm delivery, 6 born term and studied 7 days after birth, and 5 born preterm and studied 4 days after birth. The group G28+7 was raised in 95% oxygen and treated with amikacin. A sagittal section of the kidney was used for morphometric determination of relative glomerular number, glomerular size and maturation, and urine protein and creatinine concentration. The results show that there was no difference between groups in kidney weight, kidney volume, or renal corpuscle surface area. Size of the nephrogenic zone was greater and proportion of immature glomeruli was lower in G31+4 vs. G28, but not different between G31+4 and G28+7. Urine protein concentration was increased (but with great variation) in G28+7 vs. G31+4 (with little variation). Table I is truncated, missing data for G31+4. An inverse relation was demonstrated between relative glomerular number and glomerular size.”

• In fact, both G28 + 7 and G31 + 4 were raised in an incubator in 21% oxygen (normoxic) and were treated with amikacin from postnatal day 2 onwards. Preterm and term pups were housed at 95% oxygen during the first hour after birth only. This might not have been completely clear from the original Methods description. In the revised manuscript we provide a more detailed description.

• We apologize for Table 1 being truncated in the current version, as has been noted by multiple reviewers. Table 1 has been adapted to fit in ‘Landscape’ view, hopefully this will solve the issue and provide you with all the relevant information.

3. “The discussion admits that morphometric techniques utilized in the study may not be sensitive enough to detect differences between groups G31+4 and G28+7. This may explain the lack of increase in glomerular size in the G28+7 group demonstrated in the preterm mouse model cited in reference 24. P values between 0.05 and 0.15 suggest that type 2 error resulting from the small number of animals in each group (and high variation demonstrated in urine protein concentration in the G28+7 group) would support this. Stereologic approaches, including MRI image analysis, could circumvent this (Charlton JR et al. Pediatric Nephrology 2020 online publication https://doi.org/10.1007/s00467-020-04534-2). The MRI technique to study nephron number and structure has been employed in a rabbit model of neonatal acute kidney injury (Pediatric Research (2020) 87:1185–1192; https://doi.org/10.1038/s41390-019-0684-1).”

• Indeed, morphometric techniques that have been used in this experiment are sensitive to confounding. We have tried to minimize these effects by consistently using the same section thickness and measuring cross-sectional renal corpuscle surface area in all cohorts. Stereologic approaches could indeed be more sensitive to detect more subtle changes between groups as would larger cohorts. For the latter we are bound by the European and Belgian legislation and ethical restrictions on animal research which do not allow large scale experiments. As there was very little published literature on this topic to guide a power calculation for the outcome parameters, we could not accurately predict the necessary sample size.

• MRI image analysis as described by Charlton et al. provides a novel and promising tool to quantify perfused glomeruli and might be of great use in the future. In the current setting we were unable to perform the experiment using MRI image analysis. We have altered the manuscript in ‘Discussion’ and described MRI-analysis as a promising alternative.

4. “Later study time points would also be highly desirable, but the authors indicate that these are not feasible in the model, because the preterm animals do not survive longer than a week with tube feeding. Additional concerns that limit the utility of the model are the exposure of the preterm neonatal rabbit to a nephrotoxin (amikacin) and a hyperoxic environment.”

• Indeed, later study time points would be preferable as this would allow us to determine whether the glomerular developmental delay and proteinuria in the preterm rabbit are persistent or whether these changes resolve over time. At present, we are working on experiments to prolong the rabbit pup survival using different artificial milk substitutes, some of which show promising results.

• The rabbit pups were treated with amikacin from postnatal day 2 onwards in order to prevent infectious complications in these fragile pups. In previous experiments performed in our research group the rabbit pups were prone to dermal infections, and thus we chose to provide all experimental groups with prophylactic antibiotics. Mingeot-Leclercq et al. have described amikacin to be toxic to the epithelial lining of the proximal tubules, after glomerular filtration (and as such have an effect on kidney development/maturation). In a clinical context, an antibiotic regime of benzylpenicillin and amikacin is a often used in current neonatal care. As all the groups in our experiments receive this combination, all possible effects of amikacin would be equally present in all groups.

• The housing of the pups might not have been clearly described in the Methods section. In order to clarify this for the readers we have adapted the Method section. Briefly, all term and preterm pups were housed in an incubator at 95% O2 during the first hour after birth. Afterwards all one-hour survivors were housed in an incubator at 21% O2. We do appreciate that hyperoxia is no longer the most important risk factor in the etiology of BPD or other neonatal conditions, but it is an integral part of the model to mimic the lung injury. It is in this context that we intended to investigate the nephrological changes, not merely based on prematurity.

5. “Despite the lack of documented glomerular hypertrophy, the authors account for increased urine protein concentration on the basis of hyperfiltration, citing the paper by Tsukahara (reference 26). However, albumin and beta-2-microglobulin were measured by Tsukahara et al., and changes in B2M were more pronounced than those in albumin, suggesting that tubular immaturity is a greater determinant than glomerular injury in preterm neonates. An additional factor to be considered is the backflow of creatinine across the immature renal tubule that artificially increases the urine protein/creatinine concentration in preterm urine (Guignard JP & Drukker A. Pediatrics, 1999 http://www.pediatrics.org/cgi/content/full/103/4/e49).”

• Unfortunately we have not been able to perform specific protein analyses on albumin and beta-2-microglobulin in the current setting. It is certainly a factor, as a determinant for the origin of the proteinuria, to take into account in further research.

• Guignard JP & Drukker A. have shown that the preterm neonatal infant shows tubular resorption of creatinine through backflow of creatinine along ‘leaky’ and immature tubules. In the current experiment we were unfortunately not able to determine whether this was the case in our preterm rabbits. We have thus adapted the Discussion accordingly and hypothesize that the difference in protein/creatinine ratio found in the preterm rabbit may be caused by changes in glomerular structure or through tubular backflow of creatinine.

Reviewer #3:

1. “As you point out, human nephrogenesis is completed by 34-36 wk EGA. What gestational age in humans corresponds to d28 in the rabbit? Is this a model for previable human fetuses, or 28 wk ones? Also, post-natal growth restriction is common in small premature infants. This corresponds with the fact that your 28+7 group weighed less than your 31+4 group. Since absolute kidney size was not different significantly, could the kidney-to-bodyweight ratio just reflect poor growth of the rest of the body, and not say anything about the kidney per se? I suspect providing adequate nutrition to these premie bunnies was very difficult, and like human ELBW, their somatic growth rate would not mimic that seen in utero.”

• To our knowledge, the current literature provides no exact reference as to what gestational age in humans corresponds to a gestational age in rabbits as a whole. It is often provided for lung development (where it indeed corresponds to the canalicular / early glandular stage) yet there are no detailed data on comparative kidney development. We have adapted the Discussion accordingly to provide the reader with more insight in the transferability of the neonatal rabbit kidney model.

• Yes, the difference found in kidney-to-bodyweight ratio between the G31 + 4 and G28 + 7 experimental groups can certainly be explained by post-natal growth restriction that is commonly seen in both preterm human infants as well as in animal studies that study prematurity. Also in rabbits ex-utero growth of the premature pups does not mimic the growth seen in utero, as can be seen in Supplementary Table 1. G28 + 7 tended to have a lower harvest weight compared to G31 + 4. We have previously used foster animals (only possible in normoxia settings) and found that compared to gavage fed animals, the mother fed animals gained significantly more weight. So our model definitely involves a ‘failure to thrive’ postnatally.

2. “Of note, on my copy, Table 1 was too wide for the page, and much of it was missing and could not be read.”

• Indeed, Table 1 appears to be truncated in the current version of the manuscript. We have adapted this to be in ‘Landscape’ view.

3. “Also, consider an additional reference with relevance to your discussion: Li J, et al. Nephrology 25 (2020) 116–124. doi: 10.1111/nep.13623 . They report that even though premies were small & lighter at follow up, kidney volume & length were not different from mature controls. How do your data fit with these human findings?”

• Li J et al. have performed a retrospective study that compares the renal ultrasonographic ultrasounds between preterm infants and term infants. They compared ultrasound images at 32 weeks (preterm), 37 weeks and at 6 months of age. They measured kidney volume, length and also renal cortex and medulla thickness. They found that all kidney parameters were smaller compared with term babies, however by 6 months of age kidney volume and length were no longer significantly different between preterm and term infants. The catch-up growth was mainly attributable to hypertrophic growth of the renal cortex, whilst the renal medulla growth was impaired.

In respect to our data we found no significant differences in renal cortex surface area of G28 +7 compared to G31 + 4. Of note, however, nephrogenesis was still ongoing in both experimental arms, whilst in the study by Li J et al. nephrogenesis was finished in the term infants as well as in the follow-up period. To adequately compare the rabbit to the human data we would need to extend the experimental time frame.

4. “Keeping preterm rabbits alive is much trickier than term rabbits. Could term rabbits be used as a model for preterm human kidneys? Human nephrogenesis stops by 36 wks, but rabbits apparently continues postterm. Maybe term rabbit kidneys are a good model for premie human kidneys. Please comment on that.”

• This is an excellent suggestion. Indeed, term rabbits, in which nephrogenesis has been shown to continue postterm, could theoretically be used as a model for preterm human kidneys. However, a significant factor to take into consideration (and a factor that would be absent if term rabbits were used), is the changes resulting because of preterm birth itself (intrauterine development and its limited exposure to stressors and toxins is significantly different from postnatal development).

5. “Reviewer #3 made additional minor comments on the attached PDF Mark-up.”

• We have taken the comments in the attached PDF Mark-up under consideration and have adapted the manuscript where needed.

• We have adapted the text in the section ‘Glomerular Maturity’ to clarify the results of the overall immature and mature glomerular stages in the experimental groups.

• Also, we have altered Table 1 to ‘Landscape’. Hopefully, this will resolve these issues.

• Lastly, Mingeot-Leclercq et al. have indeed described amikacin to be nephrotoxic, after glomerular filtration, in the epithelial cells of the proximal tubules and thus limits the negative effect of amikacin on our experiment that studies the glomerular development. We have adapted the ‘Discussion’ based on this information.

Reviewer #4:

1. “Animal models of kidney disease are reasonable to use to understand the pathogenesis of renal disease associated with the developmentally regulated changes in nephrogenesis and will aid in developing strategies to mitigate preterm kidney injury, however there are many inherent difficulties in bridging the gap from bench to bedside. The preterm rabbit model is an acceptable model as it the smallest model that includes a factor of prematurity and these animals are most likely to survive following surgical manipulation.”

• Indeed, the rabbit provides an experimental model that includes prematurity in a relatively small species. In current neonatal research data from human subjects are lacking and thus we often rely on animal studies (and the availability of detailed histological analysis). Through this way we hope to bridge the gap from bench to bedside.

2. “Translational research involving animals require careful experimental design, therefore the authors should explain why amikacin, a nephrotoxic agent, was used in the study design which aimed to describe normal development of the kidney with regards to glomerular development and renal function. Amikacin has been shown to increase creatinine level and the effect can persist for several days after cessation of therapy.”

• Indeed, pups were treated with amikacin, an antibiotic known to be nephrotoxic. This is definitely a limitation of the model, however the decision to use a regiment of benzylpenicillin and amikacin was based on previous experiments in which the rabbit pups were prone to develop severe dermal infections (with even increased mortality). Benzylpenicillin/amikacin is a commonly used combination of antibiotics in clinical settings and thus may represent in this context the current clinic scenario (yet we do not claim that all preterm infants receive this drug regimen). Also, all experimental groups (G28 +7 and G31+4) were treated with a dosage adjusted for body weight. On top of that, Mingeot-Leclercq et al. have previously described amikacin to be nephrotoxic in the epithelial cells of the proximal tubules, after glomerular filtration. In order to provide the reader with more insight in the use of amikacin and its limitations to our experiment we have altered the Discussion.

3. “The authors should explain the respiratory state of those pups delivered at G28 and maintained for 7 days prior to being euthanized. It would seem intuitive that if there is a component of respiratory insufficiency, then hypoxia (a pathologic state) would be a contributory factor to the findings reported in these animals and do not adequately represent the real-life scenario where respiratory support and oxygen therapy may mitigate some of the changes described.”

• This might not have been completely clear from the ‘Methods’ section. We have adapted this section to provide more clarity on this part of the experimental procedure. Briefly, one-hour survivors (both term and preterm) were housed in an incubator at 21% O2 until euthanisation. The animals are never subjected to an episode of hypoxia (only the feeding moments are under normoxic conditions, but these are very limited in time – measured in seconds).

4. “Authors should state whether the does are genetically identical – if not, how does that affect the model?”

• In this model the does were not genetically identical, as is often the case in inbred mouse/rat models. This comes with both its advantages as well as drawbacks. The fact that the does were genetically diverse could prove the results to be less consistent than if the does were to be genetically identical, however it does provide a finer reflection of the clinical setting: a genetically diverse setting.

5. “Authors should clarify how the pups from each mother was assigned to each arm of the study (that is equal distribution of pups from different mothers, especially between G28 and G28+7 arms of the study), because a similar in-utero environment may contribute to the ultimate developmental changes seen and may not represent the average effect seen across mothers and pups.”

• From each mother allocated to preterm delivery, at a gestational age of 28 days, the first 3 fetuses were randomly selected for fetal harvest. This was based on the order that the pups were delivered through hysterotomy. Meaning that the pups from the experimental arm G28 and the experimental arm G28 + 7 originated from the same does.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

8 Oct 2020

PONE-D-20-11602R1

Glomerular developmental delay and proteinuria in the preterm neonatal rabbit.

PLOS ONE

Dear Dr. de Winter,

Thank you for submitting your manuscript to PLOS ONE.

After careful consideration, we invite you to perform an additional, minor's revision of your manuscript.

Indeed, reviewer No 2, while noting that many of his/her concerns have been addressed in your former revision, still has made two remarks that need to be taken into account in your final manuscript.

Please submit your revised manuscript by Nov 22 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Umberto Simeoni

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: 2 concerns remain:

1. The small number of animals in each group likely resulted in type 2 error in the statistical analyses, and in a lack of significant differences where there may be biologic effects. This should be indicated in the discussion. The comment in the author's response to reviewers, "morphometric techniques that have been used in this experiment are sensitive to confounding" would be magnified by the small number of animals.

2. In the discussion, lines 344-348, the authors hypothesize that increased proteinuria may be caused by podocyte pathology or tubular backflow of creatinine, but do not mention reduced tubular protein reabsorption that was proposed by Tsukahara based on their beta-2 microglobulin data. This should be included.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

9 Oct 2020

Reviewer #2:

1. “The small number of animals in each group likely resulted in type 2 error in the statistical analyses, and in a lack of significant differences where there may be biologic effects. This should be indicated in the discussion. The comment in the author's response to reviewers, "morphometric techniques that have been used in this experiment are sensitive to confounding" would be magnified by the small number of animals.”

• Indeed, the small number of animals in each experimental arm make it possible to create a type II statistical error, thus accepting a possibly false null hypothesis. We have adapted the manuscript in accordance and can be referenced on lines 355-357.

2. “In the discussion, lines 344-348, the authors hypothesize that increased proteinuria may be caused by podocyte pathology or tubular backflow of creatinine, but do not mention reduced tubular protein reabsorption that was proposed by Tsukahara based on their beta-2 microglobulin data. This should be included.”

• The reviewer is right. Tsukahara et al. have evaluated the presence of albumin and beta-2 microglobulin in urine samples during the neonatal period. They have found that both albumin and beta-2 microglobulin were increasingly elevated with increasing degrees of prematurity. In fact, changes in beta-2 microglobulin were more noticeable in relation to gestational age. Thus, we have adapted the manuscript to include this explanation. The data and findings from Tsukahara et al. are described in lines 337-342.

• In addition, we have adapted our hypothesis to fit the abovementioned findings by Tsukahara et al. The hypothesis is described from lines 344-349: “From this, we hypothesize that the increase in proteinuria in the absence of glomerular hypertrophy, found in preterm rabbits, may be caused by changes in the glomerular structure, such as podocyte immaturity or an “absolute” podocyte depletion (a decrease in the total number of healthy podocytes per glomerulus [29]) or through premature tubular processes such as tubular backflow of creatinine [28] and reduced tubular resorption [27].”

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

14 Oct 2020

Glomerular developmental delay and proteinuria in the preterm neonatal rabbit.

PONE-D-20-11602R2

Dear Dr. de Winter,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Umberto Simeoni

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

19 Oct 2020

PONE-D-20-11602R2

Glomerular developmental delay and proteinuria in the preterm neonatal rabbit.

Dear Dr. de Winter:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Umberto Simeoni

Academic Editor

PLOS ONE