The problem begins with the diagnostic markers themselves. Serum creatinine, the workhorse of kidney assessment in adults and older children, is nearly useless in the first days of a newborn’s life because it largely reflects the mother’s creatinine, transferred across the placenta, rather than the infant’s own kidney function. It then fluctuates with muscle mass, fluid balance, and renal perfusion, all of which swing wildly in preterm infants. Urine output, the other pillar of AKI definitions, is notoriously difficult to measure accurately in babies weighing less than a kilogram, and many preterm infants paradoxically produce large volumes of urine during injury episodes. Clinicians are, in effect, navigating with broken instruments.

Enter neutrophil gelatinase-associated lipocalin, or NGAL. This 25-kilodalton protein, produced by neutrophils and by the tubular cells of the kidney itself, surges in the urine within hours of tubular injury—far earlier than creatinine can respond. In adult and pediatric populations, urinary NGAL has earned a solid reputation as an early warning system for AKI. But in preterm neonates, its promise has been throttled by a deceptively simple gap: nobody knows what normal actually looks like. Without well-defined reference ranges for healthy preterm infants, a uNGAL value is a number without a yardstick, and clinicians cannot tell whether a given concentration signals injury or simply reflects the biology of extreme prematurity.

A research team led by Nicole F. Asdell, Jonathan L. Slaughter, Elizabeth M. Bonachea, and Tahagod Mohamed of Nationwide Children’s Hospital in Columbus, Ohio, together with Shivam Joshi of Ohio State University, set out to build that yardstick. Drawing on archived urine samples from the Ohio Perinatal Research Network biorepository, collected between 2010 and 2020 at the hospital’s level IV neonatal intensive care unit, the researchers assembled a cohort of 100 infants born before 32 weeks of gestation. Crucially, these were babies who remained free of AKI, sepsis, necrotizing enterocolitis, urinary tract infection, and patent ductus arteriosus requiring treatment throughout their initial hospital admission—conditions known to elevate NGAL independently of kidney injury.

The exclusion criteria were applied with unusual rigor. Each infant’s medical record underwent an individualized chart review, with AKI defined by creatinine-only neonatal modified KDIGO criteria, urinary tract infection confirmed by treated positive urine cultures, and sepsis identified through antibiotic exposure beyond 48 hours, diagnostic problem lists, or positive blood cultures. This painstaking filtering was essential: because uNGAL rises in response to systemic inflammation and infection, even a single confounding illness would contaminate the normative data the study was designed to establish. Each infant contributed one urine sample, collected within the first seven days of life using sterile cotton balls placed in the diaper and stored at minus 80 degrees Celsius.

In the laboratory, the team quantified uNGAL concentrations using particle-enhanced immunoturbidimetry with BioPorto Diagnostics reagents on a Roche Cobas analyzer, and measured urinary creatinine enzymatically. Because both uNGAL and the uNGAL-to-creatinine ratio were heavily right-skewed, the researchers natural log-transformed the data before running correlations and regression models, and used both linear regression and LOESS smoothing to check for nonlinearity. The statistical architecture mattered: gestational age was analyzed in three prespecified bands—23 to 25 weeks, 26 to 28 weeks, and 29 to 31 weeks—with adjusted models incorporating infant sex and birth weight.

The results were striking in their consistency. Gestational age showed a significant inverse association with uNGAL: the earlier the baby was born, the higher the baseline concentration. The Pearson correlation coefficient between gestational age and uNGAL was minus 0.503, and it strengthened to minus 0.606 when the biomarker was normalized to urinary creatinine, with p-values below 0.0001 for every analysis. Birth weight told the same story, with correlations of minus 0.483 for raw uNGAL and minus 0.567 for the creatinine-normalized ratio. In the regression models, infants born at 23 to 25 weeks had dramatically higher uNGAL than those born at 29 to 31 weeks—a log-scale difference of 1.58 unadjusted and 1.32 after adjustment for birth weight and sex, both statistically robust.

Sex left its own independent imprint on the data. Female infants had significantly higher mean uNGAL concentrations than males—225 versus 165 nanograms per milliliter, a difference that persisted in multivariable models adjusting for gestational age and birth weight. The researchers point to several plausible mechanisms: sex-based differences in tubular enzyme expression, hormonal effects, and differences in urinary white blood cell excretion, including subclinical or asymptomatic leukocyturia, which previous work suggests is more prevalent in female infants. Earlier studies, including De Mul and colleagues’ reference value work in very low birth weight infants, had hinted at these patterns, but small samples and inconsistent methods had prevented the field from consolidating them into usable clinical thresholds.

Why would the youngest preterm babies carry the highest baseline NGAL? The authors favor an explanation rooted in developmental biology: immature tubular function, increased permeability of the glomerular filtration barrier, and physiologically elevated NGAL expression during active renal development all likely contribute. But they are careful to note that alternative factors may be at play in extremely preterm infants, including a heightened inflammatory milieu, subclinical infection or leukocyturia, and the medication exposures that are routine in this population—any of which could nudge uNGAL upward independent of structural immaturity. The study’s normalization strategy also adds credibility: by expressing NGAL relative to urinary creatinine, the researchers partially controlled for the dramatic fluctuations in fluid status and urine output that make raw concentrations unreliable in preterm infants.

The study is not without limitations, and the authors are candid about them. It was conducted at a single center, relied on spot urine samples rather than timed collections, and faced uncertainty about the long-term stability of frozen uNGAL specimens. Excluding infants with AKI, necrotizing enterocolitis, sepsis, and treated patent ductus arteriosus was necessary to establish normative data, but it narrows generalizability to the broader preterm population, where such conditions are common. Still, the core message stands: NGAL is detectable in the urine as early as 23 weeks of gestation, its concentration falls with increasing gestational age and birth weight, and it runs higher in female neonates. Any clinical interpretation of uNGAL must therefore be anchored to developmental maturity and sex.

The payoff, if subsequent studies validate these findings in larger and more diverse cohorts, could be substantial. A reliable, non-invasive, blood-sparing biomarker would allow clinicians to identify kidney injury in preterm infants before urine output changes or creatinine shifts become apparent—opening a window for timely interventions such as nephrotoxic drug adjustment, fluid optimization, and blood pressure support during the earliest, most treatable phase of injury. In a population where every milliliter of blood is precious and every day of undetected organ injury compounds lifelong risk, gestational age- and sex-specific reference ranges for uNGAL could transform one of neonatology’s most stubborn diagnostic blind spots into a manageable, measurable target.

Subject of Research: Establishing baseline urinary NGAL reference ranges for acute kidney injury detection in preterm neonates

Article Title: Urinary neutrophil gelatinase-associated lipocalin concentration: a baseline study for establishing reference ranges in preterm neonates

Article References: Asdell, N. F., Slaughter, J. L., Joshi, S., Bonachea, E. M., & Mohamed, T. (2026). Urinary neutrophil gelatinase-associated lipocalin concentration: a baseline study for establishing reference ranges in preterm neonates. Journal of Perinatology. https://doi.org/10.1038/s41372-026-02848-x

Image Credits: AI Generated

DOI: 10.1038/s41372-026-02848-x

Keywords: urinary NGAL, acute kidney injury, preterm neonates, biomarkers, serum creatinine, gestational age, birth weight, neonatal intensive care, kidney development, reference ranges, Journal of Perinatology, neonatology

The research team, led by Taiji Matsusaka of Tokai University School of Medicine, built a transgenic mouse platform designed to force podocytes back into the cell cycle on demand. The system exploits the Tet-On inducible expression technology, in which a tetracycline-responsive element drives transgene expression only when the antibiotic doxycycline is present. The authors microinjected a construct containing the Simian virus 40 large T antigen (SV40T) coding sequence, together with a lacZ reporter, into fertilized mouse eggs and established transgenic lines. SV40T is a viral oncoprotein best known for promoting cell-cycle entry by binding and functionally inactivating two key tumor suppressors, p53 and the retinoblastoma protein (Rb). Because it can push otherwise quiescent cells past the G1 restriction point, it is a standard tool for immortalizing podocytes in culture, making it a logical choice for attempts to stimulate division in vivo.

The transgenic line was crossed with Nphs2-rtTA mice, in which the reverse tetracycline transactivator is expressed specifically in podocytes under the podocin promoter. In double-transgenic offspring, doxycycline in the drinking water therefore triggered SV40T production exclusively in podocytes. The specificity was confirmed by immunostaining: after four to fourteen days of treatment, roughly twenty to thirty percent of podocytes per glomerular section expressed the viral antigen, while untreated animals and single-transgenic controls showed nothing. Critically, the forced cells did more than merely turn on the transgene. Between 9.3 and 17.7 percent of podocytes became positive for Ki67, a marker spanning late G1 through mitosis, incorporated bromodeoxyuridine to indicate DNA synthesis, expressed Aurora kinase B, and occasionally displayed mitotic figures. At the same time, p57Kip2, a cyclin-dependent kinase inhibitor that typifies the mature podocyte state, was downregulated in the SV40T-positive cells. The quiescent filter cells had, by every molecular criterion, re-entered the cell cycle.

In adult mice, however, this proliferative push came at a steep price. Cytokinesis frequently failed, leaving podocytes multinucleated or bearing enlarged nuclei. Within the first week of doxycycline exposure, kidney morphology was largely preserved apart from microvillous transformation of the podocytes, but the urinary albumin-to-creatinine ratio climbed dramatically, from control values near 0.02 to 15.76 by day seven and 40.27 by day fourteen. By two weeks, the slit diaphragm proteins nephrin and podocin were reduced and the injury marker desmin appeared in glomerular segments. After four or more weeks of continuous induction, every treated mouse developed focal segmental glomerulosclerosis of the usual type, complete with secondary tubulointerstitial damage in the most severely affected kidneys. Intriguingly, the damaged podocytes no longer expressed SV40T, apparently because podocyte injury extinguishes Nphs2-driven rtTA expression, an automatic brake on the system. Repeated short treatment cycles combined with the angiotensin receptor blocker losartan produced only a modest, marginal increase in podocyte counts, confirming that adult podocytes resist expansion even under carefully titrated conditions.

That failure redirected the strategy toward embryogenesis, a period when podocyte progenitors are naturally proliferative and the glomerulus has not yet been exposed to high filtration pressures. Pregnant dams carrying SV40T/rtTA embryos received doxycycline from embryonic day 14.5 until birth. The team also incorporated a third genetic element: Bcl2 knockout alleles. Homozygous Bcl2-deficient mice are a classical model of oligomeganephronia, a condition in which apoptosis of undifferentiated mesenchymal cells between embryonic days 13 and 16 drastically reduces nephron number, forcing the surviving glomeruli to enlarge compensatorily. This provided a natural test bed for whether extra podocytes could augment, or even drive, glomerular hypertrophy under strong physiological pressure.

The results at birth were striking. Newborn double-transgenic pups, on either Bcl2 background, carried markedly more podocytes than their littermate controls, with nearly all inner-cortical podocytes expressing SV40T and most of those positive for Ki67. On average, the number of WT1-positive podocytes per glomerulus rose by 54 percent. Importantly, these surplus cells had not simply dedifferentiated into a pathological mass: they retained robust expression of podocin, WT1, and podocalyxin, the molecular signature of committed podocytes, and they did not resemble the crescents seen in collapsing glomerulopathies. Yet the glomerular tuft area, measured by painstaking morphometry of all non-tangential glomeruli across entire kidney cross-sections, was not significantly larger than in controls. Even individual glomeruli loaded with excessive podocytes never exceeded the size of ordinary control glomeruli. The correlation between podocyte number and glomerular volume, so reliable in normal growth, had been decisively broken from the other direction.

Rather than integrating neatly into the filtration barrier, the extra podocytes misbehaved structurally. They formed multilayers instead of the normal monolayer, detached from the glomerular basement membrane, and scattered into Bowman’s space or even into tubular lumens. Double immunostaining showed that most cells remained mononuclear, with WT1-positive nuclei enclosed by podocalyxin, arguing against rampant failed division. Markers of DNA damage (gammaH2AX) and apoptosis (cleaved lamin A) appeared in only a small fraction of cells, averaging 0.26 and 0.39 per glomerulus respectively. The authors interpret this pattern as evidence that adhesive failure during mitotic rounding, rather than programmed cell death, is the dominant mechanism of loss. During mitosis, epithelial cells normally round up and transiently weaken integrin-mediated adhesion before reattaching; podocytes, with their intricate foot-process architecture and dependence on precise attachment to the basement membrane, appear exceptionally vulnerable to this structural upheaval.

The Bcl2-deficient experiments sharpened the conclusion. All Bcl2 knockout mice, regardless of transgene status, showed severe renal hypoplasia with few, enlarged glomeruli. In SV40T-expressing Bcl2-deficient pups, podocyte numbers rose as in the wild-type background, but many cells again detached from the glomerular basement membrane, and the tuft area was actually significantly smaller than in Bcl2-deficient littermates lacking the transgene. In other words, even when compensatory hypertrophy should have been pushing glomeruli to expand, the surplus, poorly anchored podocytes appeared to exert a squeezing effect that constrained tuft growth. Structural integration, not cell supply, proved to be the limiting factor.

Postnatal follow-up sealed the outcome. Among 26 pups analyzed at postnatal day 7, the previously excessive podocyte populations had vanished: podocyte number and tuft area in transgenic animals were indistinguishable from controls, and glomeruli containing more than thirty podocytes, common at birth, were no longer observed. The authors propose several non-exclusive explanations for this elimination. Podocytes may lack the stringent planar-division orientation control that keeps renal tubular epithelia in a monolayer, dooming misplaced daughters. Alternatively, a quality-control mechanism known as cell competition, in which fitter neighbors actively expunge less fit cells, may recognize SV40T-induced podocytes as losers and remove them, effectively imposing an occupancy-management system on the glomerular tuft.

The broader implication is a reframing of how glomerular size is set. Because the Nphs2 promoter only activates once podocytes have passed the S-shaped body stage, the study could not boost progenitor pools at earlier stages, and the authors conclude that glomerular size is likely determined at or before that point in nephrogenesis. Adding podocytes afterward, even during the permissive embryonic environment, cannot override the architectural constraints of the developing tuft. For regenerative medicine, the message is sobering but precise: future strategies to expand the podocyte population must either target earlier progenitor stages or solve the biophysical problems of division orientation and cell-matrix adhesion so that new cells can stably integrate into the filter. Simply forcing mature or nearly mature podocytes to cycle, whether by viral oncoproteins or other means, produces cells that the glomerulus cannot accommodate and ultimately discards.

Subject of Research: The effect of experimentally induced podocyte proliferation on glomerular size in mice

Article Title: Increase in podocyte number induced by SV40T antigen during the fetal stage does not expand glomerular size in mice

Article References: Matsusaka, T., Ichimura, K., Miyaki, T., & Koizumi, M. (2026). Increase in podocyte number induced by SV40T antigen during the fetal stage does not expand glomerular size in mice. Physiological Reports, 14(19), Article e71114. https://doi.org/10.14814/phy2.71114

Image Credits: AI Generated

DOI: 10.14814/phy2.71114

Keywords: podocytes, glomerulus, SV40 large T antigen, kidney development, Tet-On system, focal segmental glomerulosclerosis, Bcl2 deficiency, oligomeganephronia, mitotic catastrophe, cell competition, glomerular basement membrane, chronic kidney disease