Preterm birth was previously identified as a high-risk factor for the development of long-term chronic kidney disease (CKD) (Hoy et al., 1999; Crump et al., 2019; Ding et al., 2021a; Gao et al., 2022). The most widely accepted mechanism by which preterm birth could increase the risk of developing long-term CKD is the “nephron number hypothesis”, which holds that preterm infants or low-birth-weight infants who are born with fewer nephrons have an increased risk for renal disease later in life (Brenner et al., 1988; Luyckx et al., 2013; Luyckx, 2017; Perico et al., 2018). Both human and animal studies have confirmed that premature birth has adverse effects on kidney development and nephron numbers (Rodriguez et al., 2004; Gubhaju and Black, 2005; Sutherland et al., 2011; Stelloh et al., 2012; Ryan et al., 2018). In animal studies, a preterm mouse model in which pups were delivered 1 or 2 days early by cesarean delivery was established, and after a few weeks of feeding, the glomeruli were counted. Fewer nephrons were present in preterm mice than in full-term animals (Stelloh et al., 2012). This study directly confirmed the effect of preterm birth on nephron numbers. In human studies, although there is little direct evidence concerning nephron numbers in the context of preterm birth, researchers have examined kidney tissue collected at autopsy from preterm neonates and showed that preterm kidneys had a greater percentage of morphologically abnormal glomeruli, which suggested that the preterm kidney may have fewer functional nephrons, thereby increasing vulnerability to impaired renal function both in the early postnatal period and later in life (Sutherland et al., 2011; Ryan et al., 2018).

In addition to the nephron number hypothesis, in our recent study, we put forward the involvement of podocyte (PD) depletion hypothesis during this process (Ding et al., 2021a; Gao et al., 2022), which holds that preterm birth accelerates PD depletion during growth and in turn contributes to a high risk for future CKD development. In our human study, a more than 5-fold increase in urinary PD excretion was detected in preterm infants when the corrected gestational age matched that of full-term infants (Gao et al., 2022). In an animal study, higher urine PD excretion was detected at the cross-section time point, and persistently higher PD depletion was detected during long-term follow-up until the age of 12 months in rats, which corresponded to middle adulthood in humans. More importantly, gradually accelerated PD depletion was observed during this follow-up. The same finding was also confirmed in kidney biopsy samples in an animal study (Ding et al., 2021a). According to the PD depletion hypothesis, if progressive PD depletion occurs over time, it leads to proteinuria, progressive glomerulosclerosis and progressive loss of renal function culminating in end-stage kidney disease (Kim et al., 2001; Kriz, 2002; Wiggins, 2007; Ding et al., 2017). Thus, our previous studies confirmed the important role of PD and PD depletion in the risk of developing CKD after preterm birth.

During normal kidney development, nephron development begins in the 9th gestational week, with rapid proliferation in the last trimester, and ceases in approximately the 36th week (Brenner and Milford, 1993; Hoy et al., 2005; Faa et al., 2010; Ryan et al., 2018). Most preterm infants are born from 21 to 37 gestational weeks, corresponding to the second and third trimesters, which indicates that prematurity interrupts the normal physiological development of the kidneys (Patel et al., 2015; Stoll et al., 2015). Compared with full-term kidney, the preterm kidney begins performing functions such as waste filtration independently very early after birth, while most work of the full-term kidney is done by the maternal placenta. Thus, the development of the preterm kidney includes two stages: the intrauterine stage before preterm birth and the abnormal extrauterine stage. In full-term infants, the kidneys experience only the intrauterine stage before birth. The major difference between the preterm and full-term kidney is that the preterm kidney experiences an extrauterine stage of independent work. This abnormal stage may have an effect on the development and differentiation of PDs and subsequent outcomes. To thoroughly understand and investigate the possible mechanism during this process, in the present study, a preterm rat model was established by delivery of pups 2 days early. After 2 days, when the preterm rats had experienced the extrauterine stage of independent work for 2 days, the kidneys from these preterm rats and from full-term rat pups were collected. Then, single-cell RNA sequencing (scRNA-seq) and subsequent bioinformatic analysis were performed. The aim of the current study was to provide a new understanding of the mechanism of long-term CKD caused by preterm birth.

Preterm birth is a risk factor for the future development of CKD (Hoy et al., 1999; Crump et al., 2019; Ding et al., 2021a; Gao et al., 2022). In our previous (Ding et al., 2021a) and present study, a reduction in the number of differentiated PDs was found to be involved in this process, and accelerated PD depletion was proven to be one of the causes of future CKD development. Thus, exploring the effect of preterm birth on PDs is important for clarifying the mechanism of CKD development caused by preterm birth. Based on this background, we performed scRNA-seq of kidneys collected from the same period in preterm (postnatal day 2) and full-term rats (postnatal day 0). The main findings of this study suggest that accelerated differentiation of PDs occurs in preterm individuals.

In humans, the gestational age of most preterm infants is 21–37 weeks, corresponding to the second and third trimesters (Patel et al., 2015; Stoll et al., 2015). Preterm birth interrupts the normal development of the kidney. Thus, the mechanism underlying the high risk of CKD development caused by preterm birth is closely related to abnormal development of the kidney after preterm birth. In recent years, scRNA-seq has made a massive contribution to elucidating developmental processes as well as molecular mechanisms during kidney development (Adam et al., 2017; Lindstrom et al., 2018a; Lindstrom et al., 2018b; Wang et al., 2018; Ding et al., 2021b). However, most of these studies have focused on early fetal kidney development (Adam et al., 2017; Lindstrom et al., 2018a; Lindstrom et al., 2018b; Wang et al., 2018). Few studies have focused on the middle and late stages of kidney development (Ding et al., 2021b). These two stages of kidney development correspond to the stages of abnormal kidney development in preterm individuals. In the early stage of kidney development, nephron progenitor cell dynamics and lineage specification are very important, and most studies have focused on these events (Lindstrom et al., 2018b). The fate of most cells in the kidney is determined in that stage. During middle- and late-stage development of the kidney, differentiation of the renal lineages leads to maturation, and most of the functional structures, namely, nephrons, form. The large number of functional structures formed requires a large amount of functional protein synthesis. As shown in Figures 4, 5, ribosome-related genes were enriched in the PDs of full-term rats compared to preterm rats. It is well known that ribosomes are organelles for protein synthesis (Dai and Zhu, 2020; Gabut et al., 2020; Liutkute et al., 2020). Previous studies have reported that mature cells and developing cells have different protein synthesis rate, this rate is low in mature cells but high in developing cells (Buszczak et al., 2014; Tahmasebi et al., 2018). Protein synthesis could be indicated by ribosome assembly. In present study, ribosome-related genes were enriched in the PDs of full-term rats (postnatal day 0) compared with preterm rats (postnatal day 2). Enriched ribosome-related genes indicated high rate of protein synthesis during the normal development of PDs in full term rats (postnatal day 0). In addition, previous studies also identified an increase in translation of ribosome-related mRNAs at early stages of differentiation, whereas translation of ribosome-related mRNA was inhibited at later time (Ingolia et al., 2011). This could account for PD specific genes highly expressed in preterm rat PDs. PDs were terminally differentiated cells. High expression of PD specific genes indicated that this cell population was already at the late stage of differentiation. Thus, protein synthesis rate could be low since ribosomal related genes are less abundant in preterm PDs (postnatal day 2) than its in full-term PDs (postnatal day 0). Both of these findings suggest that preterm birth could accelerate the differentiation of PDs.

As shown in Figure 4D, GO functional analysis revealed that kidney development-related genes were enriched in preterm rat PDs (postnatal day 2); these genes include PD-specific genes and some PD precursor genes, which play very important roles in PDs (Boute et al., 2000; Lindstrom et al., 2018a; Lindstrom et al., 2018b). In full-term rats, the major function of the kidney as well as PD is assisted by the maternal placenta, while preterm birth separates the kidney from the mother; thus, the preterm kidney as well as PDs begin working independently. PDs in preterm individuals must promptly adapt to the extrauterine environment and commit to kidney functions. Thus, PD-specific molecules should be appear and be enriched to meet the situation of preterm kidneys. These findings were confirmed by significantly increased expression of PD and PD precursor genes in PDs from preterm rats (postnatal day 2) (Figure 6A). In addition, based on the results of pseudotime trajectory analysis (Figures 6B–D), the appearance of PDs in preterm rats (postnatal day 2) preceded the appearance of PDs in full-term rats (postnatal day 0). Interestingly, most highly expressed genes were PD precursor specific genes. As identified by previous studies, Mafb, Cldn5, and Bmp7 are the first genes to accumulate in the PD lineage, and these genes are overall enriched in preterm PDs instead of one or in part (Lindstrom et al., 2018b; Lindstrom et al., 2018c). To deeply explore mechanism involved in this change, we should not focus on one of these genes. We should examine earlier events before appearance of PDs specific genes. The appearance of PD-specific genes suggests that terminally differentiated cells are present and that all of the differentiation process is at a late stage. Thus, this limiting factor may be events that interrupt normal overall ribosome biogenesis as well as overall protein synthesis. In present study, it is pity that we have not confirmed the detailed mechanism that regulates overall less ribosome biogenesis as well as protein synthesis in preterm PDs. This is a limitation of present study. Further studies could focus on the mechanism involved in the regulation of protein synthesis process. Nevertheless, we could also conclude that preterm birth accelerates PD differentiation. In addition, prior to the present study, Sutherland et al. reported that preterm birth could accelerate kidney maturation based on pathological examination (Sutherland et al., 2011). In that study, researchers examined kidneys from preterm neonates who survived several days. A reduced percentage of immature glomeruli was found in the preterm group compared with postconceptional age-matched gestational controls (Sutherland et al., 2011). The present study not only revealed accelerated PD differentiation but also provided evidence at the molecular level for accelerated maturation of the kidney after preterm birth.

As shown in Figure 1, preterm birth can cause a reduction in the number of differentiated mature PDs. In addition, our major findings suggested that preterm birth accelerates PD differentiation. What is the relationship between these processes? Does accelerated differentiation of PDs lead to a reduction in the final number of mature PDs? PDs are lost in both normal and diseased kidneys (Wiggins, 2007; Buszczak et al., 2014; Ding et al., 2017; Dai and Zhu, 2020). In normal kidneys, PDs are lost at a very slow speed, while PDs are excreted into the urine at a high rate in most glomerular diseases (Wiggins, 2007; Hodgin et al., 2015; Ding et al., 2017; Kikuchi et al., 2017). If we assume that the rate of PD depletion and the number of nephron progenitor cells are the same before delivery in the two groups, then after establishment of the preterm rat model by cesarean delivery 2 days early, nephron progenitor cells would immediately differentiate into PD precursors and then PDs in preterm rats. However, the overall rate of protein synthesis indicated by ribosome biogenesis could be lower in preterm PDs (postnatal day 2) than in full term PDs (postnatal day 0). Then, PD endowment could be reduced in preterm kidneys. Thus, according to our deductions, acceleration of PDs caused by preterm birth should be an intermediate factor of reduction of mature PDs instead of critical cause.

Present study showed that the developmental stage of PDs in preterm individuals is at a critical stage of protein synthesis. This process is also an important process for epigenetic modifications (Buszczak et al., 2014; Tahmasebi et al., 2018). Preterm kidney starts to independently filtrate waste as well as other kidney work after birth. Any adverse postnatal factors could affect epigenetic modification. Especially in preterm infants, different from preterm rats, preterm infants are exposed to oxygen, infection, drugs and so on. All of these adverse factors may disrupt epigenetic modifications and could lead to dysregulation of gene function without altering the DNA sequence. Both the gene and environment interactions could be determinations of final PD endowment. Further studies could also focus on epigenetic mechanisms during kidney and PD development.

In previous studies, nephron progenitor cells were identified in postnatal day 1 mouse kidney (Adam et al., 2017). However, in the present study, cluster analysis did not identify this special cell population in rat postnatal day 0 or postnatal day 2 kidneys. There were no clusters that highly expressed both classical nephron progenitor markers, such as Six2 and Cited1. As shown in Supplemental Figure S1, most of the expression features of known nephron progenitor markers have been shown (Combes et al., 2019; Naganuma et al., 2021). Interestingly, most of these nephron progenitor markers were present in cluster of PC 1 or PC2. As reported in Alexander et al., in nephron progenitor subpopulations, these marker genes in PCs were also highly expressed in the NP cell cycle cluster (Combes et al., 2019). Above all, the PC cluster may be the NPC cluster. However, PC did not differentially express the classical NPC markers Six 2 and Cited 1. Then, we still annotated these two clusters as proliferating cells.

Our study focused on PDs, and we did not analyze other inherent kidney cell types. However, as shown in Figure 2, except for PT cells, the numbers of most types of kidney cells in preterm rats were smaller than those in full-term rats. Except for PC cells, most types of cells are major constituents of nephrons. The decreased number of these cells suggested a smaller number of nephrons in preterm rats. This finding confirmed the nephron number hypothesis. Although we have not thoroughly explored pattern changes in other types of kidney cells, the present study could provide a resource for researchers to achieve a deep understanding of molecular and regulatory events in preterm and full-term kidneys.