Twin glomeruli: a newly discovered marker of neonephrogenesis in the ischemia-reperfusion injured adult mouse kidney

Twin glomeruli: a newly discovered marker of neonephrogenesis in the ischemia-reperfusion injured adult mouse kidney | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Twin glomeruli: a newly discovered marker of neonephrogenesis in the ischemia-reperfusion injured adult mouse kidney Mae-Ja Park, Hanguk Hwang, dongju woo, You Ri Park, Min Jung Kong, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6399428/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The renal glomerulus, a capillary plexus between two arterioles, is crucial for urine production in mammals. While partial glomerular regeneration after renal injury is well recognized, its precise mechanisms remain unclear. However, stereological studies on post-injury glomerular structural changes are limited. This study investigated three-dimensional alterations in the glomerulus over time following ischemia-reperfusion injury (IRI) in adult mouse kidneys. We identified a unique "twin glomeruli" structure between three arterioles and connected by an atypical “aefferent” arteriole. This structure appeared between 3 and 21 days post-IRI, peaking at day 9. The twin glomeruli exhibited distinct features, differing from both degenerating and developing glomeruli. Synchrotron radiation micro-computed tomography revealed a time-dependent nephron increase between 1 and 21 days post-IRI. Immunohistochemical analysis also revealed significant increases in glomerular and tubular densities from days 9 to 21. These findings suggest that twin glomeruli are a transient structure induced by IRI and may be associated with an increase in nephron numbers. Our study challenges prevailing views, revealing that twin glomeruli represent an unconventional glomerular structure occurring during kidney repair and suggesting the possibility of neonephrogenesis in the adult mouse kidney following IRI, a process previously considered impossible postnatally. Health sciences/Anatomy/Kidney/Nephrons/Glomerulus Biological sciences/Structural biology/Electron microscopy twin glomeruli renal ischemia/reperfusion injury neonephrogenesis glomerulogenesis adult mouse kidney synchrotron radiation micro-computed tomography Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights 1. A unique twin glomeruli structure was observed in the kidneys of IRI mice 2. Twin glomeruli, arranged in pairs, were connected by a single afferent arteriole 3. The number of nephrons significantly increased in adult mouse kidneys after IRI Introduction The nephron, the functional unit of the kidney, consists of a glomerulus and a tubular component. The glomerulus is an anastomosing network of fenestrated capillaries located between two arterioles and enclosed by Bowman’s capsule, which marks the beginning of the renal tubule. This intricately structured nephron comprises numerous specialized cells with distinct shapes, locations, and arrangements, all of which are essential for normal kidney function. However, these characteristics also render the nephron highly susceptible to various forms of damage. It is commonly believed that the number of nephrons is established around birth and declines throughout life due to damage, disease, or natural aging 1 . Despite the inability to generate new nephrons and their vulnerability to injury, nephrons exhibit a remarkable capacity to survive damage and partially restore function. This regenerative ability is largely attributed to the robust regenerative capacity of tubular cells and, to a lesser extent, the limited regeneration capacity of glomeruli 2 . Damage to glomerular capillaries is a critical factor in kidney disease progression 2 – 5 . Successful capillary repair can restore the glomerular structure in damaged nephrons, whereas failure of this process often leads to glomerular sclerosis and subsequent renal dysfunction 1 . For many years, it was believed that glomeruli could not regenerate after injury. However, this notion was challenged by Fioretto’s groundbreaking report of glomerular regeneration in diabetic patients 6 . This discovery paved the way for further research, demonstrating the potential for glomerular regeneration in adult mammalian kidneys. More recently, regression of acute kidney disease (AKD) and even glomerulosclerosis under specific conditions has been attributed to glomerular regeneration 7 , 8 . The identification of renal stem cells or renal progenitor cells (RPCs) has been another significant milestone in understanding the mechanisms underlying glomerular repair 9 , 10 . Renal ischemia-reperfusion injury (R-IRI), a severe form of acute kidney injury, is characterized by proximal tubular necrosis, endothelial damage, and an inflammatory response, leading to sudden loss of renal function and progressive fibrotic changes. While the damaging and reparative effects of R-IRI on tubular and glomerular cells have been extensively studied 11 , the overall architectural changes in glomeruli induced by R-IRI remain largely unexplored. Since the precise restructuring of glomerular cells is essential for restoring full functionality, understanding how the entire glomerular architecture responds to vascular injury could provide valuable insights into the mechanisms of glomerular repair. In this study, we investigated the morphological response of glomerular architecture to severe vascular damage over time using an in vivo mouse model involving bilateral clamping of the renal arteries. Scanning electron microscopy (SEM) images of intravascular corrosive resin casts revealed that IRI induced dramatic morphological changes in the glomeruli, including the appearance of a unique “twin glomeruli” structure. This structure, in which two glomeruli are interconnected by a single, highly unusual arteriole (Fig. 3 c, d), is distinctly different from normal glomeruli (Fig. 3 a, b). The twin glomeruli were observed exclusively at 3, 9, and 21 days post-injury and displayed a configuration clearly distinct from both degenerating and regenerating glomeruli. Synchrotron radiation micro-CT image analysis revealed a significant increase in the average number of glomeruli in the IRI group compared to the control group. This suggests that twin glomeruli represent a temporary, acquired structure associated with the increase in glomerular numbers following IRI in adult mouse kidneys. Remarkably, neonephrogenesis appears to be reactivated by IRI in a manner distinct from embryonic nephrogenesis, challenging the long-held belief that nephrogenesis ceases entirely after birth in mammals. Results Gross and histological characteristics of IRI kidneys in adult mouse We examined morphological changes in the glomerular architecture of kidneys from 7-week-old mice following IRI. As shown in Fig. 1 a, kidneys were harvested at 1, 3, 9, and 21 days post-injury, referred to as the day 1, 3, 9, and 21 groups, respectively, after which the size, color, and surface texture of the kidneys were observed for each group. Macroscopic observations revealed that kidneys from the day 1, 3, and 9 groups appeared slightly larger than those from the control group. Unlike the control kidneys, which displayed uniform size and color, the IRI-affected kidneys exhibited variability within each group. Subcapsular hemorrhage and blood engorgement were evident in the day 1, 3, and 9 groups, indicating severe vascular damage in the renal parenchyma. In contrast, kidneys from the day 21 group appeared slightly smaller and paler than those of the control group, possibly due to reduced residual blood at the time of organ harvesting. This reduction may be attributed to the formation of arteriovenous shunts or collateral circulation, which could have increased blood flow velocity and decreased blood retention within the tissue. As illustrated in Fig. 1 b, the mean kidney weight in all IRI groups was significantly higher than that of the control group, likely due to fluid accumulation and hemorrhage following injury. Glomerular damage was further assessed through urine analysis, with urine protein concentration, an indicator of acute kidney damage and glomerular injury, significantly elevated in all IRI groups compared to controls (Fig. 1 c). Interestingly, the day 9 group exhibited a significantly lower urine protein concentration (69.97 mg/dL) compared to the day 1, 3, and 21 groups, although it remained slightly above control levels (Supplementary Table 1). Similarly, blood urea nitrogen (BUN) and plasma creatinine levels increased sharply one day after IRI, followed by a gradual, time-dependent decline (Fig. 1 d and e, Supplementary Tables 2 and 3). These findings suggest that while glomeruli sustained severe damage from IRI, the kidneys exhibited partial recovery over time. To further characterize histopathological changes, kidney sections were stained with periodic acid-Schiff (PAS) reagent (Fig. 2 a-r). Despite the severe injury indicated by the aforementioned biochemical markers, many glomeruli in IRI kidneys retained a relatively normal configuration (Fig. 2 b-e). However, distinct pathological changes were observed in some severely affected glomeruli (Fig. 2 g-r). The injured glomerular plexus exhibited structural disorganization, appearing either loosened or densely packed. Some glomeruli displayed capillary lumen dilation, while others showed obliteration due to capillary wall collapse (Fig. 2 g-r). Signs of inflammation were also evident, including endothelial cell swelling and inflammatory cell infiltration within the glomeruli (Fig. 2 h, k, n and q). Additionally, severe damage was observed in podocytes and the glomerular basement membrane (GBM) (Fig. 2 i, l, o and r). In some cases, Bowman’s capsule exhibited significant structural changes, including tubularization (Fig. 2 i, l, o and r). Progressive collagen deposition was evident over time, as shown in Sirius red-stained renal tissue from IRI kidneys (Fig. 2 s-w, Fig. 2 x), indicating ongoing structural remodeling in response to injury. Twin glomeruli observed in IRI adult mouse kidney To visualize the 3D structural changes in glomerular architecture following IRI, we performed resin casting using Pu4ii resin via cardiac perfusion and examined the resin-cast kidneys with SEM. As expected, the glomeruli of the control group maintained their typical shape and size (Fig. 2 a, Fig. 3 a, b). In the day 1, 3, and 9 groups, the majority of glomeruli appeared normal in shape and size, similar to those in the control group. However, a smaller proportion exhibited significant morphological changes. Some glomeruli were noticeably enlarged and consisted of two to three lobes with a slightly widened urinary pole (Fig. 3 b, c). Several glomeruli in the day 1, 3, and 9 groups were markedly enlarged, with some displaying two to three distinct lobes (Fig. 3 d–h). Among these, some showed prominently bulging lobules that were slightly separated from each other (Fig. 3 e, f and h). In contrast, tiny glomeruli (Fig. 3 g) and fragmented glomerular remnants were observed less frequently (Fig. 3 i, j). A hollow, broken, basket-shaped glomerulus appeared to be caught on a branching arteriole from the interlobular artery (Fig. 3 j). Additionally, multiple arterioles were seen entering and exiting through a damaged glomerulus (Fig. 3 i). By day 21, some glomeruli remained enlarged and densely packed, but lobulated glomeruli were less common than in the earlier time points (Fig. 3 k–m). This suggests that while glomerular volume expansion persisted, lobular expansion gradually declined by day 21. Interestingly, the damaged glomeruli appeared more crushed in the SEM images than in the PAS-stained specimens. A particularly striking finding in the SEM analysis was the identification of a unique glomerular structure in the day 3, 9, and 21 groups. This structure was completely distinct from a normal glomerulus (Fig. 4 a, b). Two glomeruli were connected by a single, unusual arteriole, hereinafter referred to as an “aefferent” arteriole, forming what we describe as “twin glomeruli” (Fig. 4 c, d, g–i). In these structures, the afferent arteriole of one glomerulus appeared to simultaneously function as the efferent arteriole for the other, and vice versa. The length of the aefferent arteriole varied from approximately 20 µm to 300 µm, significantly longer than the distance between lobules in lobulated glomeruli (Fig. 4 d–i). Additionally, the shape and size of each glomerulus within the twin glomeruli varied; in some cases, a new glomerulus appeared to be forming and projecting outward from an existing one (Fig. 4 h, uppermost image). Twin glomeruli were first observed in the day 3 group (4 twin glomeruli across 6 kidneys from 3 mice) and were most frequently detected in the day 9 group (6 twin glomeruli across 6 kidneys from 3 mice). By day 21, only a single kidney contained one twin glomerulus (1 twin glomerulus across 6 kidneys from 3 mice) (Fig. 4 e, Supplementary Table 4). No twin glomeruli were observed in the control or day 1 groups. The incidence of twin glomeruli was very low, ranging from 0.016–0.389% (Fig. 4 f, Supplementary Table 5). These findings suggest that twin glomeruli are a rare, temporary, and acquired structural anomaly induced by IRI. Increased glomerular number in IRI adult mouse kidney Although only a small number of twin glomeruli were observed, their presence suggests the possibility of previously unknown repair processes following vascular injury in the adult kidney, potentially indicative of neoglomerulogenesis or neonephrogenesis. This finding contrasts with the existing consensus 1 , which holds that nephrogenic sources are not widely recognized in the adult mouse kidney. It also raises the question of whether the glomerular number could be compensated by innate mechanisms in the IRI kidney. A significant increase in glomerular count could provide insights into the potential role of twin glomeruli in this process. To quantify glomerular number, we performed synchrotron radiation micro-CT imaging (Fig. 5 a and b). As shown in Fig. 5 c, the average glomerular count in the left kidney of 7-week-old control mice was 8,017. This number increased to an average of 11,589 in the day 1 group and 11,545 in the day 3 group. The glomerular count peaked at approximately 13,969 in the day 9 group and plateaued at around 13,898 in the day 21 group (Supplementary Table 6). Overall, these results indicate an increase in glomerular number following injury. The mean glomerular volume, a hallmark of severe nephron injury, showed a slight increase over time after IRI (Fig. 5 d), suggesting significant renal tissue damage. However, the simultaneous increase in both glomerular number and volume contradicts the well-established inverse relationship between these parameters following nephron loss 12 , 13 . This is because glomerular number and volume are considered compensatory (i.e., when nephron loss occurs, surviving glomeruli enlarge to compensate). The disruption of this relationship in IRI mice suggests that glomerular volume may no longer serve as a reliable surrogate marker for nephron deficiency. Furthermore, as shown in Fig. 5 e, the variance in glomerular volume increased following IRI, with the greatest variation observed in the day 9 group. These findings suggest that active repair processes occurred at both the histological and cellular levels (Supplementary Fig. 1 and Supplementary Tables 7 and 8). IRI appears to induce dynamic reshaping of glomeruli in both shape and number. However, the observed increase in glomerular number cannot be solely explained by glomerular regeneration in injured kidneys. Other repair mechanisms, as well as potential biological or technical factors in the experiment, should also be considered. Diversity of glomerular number according to the counting methods. The glomerular count in the left kidney of a normal 7-week-old mouse in our study differed from the findings of Cullen-McEwen et al. 14 , who reported 13,440 ± 1,275 glomeruli in the kidneys of male GDNF wild-type mice (14.0 ± 0.3 months old) using the physical dissector/fractionator combination method 15 . Meanwhile, Takiyama et al., using synchrotron radiation micro-CT, reported an average of 9,546.29 ± 1,912.79 glomeruli in C57BLKs mice, a value approximately midway between the results reported by Cullen-McEwen et al. and our study. Even when examining the same specimen, glomerular counts can vary significantly depending on the counting method and researcher. However, given that experimental animals are strictly controlled in terms of reproduction, housing, and diet, such large differences in glomerular counts are unlikely to result from biological variation alone. Instead, these discrepancies are more likely attributable to methodological differences, researcher variability, or other uncontrollable factors. To better understand these discrepancies in glomerular counts in control mice and, if possible, determine a more precise estimate of glomerular number in normal mice, we tested two additional technical approaches. First, we used Tomato lectin (from Lycopersicon esculentum ) to label vascular endothelial cells, marking capillary tufts via fluorescence, followed by a histo-transparency procedure (Fig. 5 f). Glomeruli labeled with lectin were counted using the Imaris rendering program, yielding an average of 14,003 glomeruli in wild-type mice (Fig. 5 g, Supplementary Table 9). This result closely matched those obtained using the physical dissector/fractionator combination method, suggesting that the glomerular count in normal adult mice is likely closer to 14,000 rather than the 8,724 previously reported (Fig. 5 c). Based on these findings, we concluded that Tomato lectin labeling is the most reliable and objective method for glomerular counting in normal animals. However, we were unable to obtain reliable results for IRI groups using this technique due to unresolved technical and biological challenges. As a second approach, we performed glomerular counting using immunohistochemistry with anti-synaptopodin, followed by the histo-transparency method (Fig. 5 h). Glomeruli labeled with synaptopodin were counted using the Imaris rendering program, yielding an average of 10,999 glomeruli in the control group and 11,778 in the day 9 group (Fig. 5 i, Supplementary Table 10). Although the glomerular count appeared to increase on day 9 compared to the control group, the difference was not statistically significant. However, given the practical limitations of the synaptopodin immunofluorescence procedure, such as reduced molecular penetration, our glomerular counts likely underestimate the actual numbers, particularly in the IRI group compared to the control group. Despite discrepancies between experimental methods, our findings consistently demonstrate that, regardless of the absolute glomerular count in wild-type mice, glomerular numbers tend to increase following IRI, as shown by both approaches. Increases in glomerular density with higher tubular density after IRI. Although our findings suggest that twin glomeruli may represent structures formed during the process of neoglomerulogenesis (ultimately leading to neonephrogenesis) induced by IRI, we cannot rule out the possibility that simple capillary plexuses, microhematomas, or other structures, particularly prevalent in IRI kidneys, were misidentified as glomeruli. To further support our findings regarding nephron number elevation after IRI, we conducted histochemical experiments on sectioned kidney tissues. A comparative analysis of changes in glomerular and proximal convoluted tubule density was performed using tissue sections (Fig. 6 a, b; Supplementary Tables 11a, b) to examine the relative trends in glomerular and tubular density. While measuring the number of glomeruli or tubules per unit area in tissue sections provides only relative estimates, as it does not account for kidney size or potential tissue changes due to IRI or processing, this approach remains widely used in morphometric studies due to its simplicity and reliability. Immunohistochemical staining was performed to visualize aquaporin-1 (AQP1), a marker for proximal convoluted tubules (Fig. 6 a). The density of AQP1-immunoreactive proximal convoluted tubules was compared with that of glomeruli in each group. The results demonstrated a time-dependent and gradual increase in both densities after IRI, aligning with findings from synchrotron radiation micro-CT imaging. Notably, significant increases were observed in the day 9 and day 21 groups compared to the control group (Fig. 6 b, c). In contrast, the day 1 and day 3 groups exhibited no significant density changes but did not show a decrease relative to the control group (Fig. 6 b, c). These findings indicate that IRI induces genuine increases in glomerular number rather than misinterpretation of other structures, with nephron number elevation persisting for at least three weeks (Fig. 6 b, c). To further investigate the glomerular repair process, vascular endothelial growth factor A (VEGF-A), a marker of angiogenesis 16 , was analyzed via immunostaining (Fig. 6 d). VEGF-A-immunoreactive glomeruli were detected in 8.5% (42/482) of the control group, 27.52% (120/436) of the day 1 group, 33.68% (128/380) of the day 3 group, 4.97% (22/442) of the day 9 group, and 10.95% (46/420) of the day 21 group (Fig. 6 e; Supplementary Table 12). These results suggest that glomeruli are dynamic structures undergoing continuous repair, with IRI enhancing vascular repair processes. Notably, all VEGF-A-immunoreactive glomeruli were enclosed within Bowman’s capsule, indicating that glomerular repair after IRI does not occur independently but is consistently accompanied by tubular repair. Increase in necrotizing glomeruli following IRI IRI is highly destructive to the kidneys, leading to the formation of numerous nonfunctional, degenerated glomeruli. Given the severely damaged glomeruli observed in SEM images of IRI kidneys, our findings suggest that IR injury induces glomerular degeneration, resulting in an increased number of degenerating glomeruli, referred to as atubular glomerular necrosis. Therefore, it is unlikely that all the counted glomeruli in this study correspond to functional nephrons. The presence and numerical changes of these nonfunctional, destroyed glomeruli may provide insights into the origins of twin glomeruli structure 17 – 20 . To assess changes in the number of degenerating glomeruli after IRI 17 , 19 , we performed an immunofluorescence study using synaptopodin, a marker for visceral podocytes and transdifferentiated podocytes (parietal podocytes) 21 . Synaptopodin-immunoreactive transdifferentiated parietal podocytes were detected in glomeruli across all groups, including the control group (Fig. 6 f). These degenerating glomeruli accounted for 1% of the total glomeruli in the control group (3/300), 5% in the day 1 group (15/300), 1.47% in the day 3 group (6/408), 5.31% in the day 9 group (18/339), and 3% in the day 21 group (13/427) (Fig. 6 g; Supplementary Table 13). Compared to the control group, the percentage of degenerating glomeruli in the IRI groups was 1.47 to 5.31 times higher. Nephron degeneration occurs at a consistent baseline level in normal kidneys 22 , 23 . However, IRI significantly accelerates this process. These findings indicate that degenerating glomeruli must be accounted for in glomerular quantification studies. While not all glomeruli counted in this study represent functional nephrons, the number of atubular glomeruli in the IRI group was insufficient to fully explain the total increase in nephron numbers observed following IRI. This discrepancy suggests that the simultaneous increase in nephron numbers and the presence of twin glomeruli in R-IRI can only be attributed to nephrogenesis. Although the underlying mechanisms remain unclear, these findings underscore the complexity of the repair processes involved. Discussion In the present study, twin glomeruli were observed as two glomeruli arranged in series, with the efferent arteriole of one glomerulus serving as the afferent arteriole of another. This configuration is markedly different from that of typical glomeruli, which are independently supplied and drained by separate arterioles. In a normal glomerulus, the afferent arteriole delivers blood, while the efferent arteriole carries it away after filtration. According to renal hydraulic pressure profiles 24 , a significant pressure drop occurs between the afferent and efferent arterioles, with glomerular pressure maintained at levels comparable to those in the distal afferent and proximal efferent arterioles. This structure ensures that each glomerulus functions independently to maintain proper filtration. However, in the case of sequentially connected glomeruli, the downstream glomerulus would likely experience impaired filtration. Thus, the twin glomeruli configuration does not align with established renal physiology. The term "twin glomeruli" was introduced in 1897 and has been structurally examined by several researchers. However, their exact features and roles remain undefined. Traditional histological methods have been less suitable for studying such morphological deviations, as even minor discrepancies in the plane of sectioning can render these structures invisible. Consequently, twin glomeruli have remained largely overlooked for decades, with only sporadic reports linking them to congenital anomalies or disease conditions in both animals and humans. Our findings confirm that the defining characteristic of twin glomeruli is their serial arrangement rather than a symmetrical pair supplied by a bifurcated afferent arteriole. Notably, in this study, twin glomeruli were exclusively observed in IRI-induced kidneys and only for a limited duration, suggesting that they are transient, acquired structures rather than permanent renal features. Furthermore, they exhibit morphological differences from previously described degenerating or regenerating glomeruli. These peculiar and transient formations may represent an ongoing, novel repair mechanism following vascular injury, distinct from previously understood regenerative processes. Using synchrotron radiation micro-CT, we detected a significant increase in glomerular counts in IRI kidneys, with the average number rising from 8,017 in control mice to a peak of 13,969 on day 9 post-injury. Glomerular count is known to directly reflect nephron number due to the nephron’s unique structural organization 25 . Therefore, our findings strongly suggest a corresponding increase in nephron number. This contradicts the widely accepted notion that nephron numbers can only decrease over time and do not increase after birth. While damaged nephrons have been thought to regenerate only through nephron repair following injury 5 – 7 , 26 rather than through neonephrogenesis, our findings challenge this assumption. Although data on increased glomerular numbers following renal injury are limited, some studies have reported similar trends. For instance, in response to cisplatin treatment, the total glomerular count in mice did not decrease but instead showed a slight increase, despite a concurrent rise in necrotizing glomeruli 27 . Similarly, in diabetic mice treated with SGLT2 inhibitors, glomerular numbers remained stable or exhibited a slight, albeit statistically insignificant, increase 28 . These findings suggest that, under certain pathological conditions, glomerular numbers may remain stable or even increase, contrary to the long-standing consensus. In the present study, the increase in glomerular number far exceeded what could be explained by simple glomerular regeneration alone, suggesting the presence of a novel repair mechanism following vascular injury. This substantial rise supports the notion that twin glomeruli may represent transient structures formed during neoglomerulogenesis induced by IRI. Rather than merely indicating glomerulogenesis, our findings suggest that this process reflects neonephrogenesis following IRI. The simultaneous increase in both tubular components and glomeruli supports the notion of nephron number augmentation and provides a basis for distinguishing genuine glomerular increases from potential misinterpretations of other structures in IRI. Notably, the experimental group exhibited a significant and concurrent increase in both glomerular and tubular densities on days 9 and 21 post-IRI. This correlation suggests that the observed rise in glomerular number is attributable to neonephrogenesis rather than the misidentification of vascular plexuses, as a true increase in glomerular number would naturally correspond to an increase in tubular structures. While the increase in tubular density may initially suggest the formation of new tubules, it is important to consider an alternative explanation: tubular elongation, particularly an increase in the length of proximal convoluted tubules due to regenerative processes. However, previous studies offer conflicting insights into this phenomenon. Møller et al. reported no significant changes in proximal tubule length or density in cases of chronic ureteral obstruction or severe cortical interstitial fibrosis in pigs 29 . Similarly, long-term lithium administration in rats resulted in a slight decrease in proximal tubular length, accompanied by severe cortical interstitial fibrosis 30 . Conversely, another study observed an increase in the average length of proximal convoluted tubules in chronically damaged rat kidneys, although this finding was accompanied by significant variability in total tubular length, with marked shortening in severe cases 31 . Interestingly, a study on diabetic mice treated with SGLT2 inhibitors suggested the possibility of glomerular redistribution from the juxtamedullary region to the superficial cortex, potentially involving restructuring of the convoluted and straight segments of proximal tubules. However, it remains unclear whether this redistribution was due to tubular elongation or reshaping 28 . At present, insufficient data are available to determine whether changes in nephron tubular length occur in IRI mice. Further studies are needed to clarify this aspect of nephron remodeling. Murine nephrogenesis occurs in two stages based on developmental timing: embryonic nephrogenesis and postnatal nephrogenesis. During postnatal nephrogenesis, multiple nephrons (up to five) can form at a single ureteric tip 32 , 33 , unlike embryonic nephrogenesis, where each ureteric tip gives rise to a single nephron 34 . Considering these two nephrogenic processes, it is conceivable that unexpected nephron morphologies could arise during repair processes in adult kidneys if neonephrogenesis were to resume after injury. Although embryonic stem cells and their associated developmental genetic pathways are absent in the adult kidney 35 , renal progenitor cells have been identified. These cells are key contributors to renal regeneration, though their exact mechanisms remain poorly understood. Additionally, studies have shown that stem cell-derived kidney organoids implanted into mice develop more functional nephrons than in vitro cultures, even in the absence of external additives. This evidence suggests that renal progenitor cells may play a more significant role in adult kidney repair than currently recognized, warranting further investigation. Among renal progenitor cells, juxtaglomerular cells of renin lineage (CoRL) have been shown to play a crucial role in nephrogenesis, influencing the structuring and branching of renal arterioles. CoRL exhibit remarkable plasticity, the ability to replicate, and the capacity to migrate into the glomerulus, where they can replace multiple glomerular cell types in diseased rodent kidneys 36 . Thus, CoRL can be regarded as upstream mesenchymal progenitors 9 , 37 . In this context, it is conceivable that existing arterioles or aberrant vessels formed within the injured renal corpuscle may serve as conduits for renal progenitor cells, facilitating their migration from their original location along these vessels and potentially leading to the development of a new glomerulus. This newly formed glomerulus, initially connected to the pre-existing one by an arteriole, could eventually separate, with the connecting arteriole evolving into an afferent arteriole. Alternatively, IRI could induce the lobular expansion of a glomerulus through the translocation and active repair of renal progenitor cells along pre-existing arterioles. As these progenitor cells integrate into an injured glomerulus, lobules may drift apart and separate, with an interconnecting vessel gradually elongating and transforming into an afferent arteriole. In either scenario, the two glomeruli, positioned at opposite ends of the afferent arteriole, may temporarily resemble twin glomeruli. Over time, the afferent arteriole may elongate further and split into two branches: one functioning as the efferent arteriole for the first glomerulus and the other as the afferent arteriole for the second glomerulus (Fig. 7 ). While glomerular repair processes, particularly the regeneration of glomerular capillary tufts, have been widely studied, reports on the formation of new aberrant arterioles connecting glomeruli to interstitial tissue remain limited. However, some studies have demonstrated that aberrant vessels can form between glomerular spaces and interstitial tissues during glomerulosclerosis 38 , 39 . These studies suggest that neovascularization may occur through breaches in Bowman’s capsule, extending into surrounding interstitial tissues in diseased kidneys. Similarly, our findings revealed various patterns of extraglomerular aberrant vessels connecting glomeruli with their extraglomerular spaces. This suggests that vascular regeneration following renal injury is not confined to the intraglomerular space but extends into the extraglomerular and interstitial regions. Such expansion implies that renal progenitor cells may have broader roles beyond the intraglomerular environment, potentially contributing to diverse forms of glomerulogenesis within adult kidney tissue. Conclusion The appearance of twin glomeruli observed in this study suggests a previously unrecognized glomerular repair process. This finding implies the existence of novel repair mechanisms and potential changes in glomerular number following R-IRI, which may have been overlooked in prior research. The possibility that neonephrogenesis can be reactivated in adult mice under IRI conditions challenges the long-standing belief that mammalian nephrogenesis ceases entirely after birth. Given that this conclusion contradicts many previous studies, further research is crucial to validate and elucidate the underlying mechanisms of this phenomenon. Materials and methods Experimental animals Adult male C57BL/6J mice (Koatech, Gyeonggi-do, ROK), aged 7 weeks and weighing 20–25 g, were used in this study. The mice were maintained on a standard laboratory chow diet with ad libitum access to tap water. To account for inter-animal variability in glomerular number, a minimum of three mice were assigned to each experimental group, and their data were pooled for analysis. To induce ischemia, the kidneys were exposed via an incision under anesthesia with pentobarbital sodium (60 mg/kg body weight; Hanlim Pharm., Korea). A microaneurysm clamp was used to occlude the renal pedicle for 30 minutes. In the control group, a sham operation (Sham) was performed using the same procedure, except for renal pedicle clamping. Body temperature was maintained at 36.5–37 °C during all surgical procedures using a temperature-controlled heating device (FHC, Bowdoinham, ME). After IRI, renal function was assessed by measuring urine protein, BUN, and creatinine concentrations using a Vitros 250 Chemistry Analyzer (Johnson & Johnson, Rochester, NY). Hematuria was evaluated microscopically. All animal studies were approved by the Animal Care and Use Committee of Kyungpook National University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 2011). Urine protein concentrations Urine was collected during surgery by emptying the urinary bladder using a 31-gauge syringe. Additional urine samples were obtained using a metabolic cage and a 31-gauge syringe. The collected urine was stored at −70 °C with protease inhibitors (1.2 mM sodium azide, 0.5 mM PMSF, and 1 µM leupeptin) until analysis. Protein concentrations were measured at the specified time points using a Vitros 250 Chemistry Analyzer (Johnson & Johnson, Rochester, NY, USA). Plasma creatinine and BUN concentrations Blood samples were collected from the retrobulbar venous plexus at the time points indicated in the figures. Plasma creatinine and BUN concentrations were measured using a Vitros 250 Chemistry Analyzer (Johnson & Johnson, Rochester, NY, USA). Histology Three mice per group were perfusion-fixed at predetermined time points (days 1, 3, 9, and 21 post-IRI) for kidney removal. The excised kidneys were post-fixed in PLP solution (4% paraformaldehyde, 75 mM L-lysine, and 10 mM sodium periodate; Sigma-Aldrich) overnight at 4 °C. The tissues were then embedded in paraffin and sectioned into 4-μm slices using a microtome (Leica, Bensheim, Germany). Kidney sections were stained with periodic acid-Schiff (PAS), Sirius red and fast green, and subjected to immunostaining for synaptopodin, VEGF-A, AQP, and TUNEL assay. Whole-kidney images were captured using a slide scanner, and cortical regions were photographed in 10 fields per kidney using a Nikon Fx35 (Nikon, Japan). At least four kidneys per experimental condition were analyzed, with ten fields per slide counted. Sirius red and fast green special stain Sirius red and fast green staining were performed to assess collagen fiber deposition in post-IRI kidney tissue at the specified time points. Kidney sections were initially stained with 0.04% fast green for 15 minutes, followed by an aqueous wash. The sections were then stained with 0.1% Sirius red and 0.04% fast green in saturated picric acid for 30 minutes, followed by two washes with acidified water (0.5% glacial acetic acid). The sections were rehydrated and dehydrated using graded alcohol. Collagen fibers were stained red, while non-collagen proteins appeared green. The Sirius-red-positive area was quantified using an image analysis program (i-Solution, IMT, Korea). Immunohistochemistry Immunohistochemistry was performed on paraffin-embedded tissues fixed with either 4% paraformaldehyde-PBS or Bouin’s solution, following the manufacturer’s instructions. To detect atubular glomeruli in the visceral epithelial cells of Bowman’s capsule, sections were stained with anti-synaptopodin conjugated with Alexa Fluor 488 (1:200, sc-515842, Santa Cruz Biotechnology, Santa Cruz, CA). Proximal convoluted tubules were labeled using anti-aquaporin-1 (1:200, 7D11, AQP1, Bio-Rad, Alomone Labs, Jerusalem, Israel) conjugated with Texas Red. Angiogenic glomeruli were detected using anti-VEGF-A (1:500, #ab1316, Abcam plc, Cambridge, UK) conjugated with Cy3. Immunohistochemistry was performed using the EnVision Kit (Agilent, CA, USA) according to the manufacturer’s instructions. Measurement of glomerular and proximal convoluted tubular densities The densities of glomeruli and proximal convoluted tubules were quantified by manually counting their numbers in 10 random fields per kidney under a microscope at 200× magnification (Leica DM 2500, Wetzlar, Germany). Statistical comparisons between experimental groups were conducted using a Student’s t -test. Vascular corrosion casting (VCC) and SEM analyses Sham-operated and ischemia-reperfusion (IR)-injured mice were anesthetized with pentobarbital (60 mg/kg body weight; Hanlim Pharm., Korea). The thoracic cavity was surgically opened, and the ascending aorta was cannulated via the left ventricle. To ensure complete removal of blood from the vascular lumen, the vasculature was thoroughly flushed with 100 ml of heparinized normal saline solution (20 IU/100 ml). Fixation was performed by infusing approximately 15 ml of 4% paraformaldehyde in phosphate-buffered saline (PBS), with all solutions maintained at 37 °C and infused at a rate of 5 ml/min. Immediately after fixation, a polyurethane resin (PU4ii, VasQtec, Switzerland) was mixed with ethyl methyl ketone (EMK, Merck) at a 6:1 ratio, with blue pigment added for contrast. This mixture was infused using a Harvard syringe pump at a constant rate of 5 ml/min until polymerization began. To prevent air bubble formation, the resin/solvent mixture was continuously injected. The polymerized casts maintained high reproduction quality without shrinkage. Full polymerization was achieved by placing the injected kidneys in a hot water bath (50–60 °C) for 24 hours. After polymerization, soft tissues were removed through maceration in 7.5% potassium hydroxide (KOH) at 50–60 °C for 1–2 days. The corrosion casts were washed with tap water to remove lipid-rich saponified material, then further cleaned in 5% formic acid for 10 minutes, followed by multiple rinses with distilled water. The casts were dried using a freeze-drying method. For SEM analysis, the vascular casts were rendered electron-conductive by gold coating for 90 seconds using a sputter coater. The casts were then examined using a scanning electron microscope at an accelerating voltage of 15 kV (Hitachi S-4300, Japan). Synchrotron radiation micro-CT Kidney sample preparation Resin-injected kidneys were collected from mice in each experimental group before the corrosion procedure. To prevent dehydration, each whole kidney was immersed in a microtube (Axygen, CA, USA) filled with an optimal cutting temperature (OCT) compound (Scigen, CA, USA) during mounting on the synchrotron radiation micro-CT unit. Experimental layout and image acquisition Synchrotron radiation micro-CT imaging was performed at the Wiggler 6C biomedical imaging beamline of the Pohang Light Source-II (PLS-II) in Pohang, Korea, following the protocol described in a previous study 40 . Synchrotron X-rays were generated from the electron storage ring, operating at an electron energy of 3 GeV with a typical current of 320 mA. The X-rays passed through two beryllium windows, with the X-ray source positioned 28 m from the experimental hutch. The electron beam traversed a double-crystal monochromator composed of silicon (Si111) multilayers and a beryllium window. To optimize high-resolution phase-contrast detection, the kidney samples were positioned 200 mm upstream of the detector. Microtomography was performed by rotating the sample in 0.2° increments over a 180° range using a computer-controlled precision stage. Each projection had an exposure time of 100 ms. The X-ray shadow of the specimen was converted into a visible image on the surface of CdWO 4 and YAG:Ce scintillation crystals. This image was magnified using a 5× microscopic objective lens and captured with a PCO 4000 charge-coupled device (CCD) camera at a resolution of 4,008 × 2,672 pixels. Three-dimensional volume images were reconstructed using a filtered back-projection algorithm applied to the projection images with the OCTOPUS software package (CT, Belgium). Surface reconstruction, volume segmentation, and rendering were conducted using Amira software (Visualization Sciences Group, Burlington, MA, USA). Morphometry Total glomerular number, glomerular tuft volume, and whole kidney volume were quantified from the three-dimensional reconstructed synchrotron radiation micro-CT images using Amira software. Lectin injection and tissue clearing After anesthetizing the mice, Lycopersicon esculentum agglutinin (tomato lectin, FSD Fluor™ 647, 50–100 μg/100 μl; Vector Laboratories, Burlingame, CA, USA) was injected directly into the left ventricle to stain functional blood vessels. Two minutes later, the mice were perfused through the left ventricle-aorta with rinse and fixation solutions. Following a pre-rinse with the rinse solution, the mice were fixed with 4% paraformaldehyde (PFA) and post-fixed overnight in the same fixative before being stored in 1× PBS (pH 7.4) overnight. The kidneys were removed, halved, and rendered transparent using a tissue clearing kit (Cat. HRTC-001, Binaree, Daegu, Korea). Briefly, PFA-fixed kidneys were immersed in Binaree fixing solution for 24 hours and incubated with tissue clearing solution in a shaking incubator at 37 °C for 4–5 days, followed by rinsing in a washing solution. Finally, the kidneys were incubated in mounting and storage solution (Cat. SHMS-060, Binaree) for 24 hours to achieve further clearing. Lightsheet fluorescence microscopy (LSFM) and 3D imaging Mouse kidney imaging was performed at 5× magnification using a Lightsheet Z.1 fluorescence microscope (Zeiss Corporation, Jena, Germany). Three-dimensional image rendering was conducted using the Imaris software (version 9.5.1, Oxford Instruments, Abingdon-on-Thames, UK; https://imaris.oxinst.com). Quantitative evaluation of kidney glomeruli was performed in maximum intensity projection mode, with rendering quality set to 100%. The "Background Subtraction" option was applied to smooth the image by creating a Gaussian-filtered channel minus the intensity of the original channel (diameter = 2.02 μm). The surface module was designed from the Gaussian-filtered channel using a threshold algorithm to quantify glomerular number and volume. Statistical analysis Logistic-transformed values of appearance rates were used to assess group effects, and comparisons between each IR-injured group (day 1, day 3, day 9, and day 21) and the control group were performed. An unpaired two-tailed Student's t -test was used for comparisons between two datasets. Error bars in graphical data represent means ± standard deviation. All in vivo and in vitro experiments were conducted at least three times. P -values < 0.05 were considered statistically significant. Statistical significance was determined as follows: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****). “NS” denotes non-significance. Data analysis was performed using Prism 6 (GraphPad Software, Inc.). Declarations The authors have no conflicts of interest to declare. Acknowledgments The authors thank the Pohang Light Source-II (PLS-II) in Pohang, Korea, and the UNIST Optical Biomed Imaging Center in Ulsan, Korea, for their support. This work was funded by the National Research Foundation of Korea (NRF) through grants from the Korean government (MSIT) (2017R1A5A2015391, 2021M3A9H3016063, and 2022R1F1A1074842). Hanguk Hwang and Dongju Woo contributed equally to this work. Correspondence should be addressed to Mae Ja Park, MD, Ph.D., Department of Anatomy, School of Medicine, Kyungpook National University, Daegu, South Korea. Author contributions MJ Park conceived the ideas, designed the research, and wrote the manuscript. H Hwang and D Woo conducted the animal experiments and analyzed the data. YR Park, MJ Kong and C Lee conducted the animal experiments. H Lee and SH Lee contributed to writing the manuscript. SY Lee and D Woo handled electronic tasks and submitted the manuscript. SW Nam analyzed and interpreted the data and wrote the paper. KM Park provided technical support and materials for the study. YC Bae, EJ. Nam, S Park, H Kim and JY Choi provided technical support to improve the study and contributed to data interpretation. MJ Park approved the final version for submission and is responsible for data acquisition and analysis. References Bertram JF, Hughson MD, Puelles VG, Hoy WE (2016) In: Little MH (ed) Kidney Development, Disease, Repair and Regeneration. Academic, pp 167–175 Meyer-Schwesinger C (2016) The Role of Renal Progenitors in Renal Regeneration. Nephron 132:101–109. https://doi.org:10.1159/000442180 Bonventre JV, Weinberg JM (2003) Recent advances in the pathophysiology of ischemic acute renal failure. 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Am J Pathol 183:333–335. https://doi.org:10.1016/j.ajpath.2013.04.009 Sagrinati C et al (2006) Isolation and characterization of multipotent progenitor cells from the Bowman's capsule of adult human kidneys. J Am Soc Nephrol 17:2443–2456. https://doi.org:10.1681/ASN.2006010089 Lim BJ, Kim MJ, Hong SW, Jeong HJ (2016) Aberrant Blood Vessel Formation Connecting the Glomerular Capillary Tuft and the Interstitium Is a Characteristic Feature of Focal Segmental Glomerulosclerosis-like IgA Nephropathy. J Pathol Transl Med 50:211–216. https://doi.org:10.4132/jptm.2016.02.01 Kriz W, Hosser H, Hahnel B, Gretz N, Provoost AP (1998) From segmental glomerulosclerosis to total nephron degeneration and interstitial fibrosis: a histopathological study in rat models and human glomerulopathies. Nephrol Dial Transpl 13:2781–2798. https://doi.org:10.1093/ndt/13.11.2781 Yi E et al (2017) Synchrotron tomographic images from human lung adenocarcinoma: Three-dimensional reconstruction and histologic correlations. Microsc Res Tech 80:1141–1148. https://doi.org:10.1002/jemt.22910 Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6399428","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":449163958,"identity":"5d799e3b-ec36-4b0c-b180-d1a381a00b14","order_by":0,"name":"Mae-Ja 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05:30:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6399428/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6399428/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81635201,"identity":"bebc688d-eb3c-4556-ab07-59c64453257f","added_by":"auto","created_at":"2025-04-29 12:16:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":261699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological and physiological characteristics of induced IRI in adult mouse kidneys. \u003c/strong\u003e(a) Gross anatomical appearance of kidneys following IRI. (b) Mean kidney weights significantly increased after IRI. (c) Urine protein concentration peaked on day 1 post-IRI and gradually decreased over the subsequent 21 days. (d) BUN levels spiked and then gradually declined. (e) Plasma creatinine concentrations followed a similar trend (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6399428/v1/70791e30e61566eea1c6d747.png"},{"id":81635204,"identity":"dc0c7f08-0861-48de-8f39-a4a3e24838df","added_by":"auto","created_at":"2025-04-29 12:16:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2666325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological characteristics of renal corpuscles in IRI mouse kidneys.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a-r) Histopathological characteristics of kidneys. Kidney sections were stained with periodic acid-Schiff (PAS) reagent. (a) Sham-operated control kidneys displayed normal histology. (b-e) While most glomeruli showed no apparent pathological changes, distinct pathological findings were observed in the tubular region, which regressed over time. Tubular epithelial cells exfoliated in the cortical tubules, forming proteinaceous casts on the brush border that progressed from the cortex to the medulla (black arrowheads). (f-r) Control kidneys exhibited normal glomerular structures, whereas most glomeruli appeared unaffected following IRI. However, some glomeruli displayed distinct pathological changes. (g-i) On day 1 post-IRI, some glomeruli exhibited a porous and loose appearance due to dilated capillaries and a disrupted plexus. Severe destruction of Bowman’s capsule tubulization was noted in certain glomeruli, characterized by necrosis (black arrow) and detachment (white arrow) of the capsular high cells. Additional pathological findings included endothelial cell swelling, inflammatory cell infiltration, capillary lumen obliteration due to capillary wall collapse (yellow arrow), blurred glomerular basement membrane (GBM), binucleated podocytes (gray arrow), detached podocytes (blue arrow), and apoptotic body-like structures (black arrow). (j–l) On day 3 and (m–o) day 9 post-IRI, some glomeruli displayed porous and loose plexuses due to capillary dilation. Apoptotic bodies (black arrow), floating epithelial cells (blue arrow) in the urinary space, and occasional binucleated podocytes were observed. Endothelial cell swelling, inflammatory cell infiltration, capillary lumen obliteration (yellow arrow), and GBM blurring (red arrow) were also evident. Some glomeruli exhibited floating epithelial cells (blue arrow), GBM blurring, podocyte detachment (blue arrow), and binucleated podocytes. A few glomeruli were surrounded by pericorpuscular fibrotic bands (black arrowhead), indicative of degeneration. (p–r) By day 21, fibrosis and inflammatory cell infiltration had increased, with hepatized glomeruli (orange arrowhead) surrounded by pericorpuscular fibrotic bands. (s–w) Photomicrographs of Sirius red/methyl green-stained renal tissues. In sham-operated kidneys (s), faint collagen fiber staining was observed around nephrons and renal vessels. Following IRI, Sirius red-positive collagen deposition progressively increased (t–w). (x) Quantification of Sirius red-positive areas using image analysis software (i-solution, IMT, Korea). Statistical significance: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6399428/v1/e24621b40941fdc5bd8a1f7b.png"},{"id":81635895,"identity":"6eee4d77-5824-4c96-992b-eb77a5a0d268","added_by":"auto","created_at":"2025-04-29 12:24:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":555850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAtypical glomeruli observed at 3, 9, and 21 days post-IRI in adult mice. \u003c/strong\u003e(a) A typical glomerulus in 7-week-old mice, displaying a capillary network originating from an afferent arteriole and draining into an efferent arteriole. (b–m) Atypical glomeruli observed in each experimental group, exhibiting distinct lobulated shapes and variable sizes, ranging from exceptionally large to abnormally small. Some glomeruli displayed a hollow, fragmented, basket-like appearance.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6399428/v1/27b447427f47cc7a77d0e3d9.png"},{"id":81635203,"identity":"ceaae4fd-f6d0-4fca-b511-0321a564d261","added_by":"auto","created_at":"2025-04-29 12:16:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":831845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D structural characteristics of twin glomeruli in the kidney at 3, 9, and 21 days post-IRI. \u003c/strong\u003e(a) Schematic diagram of the nephron and its blood supply, illustrating the renal corpuscle (glomerulus and Bowman's capsule) and renal tubules (proximal tubule, distal tubule, and Henle's loop). (b) Scanning electron microscopy (SEM) image of a normal glomerulus in a sham-operated mouse, showing an afferent arteriole giving rise to the glomerular capillary network, with an efferent arteriole emerging from the glomerulus. (c) Proposed model of a twin nephron, in which two glomeruli become interconnected via an afferent arteriole following IRI. (d) SEM image of twin glomeruli at 9 days post-IRI, where an afferent arteriole gives rise to a glomerular capillary network, and an efferent arteriole from the first glomerulus supplies a second glomerulus. (e) Percentage of mice with twin glomeruli in each experimental group (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). (f) Incidence of twin glomeruli on days 3, 9, and 21 post-IRI in the experimental groups. (g) Twin glomeruli in the day 3 group, where an efferent arteriole emerging from the vascular pole of one glomerulus transitions into an afferent arteriole supplying a second glomerulus. The two glomeruli are connected by this afferent arteriole (white arrowhead). (h) Twin glomeruli in the day 9 group, where an efferent arteriole from a large glomerulus (*) transitions into an afferent arteriole that supplies a smaller glomerulus (**), with an approximate separation distance of 250 µm. The white arrowhead marks the afferent arteriole. (i) Twin glomeruli in the day 21 group, where one glomerulus (*) exhibits normal morphology while the other (**) appears immature. The two glomeruli are connected by a single afferent arteriole, indicated by the white arrowhead.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6399428/v1/f1ad061b08a470e97b8ad779.png"},{"id":81635896,"identity":"4f790ada-8e67-4043-b898-f1340546ba5c","added_by":"auto","created_at":"2025-04-29 12:24:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":564742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphometric characteristics of renal glomeruli in the kidney following IRI. \u003c/strong\u003e(a) Synchrotron radiation micro-CT image of a kidney from a sham-operated mouse. (b) Synchrotron radiation vascular image of a kidney from a sham-operated mouse, displaying a digitized representation of larger vasculature with all capillaries extracted. Small red dots indicate glomeruli. (c) Time-dependent increase in glomerular numbers in IR-injured adult mouse kidneys. The number of glomeruli increased significantly in the day 1 group after IRI, then plateaued from days 9 to 21 (**P \u0026lt; 0.01, ****P \u0026lt; 0.0001). (d) Changes in individual glomerular volumes over time following IRI, as observed in synchrotron radiation images (*P \u0026lt; 0.05). (e) Variance in individual glomerular volumes among sham and experimental groups, based on synchrotron radiation images. (f) Lightsheet fluorescence image of a transparent kidney from a sham-operated mouse, showing a digitized image of Tomato lectin-labeled glomeruli (FSD Fluor™ 647) extracted from Imaris-rendered images. Small red dots indicate glomeruli. (g) The average number of glomeruli in a mouse kidney was 14,003. “NT” indicates not tested. (h) Lightsheet fluorescence image of a transparent kidney from a sham-operated mouse, showing a digitized image of anti-synaptopodin-labeled glomeruli (Alexa Fluor™ 594) extracted from Imaris-rendered images. Small red dots indicate glomeruli. (i) The average number of glomeruli in the control group was 10,999, compared to 11,778 in the day 9 group. “NT” indicates not tested.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6399428/v1/b3c7e78ed840c66596bc1518.png"},{"id":81635902,"identity":"36ab5c36-8660-403c-aacb-8cc4bc78020e","added_by":"auto","created_at":"2025-04-29 12:24:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":851017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNumerical changes in nephrons, including regenerating and degenerating nephrons, following IRI. \u003c/strong\u003e(a–c) Increased densities of glomeruli and proximal convoluted tubules over time following IRI. (a) Proximal convoluted tubules were immunostained with anti-aquaporin 1 (a marker of proximal convoluted tubules), and glomeruli were counterstained with hematoxylin in sham and IR-injured mouse kidneys. Proximal convoluted tubular cells were intensely labeled with the aquaporin 1 antibody. (b, c) Both glomerular and proximal convoluted tubular densities gradually increased in the experimental groups (days 1, 3, 9, and 21) compared to sham-operated mice. (d, e) Incidence of VEGF-A immunoreactive glomeruli following IRI in adult mouse kidneys. (d) Regenerating glomeruli with VEGF-A-positive capillary endothelial cells were observed in the experimental groups. Scale bar = 50 µm. (e) The incidence of VEGF-A-positive glomeruli was significantly higher, nearly fivefold on day 1 and 5.31-fold on day 9 post-IRI, compared to the sham group (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). (f) Detection of atubular (degenerating) glomeruli in IR-injured mouse kidneys. Degenerating nephrons with synaptopodin-positive parietal podocytes were identified. Atubular glomeruli were observed in both IR-injured and sham-operated mouse kidneys. Synaptopodin, a marker for visceral podocytes and transdifferentiated parietal podocytes, was detected using anti-synaptopodin labeled with Alexa Fluor 488. Cortical regions from 10 tissue sections collected from 4 renal tissue blocks for each control or experimental mouse were examined. Scale bar = 50 µm. (g) Increased incidence of atubular glomeruli following IRI. The incidence of atubular glomeruli increased significantly, nearly fivefold on day 1 and 5.31-fold on day 9 post-IRI, compared to the sham group. Results are expressed as mean ± SEM (n = 24–34) (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6399428/v1/a1805b4d6bb73a1d45b0f40b.png"},{"id":81635211,"identity":"85158d41-9bb7-44af-bc89-56cf2f3b29a0","added_by":"auto","created_at":"2025-04-29 12:16:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":139432,"visible":true,"origin":"","legend":"\u003cp\u003eProposed model of the repair processes following IRI in adult mouse kidneys.\u003c/p\u003e\n\u003cp\u003eFollowing partial glomerular damage due to IRI, numerous studies have shown that renal progenitor cells within the glomerulus can facilitate fine glomerular regeneration, aiding in the repair of damaged glomeruli. However, some glomeruli may be severelyaffected, leading to their degeneration and eventual loss. During the glomerular repair process, renal progenitor cells may contribute to the formation of new glomeruli or additional lobules, which can gradually enlarge, creating a structure resembling two beads connected by a single strand, referred to as “twin glomeruli.” As the interglomerular or interlobular spaces between these glomeruligradually expand, each glomerulus or lobe may develop into an independent glomerulus. The capillary connecting the glomeruli or lobules (interlobular capillary) gradually acquires the characteristics of an arteriole (i.e., an “aefferent” arteriole) and elongates. Over time, the aefferent arteriole linking the two glomeruli may split, forming the efferent arteriole of one glomerulus and the afferent arteriole of the other. Simultaneously, one branch connects to the peritubular capillary network, while the other connects to the stem artery or interlobular artery, ultimately contributing to the formation of a neighboring new glomerulus. Because the twin glomeruli structure is transient and rare, it is unsurprising that it has not been frequently observed using conventional methods.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6399428/v1/465e4ba3ba8a40741df55501.png"},{"id":84703646,"identity":"8717fcee-23ac-4c07-8ac5-3f97499cda7f","added_by":"auto","created_at":"2025-06-16 11:58:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7299105,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6399428/v1/db357faf-8447-423d-855e-0de0887a2d8d.pdf"},{"id":81635200,"identity":"f3fedbbc-4ecc-46e3-8159-9d95f3c93ba4","added_by":"auto","created_at":"2025-04-29 12:16:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":102215,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6399428/v1/81aec5ddcbd50b41f994f939.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Twin glomeruli: a newly discovered marker of neonephrogenesis in the ischemia-reperfusion injured adult mouse kidney","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. A unique twin glomeruli structure was observed in the kidneys of IRI mice\u003c/p\u003e\u003cp\u003e2. Twin glomeruli, arranged in pairs, were connected by a single afferent arteriole\u003c/p\u003e\u003cp\u003e3. The number of nephrons significantly increased in adult mouse kidneys after IRI\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe nephron, the functional unit of the kidney, consists of a glomerulus and a tubular component. The glomerulus is an anastomosing network of fenestrated capillaries located between two arterioles and enclosed by Bowman\u0026rsquo;s capsule, which marks the beginning of the renal tubule. This intricately structured nephron comprises numerous specialized cells with distinct shapes, locations, and arrangements, all of which are essential for normal kidney function. However, these characteristics also render the nephron highly susceptible to various forms of damage.\u003c/p\u003e \u003cp\u003eIt is commonly believed that the number of nephrons is established around birth and declines throughout life due to damage, disease, or natural aging\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Despite the inability to generate new nephrons and their vulnerability to injury, nephrons exhibit a remarkable capacity to survive damage and partially restore function. This regenerative ability is largely attributed to the robust regenerative capacity of tubular cells and, to a lesser extent, the limited regeneration capacity of glomeruli\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Damage to glomerular capillaries is a critical factor in kidney disease progression\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Successful capillary repair can restore the glomerular structure in damaged nephrons, whereas failure of this process often leads to glomerular sclerosis and subsequent renal dysfunction\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. For many years, it was believed that glomeruli could not regenerate after injury. However, this notion was challenged by Fioretto\u0026rsquo;s groundbreaking report of glomerular regeneration in diabetic patients\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This discovery paved the way for further research, demonstrating the potential for glomerular regeneration in adult mammalian kidneys. More recently, regression of acute kidney disease (AKD) and even glomerulosclerosis under specific conditions has been attributed to glomerular regeneration\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The identification of renal stem cells or renal progenitor cells (RPCs) has been another significant milestone in understanding the mechanisms underlying glomerular repair\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRenal ischemia-reperfusion injury (R-IRI), a severe form of acute kidney injury, is characterized by proximal tubular necrosis, endothelial damage, and an inflammatory response, leading to sudden loss of renal function and progressive fibrotic changes. While the damaging and reparative effects of R-IRI on tubular and glomerular cells have been extensively studied\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, the overall architectural changes in glomeruli induced by R-IRI remain largely unexplored. Since the precise restructuring of glomerular cells is essential for restoring full functionality, understanding how the entire glomerular architecture responds to vascular injury could provide valuable insights into the mechanisms of glomerular repair.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the morphological response of glomerular architecture to severe vascular damage over time using an \u003cem\u003ein vivo\u003c/em\u003e mouse model involving bilateral clamping of the renal arteries. Scanning electron microscopy (SEM) images of intravascular corrosive resin casts revealed that IRI induced dramatic morphological changes in the glomeruli, including the appearance of a unique \u0026ldquo;twin glomeruli\u0026rdquo; structure. This structure, in which two glomeruli are interconnected by a single, highly unusual arteriole (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d), is distinctly different from normal glomeruli (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). The twin glomeruli were observed exclusively at 3, 9, and 21 days post-injury and displayed a configuration clearly distinct from both degenerating and regenerating glomeruli. Synchrotron radiation micro-CT image analysis revealed a significant increase in the average number of glomeruli in the IRI group compared to the control group. This suggests that twin glomeruli represent a temporary, acquired structure associated with the increase in glomerular numbers following IRI in adult mouse kidneys. Remarkably, neonephrogenesis appears to be reactivated by IRI in a manner distinct from embryonic nephrogenesis, challenging the long-held belief that nephrogenesis ceases entirely after birth in mammals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGross and histological characteristics of IRI kidneys in adult mouse\u003c/h2\u003e \u003cp\u003eWe examined morphological changes in the glomerular architecture of kidneys from 7-week-old mice following IRI. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, kidneys were harvested at 1, 3, 9, and 21 days post-injury, referred to as the day 1, 3, 9, and 21 groups, respectively, after which the size, color, and surface texture of the kidneys were observed for each group. Macroscopic observations revealed that kidneys from the day 1, 3, and 9 groups appeared slightly larger than those from the control group. Unlike the control kidneys, which displayed uniform size and color, the IRI-affected kidneys exhibited variability within each group. Subcapsular hemorrhage and blood engorgement were evident in the day 1, 3, and 9 groups, indicating severe vascular damage in the renal parenchyma. In contrast, kidneys from the day 21 group appeared slightly smaller and paler than those of the control group, possibly due to reduced residual blood at the time of organ harvesting. This reduction may be attributed to the formation of arteriovenous shunts or collateral circulation, which could have increased blood flow velocity and decreased blood retention within the tissue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, the mean kidney weight in all IRI groups was significantly higher than that of the control group, likely due to fluid accumulation and hemorrhage following injury. Glomerular damage was further assessed through urine analysis, with urine protein concentration, an indicator of acute kidney damage and glomerular injury, significantly elevated in all IRI groups compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Interestingly, the day 9 group exhibited a significantly lower urine protein concentration (69.97 mg/dL) compared to the day 1, 3, and 21 groups, although it remained slightly above control levels (Supplementary Table\u0026nbsp;1). Similarly, blood urea nitrogen (BUN) and plasma creatinine levels increased sharply one day after IRI, followed by a gradual, time-dependent decline (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and e, Supplementary Tables\u0026nbsp;2 and 3). These findings suggest that while glomeruli sustained severe damage from IRI, the kidneys exhibited partial recovery over time.\u003c/p\u003e \u003cp\u003eTo further characterize histopathological changes, kidney sections were stained with periodic acid-Schiff (PAS) reagent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-r). Despite the severe injury indicated by the aforementioned biochemical markers, many glomeruli in IRI kidneys retained a relatively normal configuration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-e). However, distinct pathological changes were observed in some severely affected glomeruli (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-r). The injured glomerular plexus exhibited structural disorganization, appearing either loosened or densely packed. Some glomeruli displayed capillary lumen dilation, while others showed obliteration due to capillary wall collapse (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-r). Signs of inflammation were also evident, including endothelial cell swelling and inflammatory cell infiltration within the glomeruli (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, k, n and q). Additionally, severe damage was observed in podocytes and the glomerular basement membrane (GBM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, l, o and r). In some cases, Bowman\u0026rsquo;s capsule exhibited significant structural changes, including tubularization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, l, o and r). Progressive collagen deposition was evident over time, as shown in Sirius red-stained renal tissue from IRI kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003es-w, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ex), indicating ongoing structural remodeling in response to injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTwin glomeruli observed in IRI adult mouse kidney\u003c/h3\u003e\n\u003cp\u003eTo visualize the 3D structural changes in glomerular architecture following IRI, we performed resin casting using Pu4ii resin via cardiac perfusion and examined the resin-cast kidneys with SEM. As expected, the glomeruli of the control group maintained their typical shape and size (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). In the day 1, 3, and 9 groups, the majority of glomeruli appeared normal in shape and size, similar to those in the control group. However, a smaller proportion exhibited significant morphological changes. Some glomeruli were noticeably enlarged and consisted of two to three lobes with a slightly widened urinary pole (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c). Several glomeruli in the day 1, 3, and 9 groups were markedly enlarged, with some displaying two to three distinct lobes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u0026ndash;h). Among these, some showed prominently bulging lobules that were slightly separated from each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f and h). In contrast, tiny glomeruli (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eg) and fragmented glomerular remnants were observed less frequently (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ei, j). A hollow, broken, basket-shaped glomerulus appeared to be caught on a branching arteriole from the interlobular artery (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). Additionally, multiple arterioles were seen entering and exiting through a damaged glomerulus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). By day 21, some glomeruli remained enlarged and densely packed, but lobulated glomeruli were less common than in the earlier time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ek\u0026ndash;m). This suggests that while glomerular volume expansion persisted, lobular expansion gradually declined by day 21. Interestingly, the damaged glomeruli appeared more crushed in the SEM images than in the PAS-stained specimens.\u003c/p\u003e \u003cp\u003eA particularly striking finding in the SEM analysis was the identification of a unique glomerular structure in the day 3, 9, and 21 groups. This structure was completely distinct from a normal glomerulus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Two glomeruli were connected by a single, unusual arteriole, hereinafter referred to as an \u0026ldquo;aefferent\u0026rdquo; arteriole, forming what we describe as \u0026ldquo;twin glomeruli\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d, g\u0026ndash;i). In these structures, the afferent arteriole of one glomerulus appeared to simultaneously function as the efferent arteriole for the other, and vice versa. The length of the aefferent arteriole varied from approximately 20 \u0026micro;m to 300 \u0026micro;m, significantly longer than the distance between lobules in lobulated glomeruli (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed\u0026ndash;i). Additionally, the shape and size of each glomerulus within the twin glomeruli varied; in some cases, a new glomerulus appeared to be forming and projecting outward from an existing one (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh, uppermost image).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTwin glomeruli were first observed in the day 3 group (4 twin glomeruli across 6 kidneys from 3 mice) and were most frequently detected in the day 9 group (6 twin glomeruli across 6 kidneys from 3 mice). By day 21, only a single kidney contained one twin glomerulus (1 twin glomerulus across 6 kidneys from 3 mice) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, Supplementary Table\u0026nbsp;4). No twin glomeruli were observed in the control or day 1 groups. The incidence of twin glomeruli was very low, ranging from 0.016\u0026ndash;0.389% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, Supplementary Table\u0026nbsp;5). These findings suggest that twin glomeruli are a rare, temporary, and acquired structural anomaly induced by IRI.\u003c/p\u003e\n\u003ch3\u003eIncreased glomerular number in IRI adult mouse kidney\u003c/h3\u003e\n\u003cp\u003eAlthough only a small number of twin glomeruli were observed, their presence suggests the possibility of previously unknown repair processes following vascular injury in the adult kidney, potentially indicative of neoglomerulogenesis or neonephrogenesis. This finding contrasts with the existing consensus\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, which holds that nephrogenic sources are not widely recognized in the adult mouse kidney. It also raises the question of whether the glomerular number could be compensated by innate mechanisms in the IRI kidney. A significant increase in glomerular count could provide insights into the potential role of twin glomeruli in this process. To quantify glomerular number, we performed synchrotron radiation micro-CT imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and b). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, the average glomerular count in the left kidney of 7-week-old control mice was 8,017. This number increased to an average of 11,589 in the day 1 group and 11,545 in the day 3 group. The glomerular count peaked at approximately 13,969 in the day 9 group and plateaued at around 13,898 in the day 21 group (Supplementary Table\u0026nbsp;6). Overall, these results indicate an increase in glomerular number following injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mean glomerular volume, a hallmark of severe nephron injury, showed a slight increase over time after IRI (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), suggesting significant renal tissue damage. However, the simultaneous increase in both glomerular number and volume contradicts the well-established inverse relationship between these parameters following nephron loss\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This is because glomerular number and volume are considered compensatory (i.e., when nephron loss occurs, surviving glomeruli enlarge to compensate). The disruption of this relationship in IRI mice suggests that glomerular volume may no longer serve as a reliable surrogate marker for nephron deficiency. Furthermore, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, the variance in glomerular volume increased following IRI, with the greatest variation observed in the day 9 group. These findings suggest that active repair processes occurred at both the histological and cellular levels (Supplementary Fig.\u0026nbsp;1 and Supplementary Tables\u0026nbsp;7 and 8). IRI appears to induce dynamic reshaping of glomeruli in both shape and number. However, the observed increase in glomerular number cannot be solely explained by glomerular regeneration in injured kidneys. Other repair mechanisms, as well as potential biological or technical factors in the experiment, should also be considered.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDiversity of glomerular number according to the counting methods.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe glomerular count in the left kidney of a normal 7-week-old mouse in our study differed from the findings of Cullen-McEwen et al.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, who reported 13,440\u0026thinsp;\u0026plusmn;\u0026thinsp;1,275 glomeruli in the kidneys of male GDNF wild-type mice (14.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 months old) using the physical dissector/fractionator combination method\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Meanwhile, Takiyama et al., using synchrotron radiation micro-CT, reported an average of 9,546.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1,912.79 glomeruli in C57BLKs mice, a value approximately midway between the results reported by Cullen-McEwen et al. and our study. Even when examining the same specimen, glomerular counts can vary significantly depending on the counting method and researcher. However, given that experimental animals are strictly controlled in terms of reproduction, housing, and diet, such large differences in glomerular counts are unlikely to result from biological variation alone. Instead, these discrepancies are more likely attributable to methodological differences, researcher variability, or other uncontrollable factors. To better understand these discrepancies in glomerular counts in control mice and, if possible, determine a more precise estimate of glomerular number in normal mice, we tested two additional technical approaches. First, we used Tomato lectin (from \u003cem\u003eLycopersicon esculentum\u003c/em\u003e) to label vascular endothelial cells, marking capillary tufts via fluorescence, followed by a histo-transparency procedure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Glomeruli labeled with lectin were counted using the Imaris rendering program, yielding an average of 14,003 glomeruli in wild-type mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, Supplementary Table\u0026nbsp;9). This result closely matched those obtained using the physical dissector/fractionator combination method, suggesting that the glomerular count in normal adult mice is likely closer to 14,000 rather than the 8,724 previously reported (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Based on these findings, we concluded that Tomato lectin labeling is the most reliable and objective method for glomerular counting in normal animals. However, we were unable to obtain reliable results for IRI groups using this technique due to unresolved technical and biological challenges. As a second approach, we performed glomerular counting using immunohistochemistry with anti-synaptopodin, followed by the histo-transparency method (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). Glomeruli labeled with synaptopodin were counted using the Imaris rendering program, yielding an average of 10,999 glomeruli in the control group and 11,778 in the day 9 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei, Supplementary Table\u0026nbsp;10). Although the glomerular count appeared to increase on day 9 compared to the control group, the difference was not statistically significant. However, given the practical limitations of the synaptopodin immunofluorescence procedure, such as reduced molecular penetration, our glomerular counts likely underestimate the actual numbers, particularly in the IRI group compared to the control group. Despite discrepancies between experimental methods, our findings consistently demonstrate that, regardless of the absolute glomerular count in wild-type mice, glomerular numbers tend to increase following IRI, as shown by both approaches.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIncreases in glomerular density with higher tubular density after IRI.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAlthough our findings suggest that twin glomeruli may represent structures formed during the process of neoglomerulogenesis (ultimately leading to neonephrogenesis) induced by IRI, we cannot rule out the possibility that simple capillary plexuses, microhematomas, or other structures, particularly prevalent in IRI kidneys, were misidentified as glomeruli. To further support our findings regarding nephron number elevation after IRI, we conducted histochemical experiments on sectioned kidney tissues.\u003c/p\u003e \u003cp\u003eA comparative analysis of changes in glomerular and proximal convoluted tubule density was performed using tissue sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b; Supplementary Tables\u0026nbsp;11a, b) to examine the relative trends in glomerular and tubular density. While measuring the number of glomeruli or tubules per unit area in tissue sections provides only relative estimates, as it does not account for kidney size or potential tissue changes due to IRI or processing, this approach remains widely used in morphometric studies due to its simplicity and reliability. Immunohistochemical staining was performed to visualize aquaporin-1 (AQP1), a marker for proximal convoluted tubules (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The density of AQP1-immunoreactive proximal convoluted tubules was compared with that of glomeruli in each group. The results demonstrated a time-dependent and gradual increase in both densities after IRI, aligning with findings from synchrotron radiation micro-CT imaging. Notably, significant increases were observed in the day 9 and day 21 groups compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c). In contrast, the day 1 and day 3 groups exhibited no significant density changes but did not show a decrease relative to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c). These findings indicate that IRI induces genuine increases in glomerular number rather than misinterpretation of other structures, with nephron number elevation persisting for at least three weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the glomerular repair process, vascular endothelial growth factor A (VEGF-A), a marker of angiogenesis\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, was analyzed via immunostaining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). VEGF-A-immunoreactive glomeruli were detected in 8.5% (42/482) of the control group, 27.52% (120/436) of the day 1 group, 33.68% (128/380) of the day 3 group, 4.97% (22/442) of the day 9 group, and 10.95% (46/420) of the day 21 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee; Supplementary Table\u0026nbsp;12). These results suggest that glomeruli are dynamic structures undergoing continuous repair, with IRI enhancing vascular repair processes. Notably, all VEGF-A-immunoreactive glomeruli were enclosed within Bowman\u0026rsquo;s capsule, indicating that glomerular repair after IRI does not occur independently but is consistently accompanied by tubular repair.\u003c/p\u003e\n\u003ch3\u003eIncrease in necrotizing glomeruli following IRI\u003c/h3\u003e\n\u003cp\u003eIRI is highly destructive to the kidneys, leading to the formation of numerous nonfunctional, degenerated glomeruli. Given the severely damaged glomeruli observed in SEM images of IRI kidneys, our findings suggest that IR injury induces glomerular degeneration, resulting in an increased number of degenerating glomeruli, referred to as atubular glomerular necrosis. Therefore, it is unlikely that all the counted glomeruli in this study correspond to functional nephrons. The presence and numerical changes of these nonfunctional, destroyed glomeruli may provide insights into the origins of twin glomeruli structure\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. To assess changes in the number of degenerating glomeruli after IRI\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, we performed an immunofluorescence study using synaptopodin, a marker for visceral podocytes and transdifferentiated podocytes (parietal podocytes)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Synaptopodin-immunoreactive transdifferentiated parietal podocytes were detected in glomeruli across all groups, including the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). These degenerating glomeruli accounted for 1% of the total glomeruli in the control group (3/300), 5% in the day 1 group (15/300), 1.47% in the day 3 group (6/408), 5.31% in the day 9 group (18/339), and 3% in the day 21 group (13/427) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg; Supplementary Table\u0026nbsp;13). Compared to the control group, the percentage of degenerating glomeruli in the IRI groups was 1.47 to 5.31 times higher. Nephron degeneration occurs at a consistent baseline level in normal kidneys\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, IRI significantly accelerates this process. These findings indicate that degenerating glomeruli must be accounted for in glomerular quantification studies. While not all glomeruli counted in this study represent functional nephrons, the number of atubular glomeruli in the IRI group was insufficient to fully explain the total increase in nephron numbers observed following IRI. This discrepancy suggests that the simultaneous increase in nephron numbers and the presence of twin glomeruli in R-IRI can only be attributed to nephrogenesis. Although the underlying mechanisms remain unclear, these findings underscore the complexity of the repair processes involved.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, twin glomeruli were observed as two glomeruli arranged in series, with the efferent arteriole of one glomerulus serving as the afferent arteriole of another. This configuration is markedly different from that of typical glomeruli, which are independently supplied and drained by separate arterioles. In a normal glomerulus, the afferent arteriole delivers blood, while the efferent arteriole carries it away after filtration. According to renal hydraulic pressure profiles\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, a significant pressure drop occurs between the afferent and efferent arterioles, with glomerular pressure maintained at levels comparable to those in the distal afferent and proximal efferent arterioles. This structure ensures that each glomerulus functions independently to maintain proper filtration. However, in the case of sequentially connected glomeruli, the downstream glomerulus would likely experience impaired filtration. Thus, the twin glomeruli configuration does not align with established renal physiology. The term \"twin glomeruli\" was introduced in 1897 and has been structurally examined by several researchers. However, their exact features and roles remain undefined. Traditional histological methods have been less suitable for studying such morphological deviations, as even minor discrepancies in the plane of sectioning can render these structures invisible. Consequently, twin glomeruli have remained largely overlooked for decades, with only sporadic reports linking them to congenital anomalies or disease conditions in both animals and humans.\u003c/p\u003e \u003cp\u003eOur findings confirm that the defining characteristic of twin glomeruli is their serial arrangement rather than a symmetrical pair supplied by a bifurcated afferent arteriole. Notably, in this study, twin glomeruli were exclusively observed in IRI-induced kidneys and only for a limited duration, suggesting that they are transient, acquired structures rather than permanent renal features. Furthermore, they exhibit morphological differences from previously described degenerating or regenerating glomeruli. These peculiar and transient formations may represent an ongoing, novel repair mechanism following vascular injury, distinct from previously understood regenerative processes. Using synchrotron radiation micro-CT, we detected a significant increase in glomerular counts in IRI kidneys, with the average number rising from 8,017 in control mice to a peak of 13,969 on day 9 post-injury. Glomerular count is known to directly reflect nephron number due to the nephron\u0026rsquo;s unique structural organization\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Therefore, our findings strongly suggest a corresponding increase in nephron number. This contradicts the widely accepted notion that nephron numbers can only decrease over time and do not increase after birth. While damaged nephrons have been thought to regenerate only through nephron repair following injury\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e rather than through neonephrogenesis, our findings challenge this assumption.\u003c/p\u003e \u003cp\u003eAlthough data on increased glomerular numbers following renal injury are limited, some studies have reported similar trends. For instance, in response to cisplatin treatment, the total glomerular count in mice did not decrease but instead showed a slight increase, despite a concurrent rise in necrotizing glomeruli\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Similarly, in diabetic mice treated with SGLT2 inhibitors, glomerular numbers remained stable or exhibited a slight, albeit statistically insignificant, increase\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. These findings suggest that, under certain pathological conditions, glomerular numbers may remain stable or even increase, contrary to the long-standing consensus. In the present study, the increase in glomerular number far exceeded what could be explained by simple glomerular regeneration alone, suggesting the presence of a novel repair mechanism following vascular injury. This substantial rise supports the notion that twin glomeruli may represent transient structures formed during neoglomerulogenesis induced by IRI. Rather than merely indicating glomerulogenesis, our findings suggest that this process reflects neonephrogenesis following IRI.\u003c/p\u003e \u003cp\u003eThe simultaneous increase in both tubular components and glomeruli supports the notion of nephron number augmentation and provides a basis for distinguishing genuine glomerular increases from potential misinterpretations of other structures in IRI. Notably, the experimental group exhibited a significant and concurrent increase in both glomerular and tubular densities on days 9 and 21 post-IRI. This correlation suggests that the observed rise in glomerular number is attributable to neonephrogenesis rather than the misidentification of vascular plexuses, as a true increase in glomerular number would naturally correspond to an increase in tubular structures. While the increase in tubular density may initially suggest the formation of new tubules, it is important to consider an alternative explanation: tubular elongation, particularly an increase in the length of proximal convoluted tubules due to regenerative processes. However, previous studies offer conflicting insights into this phenomenon. M\u0026oslash;ller et al. reported no significant changes in proximal tubule length or density in cases of chronic ureteral obstruction or severe cortical interstitial fibrosis in pigs\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Similarly, long-term lithium administration in rats resulted in a slight decrease in proximal tubular length, accompanied by severe cortical interstitial fibrosis\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Conversely, another study observed an increase in the average length of proximal convoluted tubules in chronically damaged rat kidneys, although this finding was accompanied by significant variability in total tubular length, with marked shortening in severe cases\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Interestingly, a study on diabetic mice treated with SGLT2 inhibitors suggested the possibility of glomerular redistribution from the juxtamedullary region to the superficial cortex, potentially involving restructuring of the convoluted and straight segments of proximal tubules. However, it remains unclear whether this redistribution was due to tubular elongation or reshaping\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. At present, insufficient data are available to determine whether changes in nephron tubular length occur in IRI mice. Further studies are needed to clarify this aspect of nephron remodeling.\u003c/p\u003e \u003cp\u003eMurine nephrogenesis occurs in two stages based on developmental timing: embryonic nephrogenesis and postnatal nephrogenesis. During postnatal nephrogenesis, multiple nephrons (up to five) can form at a single ureteric tip\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, unlike embryonic nephrogenesis, where each ureteric tip gives rise to a single nephron\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Considering these two nephrogenic processes, it is conceivable that unexpected nephron morphologies could arise during repair processes in adult kidneys if neonephrogenesis were to resume after injury. Although embryonic stem cells and their associated developmental genetic pathways are absent in the adult kidney\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, renal progenitor cells have been identified. These cells are key contributors to renal regeneration, though their exact mechanisms remain poorly understood. Additionally, studies have shown that stem cell-derived kidney organoids implanted into mice develop more functional nephrons than \u003cem\u003ein vitro\u003c/em\u003e cultures, even in the absence of external additives. This evidence suggests that renal progenitor cells may play a more significant role in adult kidney repair than currently recognized, warranting further investigation.\u003c/p\u003e \u003cp\u003eAmong renal progenitor cells, juxtaglomerular cells of renin lineage (CoRL) have been shown to play a crucial role in nephrogenesis, influencing the structuring and branching of renal arterioles. CoRL exhibit remarkable plasticity, the ability to replicate, and the capacity to migrate into the glomerulus, where they can replace multiple glomerular cell types in diseased rodent kidneys\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Thus, CoRL can be regarded as upstream mesenchymal progenitors\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In this context, it is conceivable that existing arterioles or aberrant vessels formed within the injured renal corpuscle may serve as conduits for renal progenitor cells, facilitating their migration from their original location along these vessels and potentially leading to the development of a new glomerulus. This newly formed glomerulus, initially connected to the pre-existing one by an arteriole, could eventually separate, with the connecting arteriole evolving into an afferent arteriole.\u003c/p\u003e \u003cp\u003eAlternatively, IRI could induce the lobular expansion of a glomerulus through the translocation and active repair of renal progenitor cells along pre-existing arterioles. As these progenitor cells integrate into an injured glomerulus, lobules may drift apart and separate, with an interconnecting vessel gradually elongating and transforming into an afferent arteriole. In either scenario, the two glomeruli, positioned at opposite ends of the afferent arteriole, may temporarily resemble twin glomeruli. Over time, the afferent arteriole may elongate further and split into two branches: one functioning as the efferent arteriole for the first glomerulus and the other as the afferent arteriole for the second glomerulus (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). While glomerular repair processes, particularly the regeneration of glomerular capillary tufts, have been widely studied, reports on the formation of new aberrant arterioles connecting glomeruli to interstitial tissue remain limited. However, some studies have demonstrated that aberrant vessels can form between glomerular spaces and interstitial tissues during glomerulosclerosis\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These studies suggest that neovascularization may occur through breaches in Bowman\u0026rsquo;s capsule, extending into surrounding interstitial tissues in diseased kidneys. Similarly, our findings revealed various patterns of extraglomerular aberrant vessels connecting glomeruli with their extraglomerular spaces. This suggests that vascular regeneration following renal injury is not confined to the intraglomerular space but extends into the extraglomerular and interstitial regions. Such expansion implies that renal progenitor cells may have broader roles beyond the intraglomerular environment, potentially contributing to diverse forms of glomerulogenesis within adult kidney tissue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe appearance of twin glomeruli observed in this study suggests a previously unrecognized glomerular repair process. This finding implies the existence of novel repair mechanisms and potential changes in glomerular number following R-IRI, which may have been overlooked in prior research. The possibility that neonephrogenesis can be reactivated in adult mice under IRI conditions challenges the long-standing belief that mammalian nephrogenesis ceases entirely after birth. Given that this conclusion contradicts many previous studies, further research is crucial to validate and elucidate the underlying mechanisms of this phenomenon.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult male C57BL/6J mice (Koatech, Gyeonggi-do, ROK), aged 7 weeks and weighing 20\u0026ndash;25 g, were used in this study. The mice were maintained on a standard laboratory chow diet with \u003cem\u003ead libitum\u003c/em\u003e access to tap water. To account for inter-animal variability in glomerular number, a minimum of three mice were assigned to each experimental group, and their data were pooled for analysis. To induce ischemia, the kidneys were exposed via an incision under anesthesia with pentobarbital sodium (60 mg/kg body weight; Hanlim Pharm., Korea). A microaneurysm clamp was used to occlude the renal pedicle for 30 minutes. In the control group, a sham operation (Sham) was performed using the same procedure, except for renal pedicle clamping. Body temperature was maintained at 36.5\u0026ndash;37 \u0026deg;C during all surgical procedures using a temperature-controlled heating device (FHC, Bowdoinham, ME). After IRI, renal function was assessed by measuring urine protein, BUN, and creatinine concentrations using a Vitros 250 Chemistry Analyzer (Johnson \u0026amp; Johnson, Rochester, NY). Hematuria was evaluated microscopically. All animal studies were approved by the Animal Care and Use Committee of Kyungpook National University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 2011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUrine protein concentrations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUrine was collected during surgery by emptying the urinary bladder using a 31-gauge syringe. Additional urine samples were obtained using a metabolic cage and a 31-gauge syringe. The collected urine was stored at \u0026minus;70 \u0026deg;C with protease inhibitors (1.2 mM sodium azide, 0.5 mM PMSF, and 1 \u0026micro;M leupeptin) until analysis. Protein concentrations were measured at the specified time points using a Vitros 250 Chemistry Analyzer (Johnson \u0026amp; Johnson, Rochester, NY, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasma creatinine and BUN concentrations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were collected from the retrobulbar venous plexus at the time points indicated in the figures. Plasma creatinine and BUN concentrations were measured using a Vitros 250 Chemistry Analyzer (Johnson \u0026amp; Johnson, Rochester, NY, USA). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree mice per group were perfusion-fixed at predetermined time points (days 1, 3, 9, and 21 post-IRI) for kidney removal. The excised kidneys were post-fixed in PLP solution (4% paraformaldehyde, 75 mM L-lysine, and 10 mM sodium periodate; Sigma-Aldrich) overnight at 4 \u0026deg;C. The tissues were then embedded in paraffin and sectioned into 4-\u0026mu;m slices using a microtome (Leica, Bensheim, Germany). Kidney sections were stained with periodic acid-Schiff (PAS), Sirius red and fast green, and subjected to immunostaining for synaptopodin, VEGF-A, AQP, and TUNEL assay. Whole-kidney images were captured using a slide scanner, and cortical regions were photographed in 10 fields per kidney using a Nikon Fx35 (Nikon, Japan). At least four kidneys per experimental condition were analyzed, with ten fields per slide counted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSirius red and fast green special stain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSirius red and fast green staining were performed to assess collagen fiber deposition in post-IRI kidney tissue at the specified time points. Kidney sections were initially stained with 0.04% fast green for 15 minutes, followed by an aqueous wash. The sections were then stained with 0.1% Sirius red and 0.04% fast green in saturated picric acid for 30 minutes, followed by two washes with acidified water (0.5% glacial acetic acid). The sections were rehydrated and dehydrated using graded alcohol. Collagen fibers were stained red, while non-collagen proteins appeared green. The Sirius-red-positive area was quantified using an image analysis program (i-Solution, IMT, Korea).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry was performed on paraffin-embedded tissues fixed with either 4% paraformaldehyde-PBS or Bouin\u0026rsquo;s solution, following the manufacturer\u0026rsquo;s instructions. To detect atubular glomeruli in the visceral epithelial cells of Bowman\u0026rsquo;s capsule, sections were stained with anti-synaptopodin conjugated with Alexa Fluor 488 (1:200, sc-515842, Santa Cruz Biotechnology, Santa Cruz, CA). Proximal convoluted tubules were labeled using anti-aquaporin-1 (1:200, 7D11, AQP1, Bio-Rad, Alomone Labs, Jerusalem, Israel) conjugated with Texas Red. Angiogenic glomeruli were detected using anti-VEGF-A (1:500, #ab1316, Abcam plc, Cambridge, UK) conjugated with Cy3. Immunohistochemistry was performed using the EnVision Kit (Agilent, CA, USA) according to the manufacturer\u0026rsquo;s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of glomerular and proximal convoluted tubular densities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe densities of glomeruli and proximal convoluted tubules were quantified by manually counting their numbers in 10 random fields per kidney under a microscope at 200\u0026times; magnification (Leica DM 2500, Wetzlar, Germany). Statistical comparisons between experimental groups were conducted using a Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVascular\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;corrosion casting (VCC) and SEM\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;analyses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSham-operated and ischemia-reperfusion (IR)-injured mice were anesthetized with pentobarbital (60 mg/kg body weight; Hanlim Pharm., Korea). The thoracic cavity was surgically opened, and the ascending aorta was cannulated via the left ventricle. To ensure complete removal of blood from the vascular lumen, the vasculature was thoroughly flushed with 100 ml of heparinized normal saline solution (20 IU/100 ml). Fixation was performed by infusing approximately 15 ml of 4% paraformaldehyde in phosphate-buffered saline (PBS), with all solutions maintained at 37 \u0026deg;C and infused at a rate of 5 ml/min. Immediately after fixation, a polyurethane resin (PU4ii, VasQtec, Switzerland) was mixed with ethyl methyl ketone (EMK, Merck) at a 6:1 ratio, with blue pigment added for contrast. This mixture was infused using a Harvard syringe pump at a constant rate of 5 ml/min until polymerization began. To prevent air bubble formation, the resin/solvent mixture was continuously injected. The polymerized casts maintained high reproduction quality without shrinkage. Full polymerization was achieved by placing the injected kidneys in a hot water bath (50\u0026ndash;60 \u0026deg;C) for 24 hours.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter polymerization, soft tissues were removed through maceration in 7.5% potassium hydroxide (KOH) at 50\u0026ndash;60 \u0026deg;C for 1\u0026ndash;2 days. The corrosion casts were washed with tap water to remove lipid-rich saponified material, then further cleaned in 5% formic acid for 10 minutes, followed by multiple rinses with distilled water. The casts were dried using a freeze-drying method. For SEM analysis, the vascular casts were rendered electron-conductive by gold coating for 90 seconds using a sputter coater. The casts were then examined using a scanning electron microscope at an accelerating voltage of 15 kV (Hitachi S-4300, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynchrotron radiation micro-CT\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKidney sample preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResin-injected kidneys were collected from mice in each experimental group before the corrosion procedure. To prevent dehydration, each whole kidney was immersed in a microtube (Axygen, CA, USA) filled with an optimal cutting temperature (OCT) compound (Scigen, CA, USA) during mounting on the synchrotron radiation micro-CT unit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental layout and image\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eacquisition\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSynchrotron radiation micro-CT imaging was performed at the Wiggler 6C biomedical imaging beamline of the Pohang Light Source-II (PLS-II) in Pohang, Korea, following the protocol described in a previous study\u003csup\u003e40\u003c/sup\u003e. Synchrotron X-rays were generated from the electron storage ring, operating at an electron energy of 3 GeV with a typical current of 320 mA. The X-rays passed through two beryllium windows, with the X-ray source positioned 28 m from the experimental hutch. The electron beam traversed a double-crystal monochromator composed of silicon (Si111) multilayers and a beryllium window. To optimize high-resolution phase-contrast detection, the kidney samples were positioned 200 mm upstream of the detector. Microtomography was performed by rotating the sample in 0.2\u0026deg; increments over a 180\u0026deg; range using a computer-controlled precision stage. Each projection had an exposure time of 100 ms. The X-ray shadow of the specimen was converted into a visible image on the surface of CdWO\u003csub\u003e4\u003c/sub\u003e and YAG:Ce scintillation crystals. This image was magnified using a 5\u0026times; microscopic objective lens and captured with a PCO 4000 charge-coupled device (CCD) camera at a resolution of 4,008 \u0026times; 2,672 pixels. Three-dimensional volume images were reconstructed using a filtered back-projection algorithm applied to the projection images with the OCTOPUS software package (CT, Belgium). Surface reconstruction, volume segmentation, and rendering were conducted using Amira software (Visualization Sciences Group, Burlington, MA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal glomerular number, glomerular tuft volume, and whole kidney volume were quantified from the three-dimensional reconstructed synchrotron radiation micro-CT images using Amira software.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eLectin injection\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003etissue\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eclearing\u0026nbsp;\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAfter anesthetizing the mice, \u003cem\u003eLycopersicon esculentum\u003c/em\u003e agglutinin (tomato lectin, FSD Fluor\u0026trade; 647, 50\u0026ndash;100 \u0026mu;g/100 \u0026mu;l; Vector Laboratories, Burlingame, CA, USA) was injected directly into the left ventricle to stain functional blood vessels. Two minutes later, the mice were perfused through the left ventricle-aorta with rinse and fixation solutions. Following a pre-rinse with the rinse solution, the mice were fixed with 4% paraformaldehyde (PFA) and post-fixed overnight in the same fixative before being stored in 1\u0026times; PBS (pH 7.4) overnight. The kidneys were removed, halved, and rendered transparent using a tissue clearing kit (Cat. HRTC-001, Binaree, Daegu, Korea). Briefly, PFA-fixed kidneys were immersed in Binaree fixing solution for 24 hours and incubated with tissue clearing solution in a shaking incubator at 37 \u0026deg;C for 4\u0026ndash;5 days, followed by rinsing in a washing solution. Finally, the kidneys were incubated in mounting and storage solution (Cat. SHMS-060, Binaree) for 24 hours to achieve further clearing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLightsheet fluorescence microscopy (LSFM) and 3D\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eimaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse kidney imaging was performed at 5\u0026times; magnification using a Lightsheet Z.1 fluorescence microscope (Zeiss Corporation, Jena, Germany). Three-dimensional image rendering was conducted using the Imaris software (version 9.5.1, Oxford Instruments, Abingdon-on-Thames, UK; https://imaris.oxinst.com). Quantitative evaluation of kidney glomeruli was performed in maximum intensity projection mode, with rendering quality set to 100%. The \u0026quot;Background Subtraction\u0026quot; option was applied to smooth the image by creating a Gaussian-filtered channel minus the intensity of the original channel (diameter = 2.02 \u0026mu;m). The surface module was designed from the Gaussian-filtered channel using a threshold algorithm to quantify glomerular number and volume.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLogistic-transformed values of appearance rates were used to assess group effects, and comparisons between each IR-injured group (day 1, day 3, day 9, and day 21) and the control group were performed. An unpaired two-tailed Student\u0026apos;s \u003cem\u003et\u003c/em\u003e-test was used for comparisons between two datasets. Error bars in graphical data represent means \u0026plusmn; standard deviation. All \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments were conducted at least three times. \u003cem\u003eP\u003c/em\u003e-values \u0026lt; 0.05 were considered statistically significant. Statistical significance was determined as follows: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (*), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 (**), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (***), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (****). \u0026ldquo;NS\u0026rdquo; denotes non-significance. Data analysis was performed using Prism 6 (GraphPad Software, Inc.).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Pohang Light Source-II (PLS-II) in Pohang, Korea, and the UNIST Optical Biomed Imaging Center in Ulsan, Korea, for their support. This work was funded by the National Research Foundation of Korea (NRF) through grants from the Korean government (MSIT) (2017R1A5A2015391, 2021M3A9H3016063, and 2022R1F1A1074842). Hanguk Hwang and Dongju Woo contributed equally to this work. Correspondence should be addressed to Mae Ja Park, MD, Ph.D., Department of Anatomy, School of Medicine, Kyungpook National University, Daegu, South Korea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMJ Park conceived the ideas, designed the research, and wrote the manuscript. H Hwang and D Woo conducted the animal experiments and analyzed the data. YR Park, MJ Kong and C Lee conducted the animal experiments. H Lee and SH Lee contributed to writing the manuscript. SY Lee and D Woo handled electronic tasks and submitted the manuscript. SW Nam analyzed and interpreted the data and wrote the paper. KM Park provided technical support and materials for the study. YC Bae, EJ. Nam, S Park, H Kim and JY Choi provided technical support to improve the study and contributed to data interpretation. MJ Park approved the final version for submission and is responsible for data acquisition and analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBertram JF, Hughson MD, Puelles VG, Hoy WE (2016) In: Little MH (ed) Kidney Development, Disease, Repair and Regeneration. Academic, pp 167\u0026ndash;175\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeyer-Schwesinger C (2016) The Role of Renal Progenitors in Renal Regeneration. Nephron 132:101\u0026ndash;109. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1159/000442180\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1159/000442180\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonventre JV, Weinberg JM (2003) Recent advances in the pathophysiology of ischemic acute renal failure. 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Microsc Res Tech 80:1141\u0026ndash;1148. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1002/jemt.22910\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1002/jemt.22910\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"twin glomeruli, renal ischemia/reperfusion injury, neonephrogenesis, glomerulogenesis, adult mouse kidney, synchrotron radiation micro-computed tomography","lastPublishedDoi":"10.21203/rs.3.rs-6399428/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6399428/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe renal glomerulus, a capillary plexus between two arterioles, is crucial for urine production in mammals. While partial glomerular regeneration after renal injury is well recognized, its precise mechanisms remain unclear. However, stereological studies on post-injury glomerular structural changes are limited. This study investigated three-dimensional alterations in the glomerulus over time following ischemia-reperfusion injury (IRI) in adult mouse kidneys. We identified a unique \"twin glomeruli\" structure between three arterioles and connected by an atypical \u0026ldquo;aefferent\u0026rdquo; arteriole. This structure appeared between 3 and 21 days post-IRI, peaking at day 9. The twin glomeruli exhibited distinct features, differing from both degenerating and developing glomeruli. Synchrotron radiation micro-computed tomography revealed a time-dependent nephron increase between 1 and 21 days post-IRI. Immunohistochemical analysis also revealed significant increases in glomerular and tubular densities from days 9 to 21. These findings suggest that twin glomeruli are a transient structure induced by IRI and may be associated with an increase in nephron numbers. Our study challenges prevailing views, revealing that twin glomeruli represent an unconventional glomerular structure occurring during kidney repair and suggesting the possibility of neonephrogenesis in the adult mouse kidney following IRI, a process previously considered impossible postnatally.\u003c/p\u003e","manuscriptTitle":"Twin glomeruli: a newly discovered marker of neonephrogenesis in the ischemia-reperfusion injured adult mouse kidney","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 12:16:35","doi":"10.21203/rs.3.rs-6399428/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f1410e15-30e8-4512-ad34-c7847a391847","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47796576,"name":"Health sciences/Anatomy/Kidney/Nephrons/Glomerulus"},{"id":47796577,"name":"Biological sciences/Structural biology/Electron microscopy"}],"tags":[],"updatedAt":"2025-06-16T11:50:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-29 12:16:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6399428","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6399428","identity":"rs-6399428","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}