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Renal Macrophages and Multinucleated Giant Cells: Ferrymen of the River Styx?

2022; Lippincott Williams & Wilkins; Volume: 3; Issue: 9 Linguagem: Inglês

10.34067/kid.0003992022

2641-7650

Mayandi Sivaguru, Bruce W. Fouke,

Pediatric Urology and Nephrology Studies

Introduction Like Charon regulating the passage of deceased souls across the River Styx in Greek mythology, it is hypothesized here that kidney tissue macrophages and multinucleated giant cells (MGCs), observed in both human and rodent models, may transport engulfed crystalline products of early-stage human renal biomineralization through the interstitium to the papilla. If experimentally confirmed in humans, this would suggest that complete dissolution of the crystals occurs within macrophages and MGCs before reaching the papilla in nonstone formers. Conversely, this would also suggest that within stone formers, crystal dissolution during macrophage and MGC transport is either incomplete or entirely prevented. Resulting crystal accumulation, aggregation, and breach of the papillary epithelium, would cause full-fledged late-stage Randall's plaque formation at the margin of the calyx. This heretical hypothesis that macrophages and MGCs may transport crystals through the interstitium has the potential to dramatically expand the scope of testing and development of novel therapeutic approaches to urolithiasis, which currently affects >1 billion people worldwide. The Hypothesis of Seven Decades Charon, oarsman of the mythical Greek underworld, ferried recently departed souls across the River Styx into Hades (1). His oar was used to slow, maintain, or accelerate the rate at which the deceased were boarded, and propel his doomed cargo. Those with a coin payment placed in their mouths during "proper" burials were allowed to embark, where the longer their passage, the deeper their delivery into Hades (1). A hypothesis is proposed here (Figure 1), founded on 70 years of previously published research on human and rodent models, that macrophages and MGCs serve in a role similar to that of Charon, in that they engulf early-stage biomineralization crystals (departed souls on the ferry) and transport them through the interstitium (the River Styx) to the papilla (2,3). If correct, in nonstone formers the crystals would completely dissolve during this transport process, whereas in stone formers the crystals would accumulate and eventually penetrate the papillary epithelia to form Randall's plaque at the margin of the calyx (the suffering of Hades; Figure 1). This theoretical mechanism would establish valuable new testable hypotheses for in vivo and in vitro experimentation on urolithiasis in human and animal models. Research over the 1930s to 1970s commonly focused on the origins of biomineralization within living and cadaver human renal tissues, and has provided plenty of evidence that the initial biomineralization process is indistinguishable between stone formers and nonstone formers (4–8). Conversely, recent studies have tended to focus more on later stage fully formed stones in humans and in stone-forming animals, such as canines (9–11). However, it remains largely unknown and highly controversial just how, and if, the crystalline products of early-stage renal biomineralization affect late-stage Randall's plaque formation at the renal papillae (6,11–13).Figure 1.: A hypothetical nine-stage mechanistic framework created from urology and nephrology research published from 1930 to 2022. This model illustrates that a patient becoming either a stone former or a non-stone former may result from a combination of differences in crystallization rate in the lumen and transportation rates across the tubular epithelium and through the renal insterstitium. These processes are primarily carried out by macrophages and multinucleated giant cells (MGCs) that engulf and transport crystals through the interstitium and dispose them at the papilla. Briefly, the specific steps of nine-stage framework are presented here. Stage 1: Hydroxyapatite (HAP) spherules, as well as calcium oxalate dihydrate (COD) and calcium oxalate monohydrate (COM)crystals precipitate directly from tubular fluid in both stone formers and nonstone formers. Stage 2: Due to a higher rate of crystallization in stone formers, the crystals could aggregate and partially or fully clog the tubule to trigger inflammatory responses within the epithelial cells of the tubules. Stage 3: Some of these may bind to crystal receptors on tubular epithelial surfaces such as osteopontin (OPN), matrix Gla protein, and feutin A, and trigger cascade of cellular signaling events including production of reactive oxygen species (ROS), endocytosis, and processes resulting in the transport of HAP, COD, and COM through the tubular epithelium across the basement membrane and eventually into the interstitium. Stage 4: In response to a proinflammatory signaling cascade, blood-derived monocytes differentiate into macrophages (such as M1 versus M2) via 1β and IL-13 cytokine signaling pathways in the interstitium that engulfs incoming HAP, COD, and COM crystals. Stage 5: HAP, COD, and COM crystals engulfed by macrophages and MGCs undergo diagenetic phase transitions as they migrate or pushed toward renal papilla and calyx to deliver their cargo. Stages 6–8: Within nonstone formers, macrophages and MGCs would completely dissolve and breakdown the phagocytosed CaP and HAP crystals, although this is not been consistently observed for COD and COM. Within stone formers, macrophages (specifically if they are crystal growth–promoting M1 macrophages) and MGCs, do not completely digest the crystal cargo but instead grow their engulfed COD crystals. This conglomeration of macrophages and MGCs at the renal papilla accumulate, aggregate, and fuse crystals of various sizes within the papilla, or breach the papillary epithelium to become partially exposed to the supersaturated urine in the calyx. Stage 9: Some of these macrophages, MGCs and broken membranes are released into the renal calyx in the stone formers, and the crystal aggregates grow when exposed to supersaturated urine in the calyx, while continuously incorporating free-floating stage 1 crystals of HAP, COD, and COM. Further details are provided in the main text. Tissue and crystals not drawn to scale.A Crystalline Cascade Prequel to Randall's Plaque Formation It has been proposed that early-stage renal biomineralization in human and rodent models begins with the precipitation of free-floating hydroxyapatite (HAP), calcium oxalate dihydrate (COD), and calcium oxalate monohydrate (COM) crystals from supersaturated tubular fluid (stage 1; Figure 1) (5,6,11,14). If correct, these suspended crystals (sediments) precipitate directly from tubular fluid as the product of direct spontaneous nucleation or heterogenous nucleation on suspended particles (e.g., cell debris, small crystals) (11,15). These crystal sediments, which occur in both stone formers and nonstone formers, could be precipitated and transported through the entire renal fluidic system and eventually dissolved or expelled into the renal calyx, as demonstrated in both human and rodent models (Figure 1) (11). These small, suspended crystals grow in size, potentially form crystalline aggregates anywhere in the renal tubules and collecting ducts, contribute to stone formation in the calyx, and undergo physical, chemical, and biologic alterations (diagenetic phase transitions) (13). All of these crystallization processes could accelerate in the tubular fluid of stone formers due to increased calcium (hypercalciuric), oxalate (hyperoxaluric), hypocitraturic, and phosphate concentrations, which result from increased fluid saturation states due to dehydration, pH, diet, and other factors (11). Furthermore, once COD crystals are formed, they have the potential at any stage of the process to undergo a diagenetic phase transition into COM that will release H2O molecules, which may help prevent tissue damage due to dehydration (11). Connecting the Dots In animal cell models, such as MDCK cells, a subset of HAP, COD, and COM crystal sediments settle directly on the intratubule epithelial cell surfaces that are coated with mucus containing glycosaminoglycon-hyaluronic acid (14). In both humans and in some stone-forming rodents, crystal growth inhibitors and promotors (e.g., osteopontin [OPN], matrix Gla protein and feutin A) are present on epithelial cell surfaces and extracellular matrices that bind to these crystals and trigger cascade of cellular signaling events (stages 2 and 3; Figure 1) (11,14). Continued crystal growth and aggregation within tubules would lead to partial or complete clogging of the tubule (stage 2; Figure 1) (13). These crystals, some of which bind to epithelial cell receptors, could trigger both signal transduction and a proinflammatory response that includes: (1) production of reactive oxygen species (12), (2) endocytosis (15), and (3) transport of the HAP, COD, and COM crystals through the epithelium and across the basement membrane, dislodging them into the interstitium (7,11). In response to this proinflammatory signaling cascade, blood-derived monocytes differentiate into induced macrophages (11) via 1β and IL-13 cytokine signaling pathways in the interstitium (stage 4; Figure 1) (2,16). In both nonstone formers and stone formers, another subset of renal epithelial cells produces membranous vesicles in response to increased reactive oxygen species signaling (12). These membranous vesicles are calcified by calcium phosphate (CaP) in the basement membrane (12), previously called "droplets of CaP" (3), and undergo a diagenetic phase transition into HAP (7,13). HAP, COD, and COM crystals, as in rodent models, delivered to the interstitium as a result of these processes (stages 2 and 3; Figure 1) are engulfed (phagocytosed) by both M1 and M2 macrophages and MGCs (11). Multiple mechanisms by which M1 and M2 macrophages influence calcium oxalate crystallization have previously been observed in animal models (3,17,18), including enhanced gene expression of crystallization modulators, such as osteopontin and CD44, and the expression of proinflammatory and crystal adhesion related genes such as IL-6, inducible NOS, TNF-α, C3, and VCAM-1. Although M1 macrophages tend to enhance crystal development, M2 macrophages are shown to suppress crystal growth (19). Stone formers have higher levels of M1 macrophages compared with M2, suggesting this may play a pivotal role in enhanced crystal accumulation in stone formers compared with nonstone formers. Although details on their specific roles are yet to be understood, it has been experimentally confirmed that mice deficient in the M2 macrophage showed enhanced renal calcium oxalate formation and retention (3,17,18). In humans, although it is yet to be tested, it is possible that stone formers versus nonstone formers differentially produce M1 versus M2 macrophages, respectively, or alternatively are unable to produce protective M2 macrophages and associated genes and protein products. In addition, in stone formers, the rate of crystal phagocytosis and transport by macrophages and MGCs may become too high (stages 1–3; Figure 1), due to M1 macrophage dominance, because this may cause the crystals to accumulate, aggregate, and form plaques within the interstitium, thin loops of Henle, and papilla within human and animal stone formers (Figure 1). Multiple small crystal aggregates could eventually combine from multiple locations and form a complex stone, as described before (19). As experimentally proven in rodents, HAP, COD, and COM crystals engulfed within macrophages and MGCs undergo diagenetic phase transitions as these cells migrate toward and deliver their crystal cargo to the renal papilla and the calyx (stage 5; Figure 1) (3). Although this is implied by the arcuate distributions of crystal aggregates observed in human cadaver kidneys (3–5) and microcomputed tomography scans of papillary biopsies (12), the driving force behind macrophage and MGC migration remains to be determined in humans and in animals that form stones, including canines and rodents (12). In the hypothesis presented here, calcium oxalate crystals would be completely dissolved, broken down, and removed (digested) within just a few days, which has been shown to occur in vivo in in some stone forming rodents (3) and in vitro for humans (20). During stages 6, 7, and 8 (Figure 1), within nonstone formers, COD and COM crystals have not been reliably described in the interstitium outside of primary hyperoxaluria or other extreme hyperoxaluric states (11–13). In this way, macrophages and MGCs would completely dissolve and breakdown the phagocytosed CaP and HAP crystals as they are emptied during programmed cell death (20). Conversely, during stages 6, 7, 8, and 9 (Figure 1) within stone-forming rodents, the macrophages and MGCs do not completely digest their crystal cargo (stage 6; Figure 1), and even grow their engulfed COD crystals (20). These crystals may then either accumulate within the papilla or breach the papillary epithelium and become partially exposed to supersaturated urine and suspended stage 1 free-floating crystal sediments at the margin of the renal calyx (stage 7; Figure 1) (11). The macrophages and MGCs contain HAP, COD, and COM crystals of multiple sizes that are then released through ruptured papillary epithelial cells and membranes into the calyx (stage 8; Figure 1) and mix with free-floating stage 1 crystals (11). In humans and in some rodent models, these events have been shown to trigger the growth of full fledged stones adjacent to the renal papilla in the collecting system (stage 9; Figure 1) (11). A combination of evidence from human biopsies and rodent experimental models, with the hypotheses presented here (Figure 1), potentially identifies a new pathway to find unexplored therapeutic interventions. Examples might include production of crystal suppressing M2 macrophages, modulation of the rate of migration of macrophages and MGCs through the interstitium, disruption of HAP spherule coalescence into COD and COM crystals, and enhancement of COD dissolution during macrophage and MGC transport through the interstitium. The resulting development of next-generation clinical therapies for urolithiasis would therefore better decipher Charon's long sought-after nautical map of the River Styx. Disclosures B.W. Fouke reports receiving research funding from Dornier MedTech and Lumenis. B.W. Fouke and M. Sivaguru report receiving research support from Dornier MedTech and Boston Scientific Lumenis. The Carl R. Woese Institute for Genomic at the University of Illinois Urbana-Champaign is a Carl Zeiss Labs @ Location Partner. Funding None.