Recruitment of Podocytes from Glomerular Parietal Epithelial Cells

Loss of a critical number of podocytes from the glomerular tuft leads to glomerulosclerosis. Even in health, some podocytes are lost into the urine. Because podocytes themselves cannot regenerate, we postulated that glomerular parietal epithelial cells (PECs), which proliferate throughout life and adjoin podocytes, may migrate to the glomerular tuft and differentiate into podocytes. Here, we describe transitional cells at the glomerular vascular stalk that exhibit features of both PECs and podocytes. Metabolic labeling in juvenile rats suggested that PECs migrate to become podocytes. To prove this, we generated triple-transgenic mice that allowed specific and irreversible labeling of PECs upon administration of doxycycline. PECs were followed in juvenile mice beginning from either postnatal day 5 or after nephrogenesis had ceased at postnatal day 10. In both cases, the number of genetically labeled cells increased over time. All genetically labeled cells coexpressed podocyte marker proteins. In conclusion, we demonstrate for the first time recruitment of podocytes from PECs in juvenile mice. Unraveling the mechanisms of PEC recruitment onto the glomerular tuft may lead to novel therapeutic approaches to renal injury.Chronic kidney disease, resulting in renal failure and the need for lifelong renal replacement therapy, has become a significant problem worldwide. In the United States, approximately 7% of the total Medicare budget is spent on the treatment of ESRD, and projections suggest that the amount spent will increase by another 50% by 2020.¹Most renal pathologies that ultimately lead to ESRD originate within the glomerulus. It has now been established that a depletion of podocytes, the visceral epithelium of the capillary convolute (Figure 1), is central in this process. As soon as damage to the glomerular podocytes exceeds a certain threshold (approximately 30%), glomerulosclerosis ensues.² Indeed, in patients with a surgical reduction of ≥75% of renal mass, a relative lack of podocytes (podocytopenia) and subsequent FSGS in the originally healthy remnant kidney can lead to renal failure.³ Glomerulosclerosis is also the common final pathway of all glomerular diseases leading to ESRD.⁴ In glomerular diseases such as diabetic nephropathy, glomerulonephritides, or preeclampsia, significant numbers of podocytes are lost as a result of apoptosis, necrosis or excretion of living cells into the urine. Even in normal individuals, low numbers of living podocytes are continuously shed into the urine.^5–7 These numbers are too high to be compatible with renal survival for 80 yr, suggesting the existence of a regenerative mechanism. Also, the reversal of early glomerular damage in animal models and humans^8–10 argues for the existence of such a mechanism; however, podocytes are postmitotic cells that cannot undergo complete cell divisions and are therefore unable to regenerate themselves.^8–10 A potential mechanism for podocyte replacement from bone marrow–derived stem cells has been described in the Alport mouse model as well as in kidney transplants.^11–13 Nevertheless, most studies concluded that regeneration occurs predominantly from an as-yet-unknown source of resident renal cells.^12,14–16Open in a separate windowFigure 1.Renal glomerulus. The glomerular epithelium consists of PECs (red) and podocytes (Pod; blue), which reside on the capillary convolute. Both epithelia adjoin directly at the vascular pole (VP; arrow). At the tubular pole (TP), the parietal epithelium is connected to the epithelium of the proximal tubule. In male mice, this transition from PECs to proximal tubular cells often occurs within the glomerulus. The glomerular basement membrane (black) forms a continuous barrier between the glomerular epithelium and the endocapillary compartment that contains mesangial cells (shaded) and endothelial cells of the glomerular capillaries (*). Primary urine is filtered across the three-layered filtration barrier (endothelial cells, glomerular basement membrane, and Pod) into Bowman''s space (BS).In this study, we tested the hypothesis that glomerular parietal epithelial cells (PECs) lining the inner aspect of Bowman''s capsule migrate onto the glomerular tuft and differentiate into podocytes. Several arguments support this hypothesis. PECs are present in all species whose kidneys contain glomeruli. They are located within the same compartment and are in direct continuity with podocytes at the glomerular vascular stalk, so PECs do not have to cross an anatomic barrier such as the glomerular basement membrane, as was suggested for bone marrow–derived stem cells.^11–13 PECs proliferate lifelong at a relatively low frequency,¹⁷ express several stem cell marker proteins, and could be transdifferentiated in vitro into other cell types such as adipocytes or neuronal cells, suggesting that these cells retain multipotency.^9,18,19 In rodents, PECs do not express any known specific marker protein, which has so far precluded a detailed analysis of the function of these cells.In this work, we provide the first evidence that PECs possess the capability to migrate onto the glomerular tuft via the vascular stalk, where they differentiate into podocytes. This establishes that PECs represent an intrinsic cell population from which podocytes can be recruited.     

Human Urine-Derived Renal Progenitors for Personalized Modeling of Genetic Kidney Disorders

The critical role of genetic and epigenetic factors in the pathogenesis of kidney disorders is gradually becoming clear, and the need for disease models that recapitulate human kidney disorders in a personalized manner is paramount. In this study, we describe a method to select and amplify renal progenitor cultures from the urine of patients with kidney disorders. Urine-derived human renal progenitors exhibited phenotype and functional properties identical to those purified from kidney tissue, including the capacity to differentiate into tubular cells and podocytes, as demonstrated by confocal microscopy, Western blot analysis of podocyte-specific proteins, and scanning electron microscopy. Lineage tracing studies performed with conditional transgenic mice, in which podocytes are irreversibly tagged upon tamoxifen treatment (NPHS2.iCreER;mT/mG), that were subjected to doxorubicin nephropathy demonstrated that renal progenitors are the only urinary cell population that can be amplified in long-term culture. To validate the use of these cells for personalized modeling of kidney disorders, renal progenitors were obtained from (1) the urine of children with nephrotic syndrome and carrying potentially pathogenic mutations in genes encoding for podocyte proteins and (2) the urine of children without genetic alterations, as validated by next-generation sequencing. Renal progenitors obtained from patients carrying pathogenic mutations generated podocytes that exhibited an abnormal cytoskeleton structure and functional abnormalities compared with those obtained from patients with proteinuria but without genetic mutations. The results of this study demonstrate that urine-derived patient-specific renal progenitor cultures may be an innovative research tool for modeling of genetic kidney disorders.     

Perivascular Mesenchymal Progenitors for Bone Regeneration

Mesenchymal progenitor cells reside in all assayed vascularized tissues, and are broadly conceptualized to participate in homeostasis/renewal and repair. The application of mesenchymal progenitor cells has been studied for diverse orthopaedic conditions related to skeletal degeneration, regeneration, and tissue fabrication. One common niche for mesenchymal progenitors is the perivascular space, and in both mouse and human tissues, perivascular progenitor cells have been isolated and characterized. Of these “perivascular stem cells” or PSC, pericytes are the most commonly studied cells. Multiple studies have demonstrated the regenerative properties of PSC when applied to bone, including direct osteochondral differentiation, paracrine‐induced osteogenesis and vasculogenesis, and immunomodulatory functions. The confluence of these effects have resulted in efficacious bone regeneration across several preclinical models. Yet, key topics of research in perivascular progenitors highlight our lack of knowledge regarding these cell populations. These ongoing areas of study include cellular diversity within the perivascular niche, tissue‐specific properties of PSC, and factors that influence PSC‐mediated regenerative potential. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1221–1228, 2019.     

The Importance of Glomerular Deposits of Von Willebrand Factor in Human Renal Allografts

Objective. The aim of this study was to evaluate the importance of glomerular expression of von Willebrand factor (vWF) in human renal allografts. Methods. We investigated graft biopsies from 72 renal transplant recipients, 40 with acute rejection (AR) and 32 with chronic allograft nephropathy (CAN). All biopsy specimens were immunostained with vWF and CD68 and graded using 3-tiered scales. The follow-up biopsies of patients with AR were reevaluated for development of glomerular sclerosis. Results. A significant difference was found between type 1 and type 2 AR with regard to glomerular vWF expression (P < 0.01). None of the patients with type 1 AR showed mesangial vWF expression, but 36.4% of patients with type 2 AR showed segmental mesangial vWF expression. In follow-up biopsies, 18 of 40 patients developed significant glomerular sclerosis, and patients with mesangial vWF expression (grade 3 GvWF) showed glomerular sclerosis earlier than did others (P < 0.01). In addition, the outcome for grafts that showed grade 3 glomerular vWF was significantly worse than was the outcome noted for grafts that showed grade 1 or grade 2 glomerular vWF (P < 0.001). Half of the biopsy specimens in the CAN group showed global mesangial vWF expression. Glomerular macrophage infiltration was correlated with degree of glomerular vWF expression both in the AR and in the CAN groups (P < 0.05, P = 0.001, respectively). Conclusion. We hypothesized that the increasing amount of glomerular vWF may be used as a marker of acute vascular rejection and may help for the evaluation of renal allograft biopsies without sufficient arteries. In addition, it can also be a marker for development of early glomerular sclerosis and may help us determine which patients are in need of further treatment.     

猪后肾帽状间充质细胞发育为肾小球足细胞的过程

目的:探讨中国实验用小型猪肾小球足细胞的发育过程。方法:应用过碘酸-希夫氏染色观察中国实验用小型猪胚胎不同时间点(胚胎28d至出生后21d,以周为单位,共17个时间点)肾小球发育过程中的形态学变化。应用免疫荧光技术检测猪胚肾不同阶段(帽状间充质、肾小囊体、逗号形体、"S"形体、毛细血管袢期肾小球、成熟肾小球)足细胞的发育过程。结果:猪在胚胎第28天(E28d)后肾已开始发育,可见典型的帽状间充质、肾小囊体、逗号形体和"S"形体;E35d可见肾小球形成,包括近皮质的不成熟肾小球以及近髓质的成熟肾小球。胚肾组织免疫荧光染色显示:胚肾早期足细胞标志物WT1表达于Six2阳性的后肾帽状间充质细胞,相继表达于肾小囊体、整个逗号形体、逗号形体尾部以及"S"形体下端,最终局限于肾小球足细胞。结论:中国实验用小型猪的足细胞来源于Six2阳性的后肾帽状间充质细胞,经过肾小囊体、逗号形体、"S"形体、毛细血管袢期肾小球阶段,发育成为成熟肾小球的足细胞。     

Evaluation of Renal Clinicopathological Changes in IgA Nephropathy by Urinary Podocytes Excretion and Podocalyxin Expression

Objective: To explore the association of urinary podocyte excretion and renal expression of podocyte-specific marker podocalyxin (PCX) with clinicopathological changes in immunoglobulin A nephropathy (IgAN). Methods: Morning urine samples from IgAN patients and healthy controls were collected. The expression of glomerular PCX was quantified in 50 IgAN patients diagnosed by renal biopsy. IgAN was classified based on the Lee’s Grading system and scored according to the Katafuchi semiquantitative criteria. Morphological evaluation of podocyte was determined by electron microscopy. Results: The amount of urinary podocytes in the IgAN patients was significantly higher than that in the healthy controls (p < 0.01). Pairwise comparison among Lee’s grades of IgAN showed that the median of urinary podocytes in Lee’s I–II group was lower than that in Lee’s III, IV, and V groups (p < 0.05); group III lower than group V (p < 0.05). The positive rate of urinary podocytes was the highest in Lee’s IV and V groups (100%), and lowest in Lee’s I–II group (55%). Multiple comparison among groups of Lee’s grades of IgAN showed that the glomerular PCX expression in Lee’s I–II group was higher than that in Lee’s III, IV, and V groups (p < 0.05); groups III and IV higher than group V (p < 0.05). The amount of urinary podocytes in IgAN patients was negatively correlated with PCX expression (r = ?0.702, p < 0.01), but positively correlated with 24-h urinary protein (r = 0.465, p < 0.01) and glomerular (r = 0.233, p < 0.01) and renal tubular pathological scores (r = 0.307, p < 0.05). The glomerular PCX expression was negatively correlated with 24-h urinary protein (r = ?0.367, p < 0.05) and glomerular (r = ?0.560, p < 0.05) and tubular pathological scores (r = ?0.377, p < 0.05). Electron microscopy showed significant changes in podocytes of IgAN, especially in the foot process. Conclusion: The amount of urinary podocyte can reflect the loss of podocytes in renal tissue, which may be a marker of IgAN progression.     

Glomerular Enlargement Assessed by Paired Donor and Early Protocol Renal Allograft Biopsies

The aim of the study was to evaluate the evolution of glomerular volume 4 months after transplantation. Mean glomerular volume (Vg) was estimated according to the Weibel and Gomez method in a donor and a protocol biopsy done at 139 +/- 58 d in 41 stable grafts. Biopsies were also evaluated according to the Banff schema. Vg increased after transplantation from 4.1 +/- 1.4 to 5.1 +/- 2.4 x 10(6) micro3 (p=0.02). In patients with chronic allograft nephropathy in the protocol biopsy (n=14), the Vg enlargement was -0.3 +/-x 10(6) micro3 while in patients without chronic allograft nephropathy (n=27), glomerular enlargement was 1.6 +/- 2.1 x 10(6) micro3 (p=0.01). There was a negative association between glomerular volume in the donor biopsy and glomerular enlargement after transplantation (R=- 0.34, p=0.03). Multivariate regression analysis confirmed that Vg in the donor biopsy and chronic allograft nephropathy in the protocol biopsy were independent predictors of glomerular enlargement after transplantation (R=0.48, p=0.01). Moreover, Vg in the protocol biopsy correlated with creatinine clearance at the time of biopsy (R=0.38, p=0.01). Glomeruli enlarge after transplantation and glomerular volume after 4 months correlates with creatinine clearance, suggesting that glomerular enlargement is a necessary condition for renal adaptation to the recipient. Glomerular enlargement is impaired in patients with chronic allograft nephropathy.     

免疫细胞化学法检测尿足细胞的临床应用

目的：免疫荧光细胞化学和免疫酶细胞化学两种方法检测尿足细胞的比较，以促其临床推广应用。方法：尿标本均为我科住院患者的肾活检日新鲜晨尿，采用抗人足细胞标记蛋白Podocalxin（PCX）单克隆抗体进行尿沉渣免疫荧光细胞化学和免疫酶细胞化学染色。14例患者均经肾活检，肾组织由光镜、免疫荧光和电镜检查做出病理诊断。结果：（1）14份尿标本应用免疫荧光细胞化学和免疫酶细胞化学方法均可检测到足细胞。（2）同一份尿沉渣采用免疫荧光细胞化学法检测足细胞均数为（16、7±15、1）／HP，而免疫酶细胞化学法检测足细胞均数为（26，1±24、3）／20HP，两组比较有统计学意义（P〈0、05）；但两种方法所测足细胞数之间有很好的相关性（r=0、969）。结论：免疫荧光细胞化学和免疫酶细胞化学两种方法检测尿足细胞，前者较简便、镜下易辨认，后者设备简单、标本可保存。     

Epithelial Progenitors in the Normal Human Mammary Gland

The human mammary gland is organized developmentally as a hierarchy of progenitor cells that become progressively restricted in their proliferative abilities and lineage options. Three types of human mammary epithelial cell progenitors are now identified. The first is thought to be a luminal-restricted progenitor; in vitro under conditions that support both luminal and myoepithelial cell differentiation, this cell produces clones of differentiating daughter cells that are exclusively positive for markers characteristic of luminal cells produced in vivo (i.e., keratins 8/18 and 19, epithelial cell adhesion molecule [EpCAM] and MUC1). The second type is a bipotent progenitor. It is identified by its ability to produce mixed colonies in single cell assays. These colonies contain a central core of cells expressing luminal markers surrounded by cells with a morphology and markers (e.g., keratin 14⁺) characteristic of myoepithelial cells. Serial passage in vitro of an enriched population of bipotent progenitors promotes the expansion of a third type of progenitor that is thought to be myoepithelial-restricted because it only produces cells with myoepithelial features. Luminal-restricted and bipotent progenitors can prospectively be isolated as distinct subpopulations from freshly dissociated suspensions of normal human mammary cells. Both are distinguished from many other cell types in mammary tissue by their expression of EpCAM and CD49f (6 integrin). They are distinguished from each other by their differential expression of MUC1, which is expressed at much higher levels on the luminal progenitors. To relate the role of these progenitors to the generation of the three-dimensional tubuloalveolar structure of the mammary tree produced in vivo, we propose a model in which the commitment to the luminal versus the myoepithelial lineage may play a determining role in the generation of alveoli and ducts.     

柴芩肾安方对IgA肾病大鼠肾小球足细胞nephrin表达的影响

目的：探讨柴芩肾安方对IgA肾病（IgAN）大鼠肾小球足细胞nephrin表达的影响。方法：通过切除单侧肾并反复静脉注射葡萄球菌肠毒素B（SEB）复制大鼠IgAN模型,分为正常组、切肾组、IgAN组、柴芩肾安方组和氯沙坦组。第12周末,检测各组大鼠24h尿蛋白量（Upr）,观察肾小球形态学和超微结构变化,并采用免疫组化和实时定量PCR法检测肾小球足细胞nephrin蛋白及mRNA的表达。结果：与正常组相比,IgAN组大鼠Upr显著升高（P〈0.01）,肾小球系膜增生,足突融合明显,足细胞nephrin蛋白及mRNA表达均明显下调（P〈0.01）。经柴芩肾安方治疗后上述指标均明显改善,与IgAN组比较差异有统计学意义（P〈0.01或P〈0.05）。结论：柴芩肾安方能减轻IgAN大鼠蛋白尿及肾小球病变,这可能与其恢复肾小球足细胞nephrin表达有关。     

Acute Renal Failure Secondary to Leukocyte-Mediated Acute Glomerular Injury

Acute glomemlonephritis can cause acute renal failure. Activated neutrophils and monocytes are major eflectors of glomerulonephritic renal failure. Adhesion molecules, granule enzymes, reactive oxygen radicals, lipid metabolites, and cytokines of activated neutrophils and monocytes mediate glomerular capillary constriction, occlusion, and destruction. Injurious products and biologically active mediators released by activated leukocytes have profound fictional eflects on mesangial cells and endothelial cells, which in turn participate in the disturbance of glomerularfinc-tion, for example, by altering capillary diameter and surface area. f i e glomerular injlammatory events result in decreased glomerular capillary ultrajiltraton coefl-cient and glomerular jiltration rate, as well as other functional perturbations.     

益肾胶囊对糖尿病肾病模型肾小球足细胞的影响

目的：观察益肾胶囊对糖尿病肾病（DN）大鼠肾组织病理改变及足细胞超微结构的影响。方法：将60只Wis-tar大鼠随机分为4组：正常对照组（对照组）、DN模型组（模型组）、苯那普利组、益肾胶囊组。于注射链脲佐菌素（STZ）后3d起,苯那普利组每日每只灌胃苯那普利3.125mg.kg-1.d-1,益肾胶囊组每日每只灌胃益肾胶囊625mg.kg-1.d-1,对照组及模型组每日给予等量的蒸镏水。各组分别干预12周,观察24h尿蛋白定量、血肌酐（Scr）、尿素氮（BUN）的变化,同时行肾脏病理检查。结果：12周末,模型组大鼠24h尿蛋白定量、Scr、BUN均高于对照组（P〈0.05）。苯那普利组及益肾胶囊组24h尿蛋白定量、Scr、BUN均低于模型组（P〈0.05）。光镜下模型组大鼠肾小球系膜基质增多,系膜区增宽;电镜下模型组大鼠肾小球基底膜增厚,足细胞排列紊乱,数目减少,足突增宽、融合。苯那普利组及益肾胶囊组肾小球基底膜病变减轻,细胞外基质减少,足细胞数目增多,足突融合减轻。结论：益肾胶囊能降低尿蛋白排泄,改善肾功能,并对足细胞有一定的保护作用,从而延缓大鼠糖尿病大鼠肾脏损害。     

Deficiency of the Planar Cell Polarity Protein Vangl2 in Podocytes Affects Glomerular Morphogenesis and Increases Susceptibility to Injury

The planar cell polarity (PCP) signaling pathway is crucial for tissue morphogenesis. Van Gogh-like protein 2 (Vangl2) is central in the PCP pathway; in mice, Vangl2 loss is embryonically lethal because of neural tube defects, and mutations in Vangl2 are associated with human neural tube defects. In the kidney, PCP signaling may be important for tubular morphogenesis and organization of glomerular epithelial cells (podocytes) along the glomerular basement membrane. Podocyte cell protrusions (foot processes) are critical for glomerular permselectivity; loss of foot process architecture results in proteinuria and FSGS. Previously, we showed a profound effect of PCP signaling on podocyte shape, actin rearrangement, cell motility, and nephrin endocytosis. To test our hypothesis that the PCP pathway is involved in glomerular development and function and circumvent lethality of the ubiquitous Vangl2 mutation in the Looptail mouse, we generated a mouse model with a podocyte-specific ablation of the Vangl2 gene. We report here that podocyte-specific deletion of Vangl2 leads to glomerular maturation defects in fetal kidneys. In adult mice, we detected significantly smaller glomeruli, but it did not affect glomerular permselectivity in aging animals. However, in the context of glomerular injury induced by injection of antiglomerular basement membrane antibody, deletion of Vangl2 resulted in exacerbation of injury and accelerated progression to chronic segmental and global glomerular sclerosis. Our results indicate that Vangl2 function in podocytes is important for glomerular development and protects against glomerular injury in adult animals.