C3aR-initiated signaling is a critical mechanism of podocyte injury in membranous nephropathy

: The deposition of anti-podocyte auto-antibodies in the glomerular subepithelial space induces primary membranous nephropathy (MN), the leading cause of nephrotic syndrome worldwide. Taking advantage of the glomerulus-on-a-chip system, we modeled human primary MN induced by anti-PLA2R antibodies. Here we show that exposure of primary human podocytes expressing PLA2R to MN serum results in IgG deposition and complement activation on their surface, leading to loss of the chip permselectivity to albumin. C3a receptor (C3aR) antagonists as well as C3AR gene silencing in podocytes reduced oxidative stress induced by MN serum and prevented albumin leakage. In contrast, inhibition of the formation of the membrane-attack-complex (MAC), previously thought to play a major role in MN pathogenesis, did not affect permselectivity to albumin. In addition, treatment with a C3aR antagonist effectively prevented proteinuria in a mouse model of MN, substantiating the chip findings. In conclusion, using a combination of pathophysiologically relevant in vitro and in vivo models, we established that C3a/C3aR signaling plays a critical role in complement-mediated MN pathogenesis, indicating an alternative therapeutic target for MN

Protective effect of human amniotic fluid stem cells in an immunodeficient mouse model of acute tubular necrosis.

Acute Tubular Necrosis (ATN) causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Stem-cell based therapies may provide alternative approaches to treating of ATN. We have previously shown that clonal c-kit(pos) stem cells, derived from human amniotic fluid (hAFSC) can be induced to a renal fate in an ex-vivo system. Herein, we show for the first time the successful therapeutic application of hAFSC in a mouse model with glycerol-induced rhabdomyolysis and ATN. When injected into the damaged kidney, luciferase-labeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN. We therefore speculate that AFSC could represent a novel source of stem cells that may function to modulate the kidney immune milieu in renal failure caused by ATN

Amniotic fluid stem cell therapy in chronic kidney disease progression: the case of Alport syndrome

Chronic kidney Disease (CKD) is a global health problem. It is associated with gradual decline in renal function, which develops into end stage renal disease and culminates into renal failure. Many different etiological factors are involved in the initiation and progression of CKD. The two most prevailing medical reasons for the development of progressive kidney disease are diabetes and hypertension. Regardless of the site of the initial insult, which may either be the glomerulus or tubules, all forms of progressive kidney diseases follow a final common pathway that is interstitial and glomerular fibrosis together with loss of podocytes, therefore, loss of glomerular integrity and renal function. Current therapeutic options for CKD are few and limited to the administration of renin-angiotensin system blockers, dialysis and renal transplantation. Although, pharmacotherapies are effective in slowing down the rate of progression, they do not prevent end stage renal disease. On the other hand dialysis even though life sustaining does not solve the problem and is associated with very poor quality of life. Renal transplantation thus remains the most effective method to treat end stage renal disease, however, the shortage of donor organs and immune complications associated with it do not solve the problem either. Hence, development of novel therapies remains an urgent necessity. Stem cell approach to Regenerative Medicine introduces vast possibilities for new therapies in acute as well as chronic diseases where current treatment options are either limited or inadequate. Stem cells derived from amniotic fluid (AFSC), which have emerged in recent years, represent a new source of stem cells with pluripotential properties, devoid of ethical issue associated with embryonic stem cells. Previous studies have proven the principle that AFSC can be used in regenerative medicine of the kidney due to their strong immunomodulatory properties and showed renoprotection when injected into an acute renal failure injury model in rodents. The purpose of the current study was to investigate the role of stem cells derived from amniotic fluid in an animal model of chronic kidney disease. In particular, cells positively selected for CD117 (c-kit) were administered to mice with x-linked Alport Syndrome (XLAS), a well established genetic model to study chronic kidney disease. Alport disease is associated with mutations in the collagen type IV family of proteins, the a3, a4 and a5 chains, the absence of which alters the normal composition and function of the glomerular basement membrane leading to proteinuria, and ultimately loss of podocytes. We hypothesized that (AFSC) would be able slow down chronic progression in Alport kidneys by modulating key processes such as the immune response, pro-fibrotic events initiated by TGF- signaling and cytokine/chemokine profile of the kidney via endocrine/paracrine mechanisms protecting the glomerular structure and preserving the filtration property of the organ. Single dose of systemic injection of AFSC at an early stage of the disease, before the onset of proteinuria, prolonged the life-span of Alport mice by 20% on average. This also resulted into amelioration of the functional parameters of the kidney, lowered serum creatinine, BUN as well as proteinuria levels at 2.5 months post treatment. Treated kidneys demonstrated better morphology with less severe glomerulosclerosis, lesser deposition of collagen type I associated with interstitial fibrosis and reduced infiltration of macrophages and inflammatory cells compared to their non-treated siblings. AFSC were detectable in glomeruli of injected mice but they did not differentiate into podocytes, and consequently, the a5(IV) chain of collagen missing in Alport mice was not replaced by AFSC treatment. Our findings revealed significant downregulation of key regulatory cytokines, such as TNFa, CCL2, CXCL2 and M-CSF involved in pro-fibrotic M1 macrophage signaling pathway favoring tissue remodeling instead of tissue injury. Finally, Alport mice receiving AFSC demonstrated preservation of podocyte numbers. In a closer look, glomerular cells stimulated with Angiotensin II showed increased Angiotensin II receptor type 1 (ANGTR1) expression, which was downregulated when treated with losartan (Angiotensin II antagonist). Similar to losartan AFSC decreased ANGTR1 expression, suggesting that AFSC may block the effects of Angiotensin II favoring glomerular survival. In conclusion, AFSC slow down Alport progression via preservation of podocyte number, M2c activation of macrophages and modulation of kidney microenvironment through endocrine/paracrine mechanisms favoring tissue preservation and function, in particular preserving podocyte number and filtration property.Negli ultimi anni l’aumento nell’incidenza della Malattia Renale Cronica (Chronic Kidney Disease, CKD) è diventata un importante problema di salute pubblica. Principale manifestazione clinica della CKD è un graduale declino della capacità di filtrazione del rene che termina con lo sviluppo di insufficienza renale terminale (End Stage Renal Disease, ESRD). Infezioni, infiammazioni acute e croniche, malattie genetiche o la presenza di patologie a carico di altri organi, come per esempio il diabete, possono concorrere o essere causa diretta di danno renale. Questi insulti possono portare ad una progressiva perdita della funzione renale fino a raggiungere lo stadio finale di ESRD. Indipendentemente dalle origini eziologiche, la CKD evolve con la progressione di fibrosi glomerulare e interstiziale, apoptosi dei podociti con conseguente distruzione della struttura morfo-funzionale del rene e della sua capacità di filtrazione. L’utilizzo di farmaci (come per esempio gli antiipertensivi ACE inibitori, o inibitori dell'anidrasi carbonica) spesso riesce a rallentare il progresso della malattia ma il trattamento farmacologico risulta essere meno efficace con il progredire dell’ESRD e può risultate nefrotossico con il passare del tempo. La dialisi, trattamento molto invasivo e costoso per la sanità, ed il trapianto con la scarsa disponibilità di organi e la crescente richiesta, hanno spinto alla ricerca di valide alternative con un minor costo sociale ed una maggior compliance da parte dei pazienti. Molti studi si sono focalizzati nella scoperta di nuove fonti di cellule staminali per il trattamento di malattie renali acute e croniche dove le terapie correnti sono insufficienti ed inadeguate. Negli ultimi anni, il liquido amniotico è stato proposto come fonte alternativa per l’isolamento di cellule staminali da poter utilizzare in futuro per la terapia cellulare e, con particolare riguardo, per la rigenerazione del tessuto renale. In particolare, in studi recentemente pubblicati, le cellule staminali da liquido amniotico (AFSC) si sono distinte per la loro potente azione antiinfiammatoria e renoprotettiva, con modulazione della risposta immunitaria in un modello murino di danno renale acuto. Lo scopo di questo lavoro è quello di investigare il ruolo delle cellule staminali da liquido amniotico in un modello animale genetico di danno renale cronico (CKD). Nello specifico, cellule positive per il recettore di membrana CD117 (c-kit) sono state infuse in un modello murino di Sindrome di Alport, riconosciuto come uno dei più utili modelli più utili per lo studio di malattie renali croniche. La Sindrome di Alport è una grave malattia ereditaria e progressiva dovuta a mutazioni che coinvolgono le catene a3, a4 and a5 del collagene di tipo IV. L’assenza di tali catene altera la composizione fisiologica della matrice extracellulare renale (GBM, glomerular basement membrane) e provoca una marcata perdita di funzionalità che conduce a proteinuria e, con il tempo, a perdita dei podociti con conseguente blocco della filtrazione glomerulare. Basandoci sui dati precedentemente ottenuti, in questo lavoro ipotizziamo che le cellule staminali da liquido amniotico abbiano la capacità di rallentare la progressione della CKD, tramite un’azione endocrina e paracrina su vari meccanismi patologici che includono la risposta immunitaria, la fibrosi indotta dall’attivazione di TGF-a . In aggiunta, ipotizziamo che l’azione positiva esercitata dalle cellule da liquido amniotico sia un effetto di mantenimento della struttura glomerulare con conseguente mantenimento della normale capacità di filtrazione. In particolare, una singola infusione di AFSC, amministrata nei primi stadi della malattia, è capace di prolungare la sopravvivenza nel modello murino di circa il 20% con un significativo miglioramento dei parametri fisiologici renali quali livelli di creatinina plasmatica, BUN e proteinuria, oltre due mesi dalla somministrazione. Inoltre, Il tessuto renale degli animali trattati presenta una morfologia normale con lieve sclerosi glomerulare ed una minore deposizione di collagene I, di solito associata a fibrosi interstiziale. A questo si accompagna una minore infiltrazione da parte di macrofagi e altre cellule del sistema immunitario rispetto agli animali non trattati. Una vasta serie di esperimenti è stata quindi effettuata per investigare i meccanismi di azione tramite cui le cellule staminali da liquido amniotico esercitano il loro effetto positivo. Gli animali trattati presentano un maggior numero di podociti se comparati con i topi di controllo. La presenza di cellule infuse nei glomeruli nel tempo, non è stata però accompagnata da differenziazione in podociti e produzione di nuovo collagene, escludendo quindi che il maggiore numero di podociti sia dovuto a proliferazione e differenziazione delle cellule amniotiche. Dati ottenuti tramite esperimenti in vitro confermano l’effetto positivo delle AFSC nella risposta delle cellule del glomerulo all’Angiotensina II , tramite diminuzione nell’espressione del suo recettore di tipo 1 (ANGTR1), esercitando un effetto benefico di protezione delle cellule del golomerulo, in particolare dei podociti. Contemporaneamente, una significativa variazione nel profilo immunostimolatorio è stata rilevata negli animali trattati, che presentavano una diminuzione significativa nell’espressione di molecole quali TNF-a, CCL, CXCL2 e M-CSF, coinvolte nella stimolazione di macrofagi di fenotipo M1, noti per la loro azione pro-fibrotica e pro-infiammatoria. Per concludere, le cellule staminali da liquido amniotico sono capaci di rallentare la progressione della Sindrome di Alport tramite mantenimento del numero di podociti, attivazione di macrofagi M2c pro-rigenerativi a discapito dei pro-fibrotici M1 e modulazione endocrina/paracrina dei segnali cellulari all’interno del rene; favorendo il mantenimento strutturale e funzionale del tessuto renale

Glomerular endothelial cell heterogeneity in Alport syndrome

Abstract Glomerular endothelial cells (GEC) are a crucial component of the glomerular physiology and their damage contributes to the progression of chronic kidney diseases. How GEC affect the pathology of Alport syndrome (AS) however, is unclear. We characterized GEC from wild type (WT) and col4α5 knockout AS mice, a hereditary disorder characterized by progressive renal failure. We used endothelial-specific Tek-tdTomato reporter mice to isolate GEC by FACS and performed transcriptome analysis on them from WT and AS mice, followed by in vitro functional assays and confocal and intravital imaging studies. Biopsies from patients with chronic kidney disease, including AS were compared with our findings in mice. We identified two subpopulations of GEC (dimtdT and brighttdT) based on the fluorescence intensity of the TektdT signal. In AS mice, the brighttdT cell number increased and presented differential expression of endothelial markers compared to WT. RNA-seq analysis revealed differences in the immune and metabolic signaling pathways. In AS mice, dimtdT and brighttdT cells had different expression profiles of matrix-associated genes (Svep1, Itgβ6), metabolic activity (Apom, Pgc1α) and immune modulation (Apelin, Icam1) compared to WT mice. We confirmed a new pro-inflammatory role of Apelin in AS mice and in cultured human GEC. Gene modulations were identified comparable to the biopsies from patients with AS and focal segmental glomerulosclerosis, possibly indicating that the same mechanisms apply to humans. We report the presence of two GEC subpopulations that differ between AS and healthy mice or humans. This finding paves the way to a better understanding of the pathogenic role of GEC in AS progression and could lead to novel therapeutic targets

Amniotic fluid stem cells prevent \u3b2-cell injury

Background aims. The contribution of amniotic \ufb02uid stem cells (AFSC) to tissue protection and regeneration in models of acute and chronic kidney injuries and lung failure has been shown in recent years. In the present study, we used a chemically induced mouse model of type 1 diabetes to determine whether AFSC could play a role in modulating b-cell injury and restoring b-cell function. Methods. Streptozotocin-induced diabetic mice were given intracardial injection of AFSC; morphological and physiological parameters and gene expression pro\ufb01le for the insulin pathway were evaluated after cell transplantation. Results. AFSC injection resulted in protection from b-cell damage and increased b-cell regeneration in a subset of mice as indicated by glucose and insulin levels, increased islet mass and preservation of islet structure. Moreover, b-cell preservation/regeneration correlated with activation of the insulin receptor/Pi3K/Akt signaling pathway and vascular endothelial growth factor-A expression involved in maintaining b-cell mass and function. Conclusions. Our results suggest a therapeutic role for AFSC in preserving and promoting endogenous b-cell functionality and proliferation. The protective role of AFSC is evident when stem cell transplantation is performed before severe hyperglycemia occurs, which suggests the importance of early intervention. The present study demonstrates the possible bene\ufb01ts of the application of a none genetically engineered stem cell population derived from amniotic \ufb02uid for the treatment of type 1 diabetes mellitus and gives new insight on the mechanism by which the bene\ufb01cial effect is achieved

Amniotic Fluid Stem Cells Inhibit the Progression of Bleomycin-Induced Pulmonary Fibrosis via CCL2 Modulation in Bronchoalveolar Lavage

<div><p>The potential for amniotic fluid stem cell (AFSC) treatment to inhibit the progression of fibrotic lung injury has not been described. We have previously demonstrated that AFSC can attenuate both acute and chronic-fibrotic kidney injury through modification of the cytokine environment. Fibrotic lung injury, such as in Idiopathic Pulmonary Fibrosis (IPF), is mediated through pro-fibrotic and pro-inflammatory cytokine activity. Thus, we hypothesized that AFSC treatment might inhibit the progression of bleomycin-induced pulmonary fibrosis through cytokine modulation. In particular, we aimed to investigate the effect of AFSC treatment on the modulation of the pro-fibrotic cytokine CCL2, which is increased in human IPF patients and is correlated with poor prognoses, advanced disease states and worse fibrotic outcomes. The impacts of intravenous murine AFSC given at acute (day 0) or chronic (day 14) intervention time-points after bleomycin injury were analyzed at either day 3 or day 28 post-injury. Murine AFSC treatment at either day 0 or day 14 post-bleomycin injury significantly inhibited collagen deposition and preserved pulmonary function. CCL2 expression increased in bleomycin-injured bronchoalveolar lavage (BAL), but significantly decreased following AFSC treatment at either day 0 or at day 14. AFSC were observed to localize within fibrotic lesions in the lung, showing preferential targeting of AFSC to the area of fibrosis. We also observed that MMP-2 was transiently increased in BAL following AFSC treatment. Increased MMP-2 activity was further associated with cleavage of CCL2, rendering it a putative antagonist for CCL2/CCR2 signaling, which we surmise is a potential mechanism for CCL2 reduction in BAL following AFSC treatment. Based on this data, we concluded that AFSC have the potential to inhibit the development or progression of fibrosis in a bleomycin injury model during both acute and chronic remodeling events.</p></div

A Novel Source of Cultured Podocytes

<div><p>Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated <i>in vitro</i> for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for <i>in vitro</i> studies. After <i>in vitro</i> differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time <i>in vitro</i>. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.</p></div

AFSC modulate AECII secreted CCL2 in BAL through proteolytic cleavage by transient MMP2 expression.

<p>(A) Representative Bis-Tris SDS-PAGE analysis of AECII fractions from control (n = 6), bleomycin-injured (n = 6) and bleomycin-injured with AFSC treatment at 2 hours post-bleomycin (n = 6), harvested at day 3, demonstrated a subtle 0.4 KDa shift of CCL2 to a putative inhibitory form. (B) Representative Bis-Tris SDS-PAGE analysis of BAL fluid containing 20 µg of total protein from control, bleomycin-injured and bleomycin-injured with AFSC treatment at 2 hours post-bleomycin, harvested at day 3 demonstrates increased MMP-2 expression in AFSC treated cohorts. (C–D) Measurement of endogenous active MMP-2 in BAL fluid at 6 and 22 hours of incubation demonstrating increased MMP-2 activity acutely following AFSC treatment, which is diminished 28 days post-bleomycin injury. (E) Representative gelatin zymography of BAL fluid containing 20 µg of total protein from control, bleomycin-injured and bleomycin-injured with AFSC treatment at 2 hours post-bleomycin, harvested at day 3 (F) Representative gelatin zymography of BAL fluid containing10 µg of total protein control, bleomycin-injured and bleomycin-injured with AFSC treatment harvested at day three as compared to BAL fractions from animals harvested at 28 days post-bleomycin injury (receiving AFSC at either day 0 or day 14 post-bleomycin injury), demonstrates transient nature of MMP2 increase.</p