Acute kidney injury (AKI) is a health problem that affects about 13.3 million people every year, 85% of whom live in developing countries. At present, there are no effective pharmacological treatments that satisfactory can restore kidney function. It is therefore urgent and necessary to identify new approaches to support and improve renal function. The aim of the present study was to evaluate and compare the protective effect of conditioned medium (CM) and EVs obtained from umbilical cord (uc)-derived MSCs in a mice model of AKI induced by cisplatin and in vitro in cisplatin-damaged proximal tubular cells. In vivo, the effects of CM and EVs treatment on improving renal function in the AKI model have been evaluated by measuring BUN (blood urea nitrogen). In cisplatin-treated mice receiving saline, at 4 days we observed a significant increased BUN associated with a severe renal function impairment. Renal damage in cisplatin-treated mice was evidenced by the presence of tubular necrotic area, basement membrane denudation and deposition of intratubular CASTs, cylindrical structures formed from proteins secreted by tubular cells in pathological conditions. When cisplatin-treated mice were intravenously injected with CM or EVs, mice exhibited a significant improvement of renal function and structure after 4 days, as indicated by the reduction of BUN levels, tubular necrosis and CAST formation. Tubular cell proliferation, evaluated by labeling with the nuclear proliferation marker Ki67, was reduced in the cisplatin-treated mice but increased significantly upon administration of CM and EVs. Since the dysregulation of both functional and structural integrity of mitochondria is a critical early event responsible for tissue injury occurring during AKI, we evaluated the mitochondrial damage by analyzing the expression of proteins involved in the metabolism, biogenesis and oxidative stress of mitochondria. At 4 days after cisplatin, we found that the expression of the antioxidant enzyme SOD2 and the ATP synthase subunit ATP5I was reduced in the kidneys of cisplatin mice and that the treatment with CM and EVs re-established their expression. As well, CM and EVs administration increased mitochondrial mass evaluated by staining of voltage-dependent anion channel (VDAC) and induced the expression of PGC1α, a transcription factor involved in mitochondrial biogenesis. Since previous studies highlighted the critical role of the mitochondrial deacetylase SIRT3, involved in energy homeostasis and antioxidant defenses, in protecting mice from AKI, we investigated the effect of CM and EVs treatments on SIRT3 expression in the kidney. While cisplatin-treated mice exhibited low level of SIRT3 expression, a remarkable increase of this protein, as assessed by immunohistochemical analysis, was found following CM and EVs administration. In search of the mechanisms underlying the renoprotective effect of ucMSCs-derived CM and EVs, we performed studies using an in-vitro model of cisplatin damage on human renal proximal tubular epithelial cells (RPTEC). CM and EVs administration protected RPTEC from cisplatin damage by improving cell viability, increasing SOD2 expression, reducing mitochondrial fragmentation and preserving mitochondrial network. In conclusion, our data demonstrate that both CM and EVs derived from ucMSCs are able to improve renal function and structure preserving from cisplatin-induced renal damage. By these results, it is likely that EVs, a fraction of CM, are the main biological element of CM exerting the beneficial effects which pass through preservation of mitochondrial functional integrity. CM and EVs from MSCs could be an important alternative to cell treatments, with the advantage to be obtained more easily than stem cells and to have reduced safety concerns.
L’insufficienza renale acuta (AKI) è un problema di salute che colpisce circa 13,3 milioni di persone ogni anno e ad oggi non esistono trattamenti farmacologici efficaci che possano ripristinare in modo soddisfacente la funzionalità renale, per questo è urgente identificare nuovi approcci per supportare e migliorare la funzione renale. Lo scopo di questo studio è stato quello di valutare e confrontare l'effetto protettivo del medium condizionato (CM) e delle vescicole extracellulari (EVs) ottenuti da MSC derivate dal cordone ombelicale (uc) in un modello murino di AKI indotto da cisplatino e in vitro in cellule tubulari prossimali danneggiate dal cisplatino. In vivo, gli effetti del trattamento con CM ed EVs sul miglioramento della funzione renale nel modello AKI sono stati valutati misurando il livello dell’azotemia serica (BUN). Nei topi trattati con cisplatino, a 4 giorni abbiamo osservato un aumento significativo di BUN associato ad una grave compromissione della funzione renale evidenziato dalla presenza di area necrotica tubulare e deposizione di CAST intratubulari. Quando i topi però ricevevano CM o EVs, mostravano un significativo miglioramento della funzione e della struttura renale, come indicato dalla riduzione dei livelli di BUN, necrosi tubulare e formazione di CAST. La proliferazione delle cellule tubulari, valutata mediante il marcatore di proliferazione nucleare Ki67, si riduceva nei topi trattati con cisplatino ma aumentava significativamente dopo la somministrazione di CM e EVs. Poiché la disregolazione dell'integrità funzionale e strutturale dei mitocondri è un evento precoce responsabile del danno tissutale che si verifica durante l'AKI, abbiamo valutato il danno mitocondriale analizzando l'espressione delle proteine coinvolte nel metabolismo, nella biogenesi e nello stress ossidativo dei mitocondri. A 4 giorni dal trattamento con cisplatino, l'espressione dell'enzima antiossidante SOD2 e della subunità dell’ATP sintasi ATP5I era ridotta nei topi trattati con cisplatino e il trattamento con CM ed EVs ne ripristinava l'espressione. Inoltre, la somministrazione di CM ed EVs ha aumentato la massa mitocondriale valutata mediante l’analisi del canale anionico voltaggio-dipendente (VDAC) e ha indotto l'espressione di PGC1α, un fattore di trascrizione coinvolto nella biogenesi mitocondriale. Poiché studi precedenti hanno evidenziato il ruolo critico della deacetilasi mitocondriale SIRT3, coinvolta nell'omeostasi energetica e nelle difese antiossidanti, abbiamo studiato l'effetto dei trattamenti con CM ed EVs sull’espressione di SIRT3 nel rene. I topi trattati con cisplatino hanno mostrato un basso livello di espressione di SIRT3, mentre mediante analisi immunoistochimiche è stato riscontrato un notevole aumento di questa proteina dopo la somministrazione di CM ed EVs. Per caratterizzare i meccanismi alla base dell'effetto renoprotettivo di CM ed EVs derivati da ucMSC, abbiamo utilizzato un modello in vitro di danno da cisplatino su cellule epiteliali tubulari prossimali renali umane (RPTEC). La somministrazione di CM ed EVs ha protetto le RPTEC dai danni del cisplatino migliorando la vitalità cellulare, aumentando l'espressione di SOD2, riducendo la frammentazione mitocondriale e preservando il network mitocondriale. In conclusione, i nostri dati dimostrano che sia CM che EVs derivati da ucMSC sono in grado di migliorare la funzione renale e la struttura preservando dal danno renale indotto dal cisplatino. Con questi risultati, è probabile che le EVs, che sono una frazione del CM, siano il principale elemento biologico del CM che esercita gli effetti benefici che passano attraverso la protezione dell'integrità funzionale mitocondriale. CM ed EVs da MSC potrebbero rappresentare un'importante alternativa ai trattamenti cellulari, con il vantaggio di essere ottenuti più facilmente rispetto alle cellule staminali e di avere minori problemi di sicurezza.
Effetto renoprotettivo del medium condizionato e delle vescicole extracellulari derivati da cellule stromali mesenchimali in un modello murino di insufficienza renale acuta
KOSOVARI, MELISSA
2020/2021
Abstract
Acute kidney injury (AKI) is a health problem that affects about 13.3 million people every year, 85% of whom live in developing countries. At present, there are no effective pharmacological treatments that satisfactory can restore kidney function. It is therefore urgent and necessary to identify new approaches to support and improve renal function. The aim of the present study was to evaluate and compare the protective effect of conditioned medium (CM) and EVs obtained from umbilical cord (uc)-derived MSCs in a mice model of AKI induced by cisplatin and in vitro in cisplatin-damaged proximal tubular cells. In vivo, the effects of CM and EVs treatment on improving renal function in the AKI model have been evaluated by measuring BUN (blood urea nitrogen). In cisplatin-treated mice receiving saline, at 4 days we observed a significant increased BUN associated with a severe renal function impairment. Renal damage in cisplatin-treated mice was evidenced by the presence of tubular necrotic area, basement membrane denudation and deposition of intratubular CASTs, cylindrical structures formed from proteins secreted by tubular cells in pathological conditions. When cisplatin-treated mice were intravenously injected with CM or EVs, mice exhibited a significant improvement of renal function and structure after 4 days, as indicated by the reduction of BUN levels, tubular necrosis and CAST formation. Tubular cell proliferation, evaluated by labeling with the nuclear proliferation marker Ki67, was reduced in the cisplatin-treated mice but increased significantly upon administration of CM and EVs. Since the dysregulation of both functional and structural integrity of mitochondria is a critical early event responsible for tissue injury occurring during AKI, we evaluated the mitochondrial damage by analyzing the expression of proteins involved in the metabolism, biogenesis and oxidative stress of mitochondria. At 4 days after cisplatin, we found that the expression of the antioxidant enzyme SOD2 and the ATP synthase subunit ATP5I was reduced in the kidneys of cisplatin mice and that the treatment with CM and EVs re-established their expression. As well, CM and EVs administration increased mitochondrial mass evaluated by staining of voltage-dependent anion channel (VDAC) and induced the expression of PGC1α, a transcription factor involved in mitochondrial biogenesis. Since previous studies highlighted the critical role of the mitochondrial deacetylase SIRT3, involved in energy homeostasis and antioxidant defenses, in protecting mice from AKI, we investigated the effect of CM and EVs treatments on SIRT3 expression in the kidney. While cisplatin-treated mice exhibited low level of SIRT3 expression, a remarkable increase of this protein, as assessed by immunohistochemical analysis, was found following CM and EVs administration. In search of the mechanisms underlying the renoprotective effect of ucMSCs-derived CM and EVs, we performed studies using an in-vitro model of cisplatin damage on human renal proximal tubular epithelial cells (RPTEC). CM and EVs administration protected RPTEC from cisplatin damage by improving cell viability, increasing SOD2 expression, reducing mitochondrial fragmentation and preserving mitochondrial network. In conclusion, our data demonstrate that both CM and EVs derived from ucMSCs are able to improve renal function and structure preserving from cisplatin-induced renal damage. By these results, it is likely that EVs, a fraction of CM, are the main biological element of CM exerting the beneficial effects which pass through preservation of mitochondrial functional integrity. CM and EVs from MSCs could be an important alternative to cell treatments, with the advantage to be obtained more easily than stem cells and to have reduced safety concerns.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/13153