Amelioration of diabetic nephropathy in mice by a single intravenous injection of human mesenchymal stromal cells at early and later disease stages is associated with restoration of autophagy

Abstract

Background and aims Mesenchymal stromal cells (MSCs) a potentially effective disease-modulating therapy for diabetic nephropathy (DN) but their clinical translation has been hampered by incomplete understanding of the optimal timing of administration and in vivo mechanisms of action. This study aimed to elucidate the reno-protective potency and associated mechanisms of single intravenous injections of human umbilical cord-derived MSCs (hUC-MSCs) following shorter and longer durations of diabetes. Methods A streptozotocin (STZ)-induced model of diabetes and DN was established in C57BL/6 mice. In groups of diabetic animals, human (h)UC-MSCs or vehicle were injected intravenously at 8 or 16 weeks after STZ along with vehicle-injected non-diabetic animals. Diabetes-related kidney abnormalities was analyzed 2 weeks later by urine and serum biochemical assays, histology, transmission electron microscopy and immunohistochemistry. Serum concentrations of pro-inflammatory and pro-fibrotic cytokines were quantified by ELISA. The expression of autophagy-related proteins within the renal cortices was investigated by immunoblotting. Bio-distribution of hUC-MSCs in kidney and other organs was evaluated in diabetic mice by injection of fluorescent-labelled cells. Results Compared to non-diabetic controls, diabetic mice had increases in urine albumin creatinine ratio (uACR), mesangial matrix deposition, podocyte foot process effacement, glomerular basement membrane thickening and interstitial fibrosis as well as reduced podocyte numbers at both 10 and 18 weeks after STZ. Early (8 weeks) hUC-MSC injection was associated with reduced uACR and improvements in multiple glomerular and renal interstitial abnormalities as well as reduced serum IL-6, TNF-α, and TGF-β1 compared to vehicle-injected animals. Later (16 weeks) hUC-MSC injection also resulted in reduction of diabetes-associated renal abnormalities and serum TGF-β1 but not of serum IL-6 and TNF-α. At both time-points, the kidneys of vehicle-injected diabetic mice had higher ratio of p-mTOR to mTOR, increased abundance of p62, lower abundance of ULK1 and Atg12, and reduced ratio of LC3B to LC3A compared to non-diabetic animals, consistent with diabetes-associated suppression of autophagy. These changes were largely reversed in the kidneys of hUC-MSC-injected mice. In contrast, neither early nor later hUC-MSC injection had effects on blood glucose and body weight of diabetic animals. Small numbers of CM-Dil-labeled hUC-MSCs remained detectable in kidneys, lungs and liver of diabetic mice at 14 days after intravenous injection. Conclusions Single intravenous injections of hUC-MSCs ameliorated glomerular abnormalities and interstitial fibrosis in a mouse model of STZ-induced diabetes without affecting hyperglycemia, whether administered at relatively short or longer duration of diabetes. At both time-points, the reno-protective effects of hUC-MSCs were associated with reduced circulating TGF-β1 and restoration of intra-renal autophagy.

1. Introduction

The prevalence of diabetes mellitus (DM) has greatly increased in the past two decades, from 151 million people (4.6% of the global population) in 2000 to 537 million (10.5%) today. It is, furthermore, estimated that the number of people globally with DM in adulthood will increase to 643 million by 2030 (11.3%) and 783 million (12.2%) by 2045 [1]. Diabetic nephropathy (DN) is one of the most detrimental microvascular complications of DM, and occurs in both type 1 DM (T1DM) and type 2 DM (T2DM). Up to 40% of people with DM will eventually develop chronic kidney disease (CKD) as a result of DN and associated causes of kidney failure.

Current therapeutic strategies for DN primarily focus on glycemic control and blockade of the renin-angiotensin system, but they do not halt disease progression. Mesenchymal stromal cells (MSCs) have emerged as a promising cell-based therapy due to their immunomodulatory and regenerative properties. However, the optimal timing of administration and the in vivo mechanisms of action remain incompletely understood, hindering clinical translation. This study investigates the reno-protective effects of a single intravenous injection of human umbilical cord-derived MSCs (hUC-MSCs) in a mouse model of streptozotocin-induced diabetes, at both early (8 weeks) and later (16 weeks) stages of disease, and explores the associated mechanisms, particularly focusing on autophagy restoration.