Anna Maestroni

New susceptibility loci associated with kidney disease in Type 1 diabetes

Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ∼2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2×10−8) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0×10−9). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-β1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1×10−7), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN.

Persistent Renal Hypertrophy and Faster Decline of Glomerular Filtration Rate Precede the Development of Microalbuminuria in Type 1 Diabetes

Soon after the onset of type 1 diabetes, renal hypertrophy and hyperfiltration become manifest, particularly among patients who will subsequently develop diabetic nephropathy. Whether these early renal dysfunctions are involved in the pathogenesis of diabetic nephropathy is currently unclear. We evaluated, during the same day, kidney volume and glomerular filtration rate (GFR) in 146 patients with type 1 diabetes and normal renal function. All the individuals were then monitored for a mean of 9.5 ± 4.4 years for the development of microalbuminuria. Kidney volume and GFR were reevaluated in a subset of 68 patients 4 years after baseline. During follow-up, microalbuminuria developed in 27 of 146 diabetic patients. At baseline, kidney volume (312.8 ± 52.6 vs. 281.4 ± 46.1 vs. 236.8 ± 41.6 ml/1.73 m2, P < 0.05) but not GFR was increased in patients predisposed to microalbuminuria. Risk of progression was higher in patients with increased kidney volume (P = 0.0058). Patients predisposed to microalbuminuria showed a stable increase in kidney volume (P = 0.003), along with a faster decline of GFR (P = 0.01). Persistent renal hypertrophy and faster decline of GFR precede the development of microalbuminuria in type 1 diabetes. These findings support the hypothesis that renal hypertrophy precedes hyperfiltration during the development of diabetic nephropathy.

The role of angiogenesis in the development of proliferative diabetic retinopathy: Impact of intravitreal anti-VEGF treatment

Although cellular and molecular bases of proliferative diabetic retinopathy are only partially understood, it is evident that this complication of diabetes is characterized by the formation of new vessels inside the retina showing abnormal architecture and permeability. This process, if not controlled by selective laser photocoagulation, leads to irreversible retinal damages and loss of vision. Angiogenesis, that is, the condition characterized by the growth of new blood vessels originated from preexisting ones, was shown to have a major role in the pathogenesis of proliferative retinopathy and, as a consequence, intravitreal antiangiogenic injection was suggested as a feasible treatment for this disease. Here, we describe the different antiangiogenic approaches used to treat this disease along with the respective advantages and limitations when compared to laser treatment. Altogether, even though further and longer studies are still needed to clarify the best possible therapeutic protocol, the antiangiogenic treatment will reasonably have a future role in the therapy and prevention of proliferative diabetic retinopathy.

PD-L1 genetic overexpression or pharmacological restoration in hematopoietic stem and progenitor cells reverses autoimmune diabetes

Restoration of a PD-L1 defect in HSPCs reverses diabetes in NOD mice and thus may represent a potential cure for T1D. Stemming attacks on the pancreas In type 1 diabetes, autoreactive CD4 T cells attack and kill pancreatic β cells, disrupting insulin production. Many approaches have been taken to inhibit this process, but few have translated into real benefit for diabetic patients. Ben Nasr et al. demonstrate that hematopoietic stem and progenitor cells from NOD mice and diabetic patients express less PD-L1, which is a T cell inhibitory molecule. Induction of PD-L1 expression on stem cells reversed diabetes in NOD mice and inhibited human autoimmune responses in vitro. Either gene therapy or pharmacological modulation of PD-L1 on stem cells could be brought into the clinic, providing a new way to interrupt the autoimmune response and help people with diabetes. Immunologically based clinical trials performed thus far have failed to cure type 1 diabetes (T1D), in part because these approaches were nonspecific. Because the disease is driven by autoreactive CD4 T cells, which destroy β cells, transplantation of hematopoietic stem and progenitor cells (HSPCs) has been recently offered as a therapy for T1D. Our transcriptomic profiling of HSPCs revealed that these cells are deficient in programmed death ligand 1 (PD-L1), an important immune checkpoint, in the T1D nonobese diabetic (NOD) mouse model. Notably, the immunoregulatory molecule PD-L1 plays a determinant role in controlling/inhibiting activated T cells and thus maintains immune tolerance. Furthermore, our genome-wide and bioinformatic analysis revealed the existence of a network of microRNAs (miRNAs) controlling PD-L1 expression, and silencing one of key altered miRNAs restored PD-L1 expression in HSPCs. We therefore sought to determine whether restoration of this defect would cure T1D as an alternative to immunosuppression. Genetically engineered or pharmacologically modulated HSPCs overexpressing PD-L1 inhibited the autoimmune response in vitro, reverted diabetes in newly hyperglycemic NOD mice in vivo, and homed to the pancreas of hyperglycemic NOD mice. The PD-L1 expression defect was confirmed in human HSPCs in T1D patients as well, and pharmacologically modulated human HSPCs also inhibited the autoimmune response in vitro. Targeting a specific immune checkpoint defect in HSPCs thus may contribute to establishing a cure for T1D.

Detergent-Enzymatic Decellularization of Swine Blood Vessels: Insight on Mechanical Properties for Vascular Tissue Engineering

Small caliber vessels substitutes still remain an unmet clinical need; few autologous substitutes are available, while synthetic grafts show insufficient patency in the long term. Decellularization is the complete removal of all cellular and nuclear matters from a tissue while leaving a preserved extracellular matrix representing a promising tool for the generation of acellular scaffolds for tissue engineering, already used for various tissues with positive outcomes. The aim of this work is to investigate the effect of a detergent-enzymatic decellularization protocol on swine arteries in terms of cell removal, extracellular matrix preservation, and mechanical properties. Furthermore, the effect of storage at −80°C on the mechanical properties of the tissue is evaluated. Swine arteries were harvested, frozen, and decellularized; histological analysis revealed complete cell removal and preserved extracellular matrix. Furthermore, the residual DNA content in decellularized tissues was far low compared to native one. Mechanical testings were performed on native, defrozen, and decellularized tissues; no statistically significant differences were reported for Youngs modulus, ultimate stress, compliance, burst pressure, and suture retention strength, while ultimate strain and stress relaxation of decellularized vessels were significantly different from the native ones. Considering the overall results, the process was confirmed to be suitable for the generation of acellular scaffolds for vascular tissue engineering.

VEGF gene variability and type 1 diabetes: evidence for a protective role

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine originally described as an angiogenic factor. A number of reports have recently demonstrated that VEGF increases pancreatic islet survival after islet transplantation by stimulating angiogenesis and improving islet revascularization. Whether VEGF can protect from the autoimmune destruction of insulin-producing beta-cells that characterizes the development of type 1 diabetes is presently unknown. To clarify this issue, we studied the association of three polymorphisms of the promoter region of VEGF with type 1 diabetes in the Italian and the Finnish populations. The polymorphisms considered [C(-2578)A, G(-1190)A, and G(-1154)A] are known to modulate in vitro and in vivo VEGF expression. We found that VEGF promoter genotypes are associated with type 1 diabetes in both populations, but with different combinations. In Italian individuals, the -2578AA and -1190AA genotypes are associated with type 1 diabetes and accelerate its onset, while in Finnish individuals, -1154GG and -1190GG protect from type 1 diabetes and delay its onset. In conclusion, because the expected functional consequence of both genotype combinations is a reduced VEGF expression in diabetic patients, we propose a protective role of VEGF in the development of type 1 diabetes.

Increased clonogenic potential of circulating endothelial progenitor cells in patients with type 1 diabetes and proliferative retinopathy

To the Editor: The identification of endothelial progenitor cells (EPCs) along with the description of their essential role in postnatal neovascularisation has been a major achievement for stem cell biology [1]. In particular the finding that the clonogenic potential of EPCs is reduced in patients with high cardiovascular risk [2] represents the first step towards the recognition of the therapeutic effect of EPC transplantation in cardiac and limbic ischaemia [3, 4]. However, two harmful side effects are expected to be associated with EPC transplantation as a consequence of their physiological function: malignant tumour neovascularisation and the induction of proliferative diabetic retinopathy [1]. The contribution of EPCs to tumour neovascularisation has been clearly demonstrated in animal models and confirmed, although to a lesser extent, also in humans [5]. Concerning diabetic retinopathy, recent reports have demonstrated that EPCs play a major role in the retinal neovascularisation of a mouse model of proliferative retinopathy [6], and that the concentration of stromalcell-derived factor 1, the most important chemokine mobilising EPCs, increases with the severity of diabetic retinopathy in vitreous samples of patients affected by type 2 diabetes [7]. These studies are in some way in contrast to two independent reports performed in non-complicated patients showing that type 1 diabetes per se is associated with a reduced number and function of EPCs [8], and that EPCs in type 2 diabetes are impaired in adhesion, proliferation and tubulisation [9]. Finally, a recent study has demonstrated an increased number of CD34+ mononuclear cells in patients affected by type 2 diabetes with both non-proliferative and proliferative retinopathy [10]. Although of interest, these results cannot be easily compared with the ones described above as EPCs represent only one of the different classes of circulating cells staining positively for CD34+ [1]. Taken together, and from a largely speculative point of view, these apparently discrepant results can be explained by a unifying hypothesis. Taking into account the fact that EPCs have a physiological role in the rescue and maintenance of the existing retinal capillary bed [11], it is possible to hypothesise that the reduced number and clonogenic potential of EPCs found in non-complicated diabetic patients [8, 9] might predispose these patients to the later development of diabetic retinopathy. Once the damage is widespread and specific chemokines are produced by the suffering retina, the bone marrow would respond by increasing the production of EPCs, which at this stage, possibly because of the high ambient glucose and/or the badly damaged retina, would give rise to an incompetent neoangiogenesis. To test this hypothesis and verify the possible association between the clonogenic potential of circulating EPCs and proliferative diabetic retinopathy, we isolated EPCs from whole blood obtained from 11 patients with type 1 diabetes and untreated proliferative retinopathy, from 12 patients without retinopathy despite similar age, gender distribution and duration of type 1 diabetes, and from 11 ageand sex-matched non-diabetic controls. Patients affected by type 1 diabetes took no medication other than insulin. Clinical characteristics of subjects included in the study are described in Table 1. Patients were classified with respect to the status of diabetic retinopathy by means of stereoscopic fundus examination and fluorescent angiography. Patients considered for this study either had no signs of diabetic V. Asnaghi . G. Mazzolari . M. R. Pastore . A. Maestroni . D. Ruggieri . L. Luzi . G. Zerbini (*) Renal Pathophysiology of Diabetes Unit, Division of Nutrition–Metabolism, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy e-mail: g.zerbini@hsr.it

Circulating IGF-I and IGFBP3 Levels Control Human Colonic Stem Cell Function and Are Disrupted in Diabetic Enteropathy

The role of circulating factors in regulating colonic stem cells (CoSCs) and colonic epithelial homeostasis is unclear. Individuals with long-standing type 1 diabetes (T1D) frequently have intestinal symptoms, termed diabetic enteropathy (DE), though its etiology is unknown. Here, we report that T1D patients with DE exhibit abnormalities in their intestinal mucosa and CoSCs, which fail to generate in vitro mini-guts. Proteomic profiling of T1D+DE patient serum revealed altered levels of insulin-like growth factor 1 (IGF-I) and its binding protein 3 (IGFBP3). IGFBP3 prevented in vitro growth of patient-derived organoids via binding its receptor TMEM219, in an IGF-I-independent manner, and disrupted in vivo CoSC function in a preclinical DE model. Restoration of normoglycemia in patients with long-standing T1D via kidney-pancreas transplantation or in diabetic mice by treatment with an ecto-TMEM219 recombinant protein normalized circulating IGF-I/IGFBP3 levels and reestablished CoSC homeostasis. These findings demonstrate that peripheral IGF-I/IGFBP3 controls CoSCs and their dysfunction in DE.

Stem Cells and the Kidney: A New Therapeutic Tool?

In the past few years, a number of studies have shown that stem cells can be found in virtually every organ of the adult organism. The kidney is not an exception, and resident stem cells have been identified both in the papilla and along the tubules. Of interest, kidney-bound stem cells have been identified also in the bone marrow. When injected, both resident and bone marrow-derived stem cells are able to reach the injured renal tissue and, once there, to differentiate into renal cells. The evidence that, in humans, some of the acute and most of the chronic renal damages lead to ESRD suggests that in normal conditions, the reservoir of stem cells (considering both resident and bone marrow-derived stem cells) is insufficient to allow a major renal regeneration. Probably the number of stem cells that are ready to intervene in an adult kidney are sufficient to compensate for the normal cell turnover but largely inadequate to counteract a major injury. This is confirmed further by the finding that, even by transplanting a syngenic bone marrow in rats with ablation of 5/6 of the renal function, it is not possible to increase the life expectancy of the animals. Altogether, this evidence suggests that, to clarify the potentiality of a stem cell therapy for renal diseases, experiments that aim to clarify the ideal concentration of stem cells to be injected and to identify the best way of administration are needed.

Improved function of circulating angiogenic cells is evident in type 1 diabetic islet-transplanted patients

Circulating angiogenic cells (CACs) are vascular‐committed bone marrow‐derived cells that are dysfunctional in type 1 diabetes (T1D). Here we studied whether restoration of normoglycemia following islet transplantation is associated with better CAC function. We carried out a cross‐sectional study of 18 T1D patients, 14 insulin‐independent islet‐transplanted patients (ITA) and 14 healthy controls (C) evaluating in vivo and in vitro CACs viability and function. We found that the percentage of CACs in vivo did not differ among the three groups while the number of CAC colonies obtained from T1D, but not from ITA, was reduced compared to C (C = 7.3 ± 1.9, T1D = 0.9 ± 0.4 and ITA = 4.7 ± 1.9; p < 0.05 T1D vs. all). In vitro CAC migration/differentiation were similar, while in vivo an improved angiogenic ability of ITA compared to T1D was shown (capillary density: C = 93.5 ± 22.1, T1D = 19.2 ± 2.8 and ITA = 44.0 ± 10.5, p < 0.05 T1D vs. all). Increased apoptosis and lesser IL‐8 secretion were evident in CACs obtained from T1D compared to C and ITA. in vitro addition of anti‐hIL‐8 reduced the number of colonies obtained from C. Finally, T1D, but not ITA, had a lower endothelial‐dependent dilatation (EDD) compared with C. These data suggest that CAC function is altered in T1D and may be improved after islet transplantation.