Erosion of Immune Regulation in Diabetic Kidney Disease

Diabetic kidney disease (DKD) is a global health problem eventually affecting 30%–40% of individuals living with type 1 diabetes (T1D).1 Despite advances in diabetes management, the prevalence of DKD in this population has been relatively unchanged.2 DKD staging often relies on surrogate markers of kidney injury such as albuminuria,3 present when the glomerular filtration barrier has been compromised. Understanding what may permit the development of kidney injury or the progression of injury is clinically meaningful and would allow for personalized intervention strategies. Beyond a metabolic disease, DKD is an immune-influenced disease, with clear evidence that heightened immune activation is associated with future progression to ESKD in T1D.4 Understanding what promotes and, in this case, regulates inflammation is key. The immune system coordinates responses through complex interactions among multiple cell types. Of these, a subset of adaptive T cells, regulatory T cells (Tregs), serve the critical role of dampening or downregulating the overall immune response.5 Tregs sit at the interface of immune tolerance, tissue repair, and chronic inflammation. We currently do not fully appreciate the role that Tregs play in the progression of DKD in T1D, but understanding how this cell type functions may reveal mechanisms of disease progression, stratify risk, or serve as pharmacodynamic readouts for immunomodulatory strategies. In this issue of Kidney360, Rojas-Canales, Joglekar, and Ekinci et al. explore the relationship between different Treg profiles and the staging of albuminuria in DKD.6 This was an exploratory, cross-sectional study in 31 individuals comparing normoalbuminuria controls with individuals with T1D with or without albuminuria. Important to note, for this study, those with albuminuria were all at least stage 2. Peripheral blood mononuclear cells were collected and Tregs isolated. Complementary flow cytometry and transcriptomic analyses were used to profile these circulating Tregs. A relatively unbiased approach using Flow Self-Organizing Map was used to cluster Tregs based on flow cytometric phenotype profiling. The authors were able to define 14 clusters, of which three exhibited differential abundance across the disease spectrum. The Treg EMRA+ (effector memory cells re-expressing CD45RA) population significantly increased in individuals with T1D, largely driven by those with albuminuria. Conversely, the Treg central memory FOXP3-lo population and the subpopulation that were PI-16+ were decreased in those with T1D, and again, this difference was largely driven by those with albuminuria. EMRA+ cells are thought to be highly differentiated with reduced proliferative capacity. In conventional T cells, EMRA+ cells are often cytotoxic and upregulate inflammatory programs.7 In Tregs, EMRA-like cells are highly antigen experienced and possibly exhausted. This suggests chronic antigen exposure, ongoing immune activation, and a potential loss of regulatory reserve. FOXP3 is the canonical transcription factor governing Treg differentiation. Tregs that are FOXP3-lo are thought to be less suppressive and less stable in their regulatory phenotype.8 The observation that PI-16+ Tregs are decreased is particularly interesting. PI-16 marks cells that are quiescent or resting but poised to respond and proliferate.9 This is consistent with a loss of regulatory reserve in T1D individuals with albuminuria. The authors then performed RNA sequencing to further understand the phenotype of the Tregs in these individuals. They observed that in albuminuric DKD, there is an activation of signaling programs related to cytokine signaling, immune response activation, and cell-to-cell communication, indicating that these cells are indeed not dormant but are active and engaged. In addition, pathways related to metabolism, stress, autophagy, and trafficking were noted. Taken together, these reinforce that Tregs in patients with DKD are activated but are not able to maintain a durable regulatory function. This leads us to question what the cells are doing and to whom they are communicating. CellChat, a tool for interrogating signaling potential based on known ligand-receptor pairs, was used with the present transcriptomic dataset and existing kidney single-cell RNA sequencing data. This analysis revealed that signaling potential through LRRC4B to be downregulated in DKD Tregs, while potential to signal through TGM2 to be upregulated. This analysis is largely hypothesis-generating, and these results should be investigated further in more targeted, mechanistic studies in the future. To further understand what may be regulating changes in transcript expression, the authors used a microRNA (miRNA) profiling array to determine whether there were any relationships between validated miRNAs in these circulating Tregs and eGFR and disease grouping, an indicator of renal function, in these individuals. There was no single dominant miRNA associated with DKD, but there were a handful that were significantly different between T1D and controls. This study used a methodologic triangulation using flow cytometry, RNA transcriptomics, and miRNA array in the same cells to provide a clear picture of the dynamic differences in Tregs in patients with T1D and how these cells may also be important in the further progression to DKD. A future replication cohort in a larger diverse population would validate key Treg clusters, the top RNA pathway enrichments, and the top-ranked miRNA signatures. It is important to note that this was a cross-sectional study and therefore difficult to directly ascribe the observed differences to causality. A longitudinal study sampling Tregs across the progression of albuminuria and the decline in eGFR would be important to determine whether these signatures can be used to predict response to standard-of-care therapies. Further mechanistic studies in preclinical or in vitro systems would be needed to truly determine causality. Despite advances in care, DKD in T1D remains a prevalent complication. This study reframes how we may qualitatively understand the role of inflammation in this context, broadening the framework beyond enhanced activation of proinflammatory pathways to include a reduction in regulatory reserve to dampen or restrain sustained immune activation. Specific and unique subpopulations associated with T1D were identified, providing a new target for further investigation. Defining immune state and the profile of circulating cells, as was done in this study, may inform risk stratification and therapeutic targeting, with further research determining whether loss of regulatory reserve marks a critical transition in DKD progression.