Diabetic kidney disease (DKD) (1, 2) is one of the most important microvascular complications of diabetic mellitus and a leading cause of renal function loss and end-stage renal disease (ESRD). Nevertheless, the mechanism of DKD is complex and not fully elucidated.

Renal parenchymal cells, resident immune cells, and infiltrating immune cells orchestrate active cell-to-cell interactions, thereby contributing to the development of DKD. Previous studies (3, 4) have revealed the significance of cell communication in the pathogenesis of DKD. Dmike et al. (5) deciphered the tubulovascular cross-talk mediated by vascular endothelial growth factor A. Wu et al. (6) found that high glucose-induced glomerular endothelial cell-derived exosomes trigger the epithelial-mesenchymal transition and podocytes dysfunctions. Nespoux et al. (7) reviewed the renoprotective mechanism of sodium-glucose cotransporter 2 (SGLT2) inhibitors, which downregulate tubular reabsorption-induced early glomerular hyperfiltration. Garson et al. (8) revealed that podocytes mediate glomerular transendothelial albumin passage via endothelin-1-regulated heparanase expression. Lai et al. (9) revealed the importance of cell-to-cell communication between different glomerular cell types in DKD using podocyte and endothelial-specific elimination of bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) expression in streptozotocin-induced diabetic endothelial nitric oxide synthase (eNOS)-deficient and control eNOS-deficient mice. Unfortunately, these studies merely highlight the limited types of cell-to-cell interactions in DKD, and detailed insight into cell communication in DKD is lacking.

Single-cell sequencing (scRNA-seq) is a technological evolution and provides unprecedented insight into cell communication (10–12). In experimental studies of renal diseases (13–16), scRNA-seq technology furthers the understanding of the mechanisms and cell-to-cell interactions involved in disease pathogenesis. In human kidneys (17–19), scRNA-seq has helped to identify novel cell types, reveal potential mechanisms, and investigate cell communication from distinct aspects. Lake et al. (17) primarily deciphered the cell types, distributions, cell differentiation, and cell-to-cell interactions based on integrins in normal human kidneys. A study on lupus nephritis (18) highlighted the immune cells, immune-associated mechanisms, and cell-to-cell interactions based on the functions of chemokines and cytokines. These studies merely described the specific patterns of cell cross-talk. Moreover, cross-talk has not been fully elucidated in DKD (19).

In this study, we provided an overall perspective of cell communication in human DKD based on a single-nucleus transcriptomic dataset. In addition, the relationships between hub genes involved in cell communication and renal function were determined. This study of cell communication between individual cells based on ligand-receptor interactions in DKD may facilitate the discovery of novel mechanisms, biomarkers, and drug targets to better serve patients.

Cell cross-talk participates in the development of DKD. Based on a snRNA-seq dataset and two bulk gene datasets, we provided new insight into cell communication and genes involved in DKD.

In 2019, Fu et al. (14) primarily performed scRNA-seq analysis in streptozotocin-induced diabetic endothelial nitric oxide synthase (eNOS)-deficient and control eNOS-deficient mice and revealed increased infiltrating macrophages in glomeruli, dynamic alterations in the pattern of expressed genes in glomerular endothelial cells and mesangial cells of DKD and control mice, and variable responses of individual cells. In addition, this study preliminarily analyzed the cell cross-talk between glomerular individual cells based on ligand-receptor analysis. In the same year, Wilson et al. (19) performed snRNA-seq analysis in DKD for the first time and revealed the significance of increased potassium secretion and angiogenic and possible ligand-receptor signaling pathways in glomerular individual cells. Regretfully, the former studies have limitations. First, only the cell-to-cell interactions between glomerular individual cells were reported. Second, the subunit architecture of ligands and receptors, which accurately represents heteromeric complexes, was not taken into account. This is crucial, as cell cross-talk interacts mediated by multisubunit protein complexes instead of the binary representation used in the previous study (19). Third, the relationship between cell communication-related genes and clinical indicators was not elucidated. In this study, we analyzed cell cross-talk in all individual cells in human kidneys using a novel method (20), which accurately represents heteromeric complexes, and revealed the relationship between hub genes involved in cell cross-talk and renal function. This study reveals further mechanisms and indicates novel biomarkers and potential therapeutic targets.

Cell-to-cell interactions in the same cell type play important roles in both nondiabetic and diabetic kidneys. Podocyte-to-podocyte interactions possibly maintain the basic function of the kidney, which needs to be further studied. In DKD, mesangial cell proliferation contributes to increased internal communication via integrin pathways. Moreover, the internal communication of glomerular endothelial cells via CD74-APP is increased. CD74 (23) is upregulated in diabetic retinopathy with proliferative lesions, and APP (24, 25) is increased in diabetic microvascular complications, indicating a potential mechanism by which the angiogenesis mediated by CD74 and APP participates in DKD development and progression.

Cell-to-cell interactions limited into glomerular or tubular individual cell types are changed in DKD. In glomeruli, podocyte-expressed FGF1-mediated cell cross-talk is decreased, which is consistent with the former report that the protein levels of glomerular FGF1 are decreased in DKD (26). We found that the levels of glomerular FGF1 are positively related to the levels of GFR, suggesting that FGF1 may contribute to the development of DKD. Previous studies (26, 27) showed that FGF1 supplementation ameliorates DKD due to anti-inflammatory and antioxidative stress mechanisms, suggesting that FGF1 is a renoprotetctive factor and an encouraging therapeutic target in DKD. In tubules, PCT-expressed NRP1-mediated cell cross-talk was increased, and NRP1 expression was upregulated (Supplementary Figure 1). We also found that FGF1 expression is negatively associated with GFR, suggesting a potential NRP1-regulated mechanism in DKD. However, a previous study (28) showed a low density of NRP1 expression and downregulated NRP1 levels in renal fibrosis, which is contradictory to our findings. To elucidate the role of NRP1 in DKD, more samples including different disease statuses need to be collected, and further studies are needed.

In the DKD groups, we deciphered active glomerulotubular cell-to-cell interactions. In a tubule-centric view (29), the upregulation of SGLT1 and SGLT2 in PCT induced the alteration of glomerulotubular communication and hyperfiltration, explaining the renoprotective mechanisms of the novel agent SGLT2 inhibitor in DKD treatment (30–32). Nevertheless, the levels of SGLT1 and SGLT2 were not significantly altered in PCT in this study. Individual differences and different disease statuses may lead to contradictory results.

Finally, cell identification revealed immune cells in kidneys. Interestingly, DKD with high IFTA (interstitial fibrosis and tubular atrophy) samples contributed all identified B cells, suggesting the crucial role of B cells in DKD (Supplementary Figures 2A,B). We performed DEG analysis in immune cells and found that CD20 expression was significantly increased in the DKD groups (Supplementary Figure 2C). Some studies have revealed that targeting CD20 achieves therapeutic effects in renal diseases. An experimental study showed the protective effects of CD20 antibodies in lupus mice (33). Clinical evidence provides that CD20 antibodies achieve therapeutic effects in recurrent focal segmental glomerulosclerosis (34) and membranous nephropathy (35). However, the role of B cells in DKD has not been fully elucidated (36). Increased IgG+ B cells were found in the glomeruli of diabetic NOD mice when compared with those in nondiabetic mice, suggesting that B cells may contribute to the pathogenesis and prognosis of DKD (37). In DM patients, Zhang et al. (38) found increased CD38+CD19+ B cell counts in the peripheral blood. Moreover, the number of CD38+CD19+ B cells was positively correlated with the 24 h urinary protein concentration and was reduced after treatment. Taken together, these findings suggest that B cells may participate in the development of DKD. We speculate that agents targeting B cells or CD20 antibodies may have promising therapeutic effects in DKD, which needs to be further studied in future research.

Regretfully, snRNA-seq has its own limitation in capturing immune cell populations due to nanodrop technology, and frozen or optimal cutting temperature compounds may lead to the loss of information. Thus, the cross-talk between immune cells and renal parenchymal cells in DKD was not fully deciphered in this study. In addition, larger sample sizes and conditional knockout models are needed to better elucidate cell cross-talk and its further mechanism in DKD.

In summary, this study revealed cell cross-talk based on snRNA-seq and the associations between genes involved in cell communication and renal function in DKD. In DKD, cell-to-cell interactions via integrin pathways are increased, mesangial cells are stimulated and glomerulotubular communication is strongly enhanced. The level of glomerular FGF1 is positively associated with the level of GFR, while the levels of glomerular NRP1, tubular COL4A1, and tubular NRP1 are negatively associated with the level of GFR. This study furthers our understanding of cell cross-talk in DKD and reveals novel mechanisms, new biomarkers, and potential therapeutic targets to benefit patients.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

YW designed the study, performed the data analysis, and wrote the first draft. XG and ZJ revised the draft. AL and ML helped improve the methodology. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.