Table 2 Application, advantages and limitations of technologies in studying glomerular cell crosstalk in DKD

CellPhoneDB: a new repository of ligands, receptors and their interactions, which is more accurate to represent heterogeneous complexes compared to others.

It has been used to analyze scRNA-seq data on a public dataset to identify the cell-cell crosstalk networks in DKD;

• Advantages:

Not mentioned.

• Limitations:

1) The difficulty of isolating glomerular cells from core needle biopsy specimens;

2) No effective standardized pipelines are available at present;

3) Unspecific available cell or unrevealed cell markers;

4) Requiring appropriate analytical and statistical methods, depending on the choice of computing tools and databases.

5) Relying on the affymetrix microarray dataset to infer the pathogenesis of diabetes nephropathy, and the human transcriptome data is limited.

[204, 205]

CellChat: a tool that can quantitatively infer and analyze cellular interaction networks from scRNA-seq data and performs well in predicting stronger interactions, which helps to narrow the range of interactions for further experimental validation.

It has been applied to discover the dysfunctional signaling and metabolic pathways in the thin endometrium, which provides insights into the mechanisms and treatment strategies of atrophic endometrium;

[206, 207]

ICELLNET: a global, versatile, biologically validated, and easy-to-use framework to analyze cell crosstalk from individual or multiple cell-based transcriptomic profiles.

1) It has been applied to three datasets generated based on RNA-seq, scRNA-seq, and microarray;

2) It has revealed autocrine IL-10 control of human dendritic cell communication with up to 12 cell types;

[208]

SMARTseq2 technology: a technology used for scRNA-seq analysis of glomerulus-associated cells.

1) It showed the proximal tubular cells were strongly affected at an early stage of IgA nephropathy and a potential glomerular-tubular cell-cell crosstalk pathway was identified;

2) Researchers also explored the early human DKD via single-cell transcriptomic landscape and showed the altered signaling networks in the diabetic glomerulus among GECs, podocytes and MCs;

[122, 202]

Spatial transcriptomics: an emerging new technology that provides quantitative gene expression data and visualization of mRNA distribution in tissue slices.

A study explored the cell-cell interactions and signaling networks in the cell subsets using ligand-receptor analysis and visualized potential interactions in different kidney cell types;

• Advantages:

It can quantify the mRNA levels of thousands of genes in the entire tissue slice and the mRNA levels in the tissue slice, allowing for the identification of direct connections between histological findings and gene expression.

• Limitations:

Not mentioned.

[136, 209]

Biomimetic systems in vitro

Kidney organoid

Kidney organoid: a self-organizing, three-dimensional aggregate of cells, derived from embryonic stem cells and induced pluripotent stem cells (iPSCs).

A study developed the first diabetic kidney organoid model to explore the potential mechanisms underlying the increased disease severity in patients with COVID-19 and diabetes;

• Advantages:

It may be the first time to allow in vitro research on Proteinuria, to realize mechanism and preclinical research.

• Limitations:

Different protocols and different measurements need to be standardized.

[210,211,212,213]

Microfluidic bioreactor

The microfluidic bioreactors: an emerging technology allowing continuous infusion of culture medium and secretion factors during the renal patterning process.

It has been used to coculture endothelial cells(ECs) with human kidney proximal tubule epithelial cells(HPTECs), which showed an increased upregulation of kidney-specific genes and suggested a potential bidirectional paracrine signaling;

• Advantages:

It shows promise in enhancing both renal organoid differentiation and cell type and also allows for the simultaneous generation of paracrine signals between two separated cell populations through shared media, allowing for the exchange of soluble factors and transient signals.

• Limitations:

Not mentioned.

[214]

Kidney on-a-chip

Glomerulus on a chip: a new in vitro organ model developed in the field of organ chip research based on microfluidic device technology.

Podocytes and endothelial cells are co-cultured in a microfluidic device in the glomerulus on the chip with physical stimuli that simulate physiological environments to enhance cell function and construct functional filtration barriers.

1) A new customized chip-based glomerular co-culture model has been developed and demonstrated that the co-culture affected the morphology and transcriptional phenotype of glomerular endothelial cells and podocytes;

2) A study used a glomerulus-on-a-chip microdevice that reconstitutes organ-level kidney functions to create a human disease model of early-stage DKD on a chip.

• Advantages:

The co-culture improves barrier function as a relevant functional readout for clinical translation, and this has been used to investigate specific glomerular paracrine pathways and unravel the role of glomerular crosstalk in glomerular path/physiology.

• Limitations:

Not mentioned.

[215, 216]