In the early stages of DKD, there is a loss of podocytes and mesangial cells with podocyturia. Secretion of Semaphorin 3C (SEMA3C) from MCs initiated by hyperglycemic conditions causes endocytosis of microtubules and increases the glomerular permeability by altering GEC permeability characteristics through the actions of Neuropilin-1 (NRP1) and Neuropilin-2 (NRP2) (17). These processes in early DKD exemplify one of the many instances of cell-to-cell cross-talk that occurs between MCs and GECs and initiates early renal damage. Persistent hyperglycemia in DKD activates NADPH oxidases in GECs, which leads to increased ROS, which triggers PC activation and mesangial expansion and contributes to proteinuria and progression of DKD. Activation of transforming growth factor beta (TGF-β) in GECs may play a role in mesangial expansion that ultimately leads to mesangial sclerosis. With continued expansion, mesangium expands into GECs, decreasing the filtration area and GFR. This constitutes a major structural change in DKD with important functional implications. Exosomes from high glucose (HG)-treated GECs upregulate the expression of genes for Fibronectin and Collagen IV in MCs that contribute to mesangial expansion (18). Another factor that contributes to mesangial expansion is the activation of the mechanistic target of rapamycin complex (mTORC) in PCs (19). Thus, a cross-talk involving all three major glomerular cells (GECs, PCs, MCs) is involved in mesangial expansion. In cell culture studies, GECs exposed to HG upregulated Endothelin 1 (ET-1) secretion, which binds to the Endothelin A receptor on MCs and promotes MC proliferation mediated by the RhoA-Rho kinase (ROCK) pathway (20). Furthermore, downregulation of genes related to the extracellular matrix (ECM) was observed in podocytes co-cultured under HG conditions along with GECs but not when PCs were cultured alone, underscoring the role of GEC-PC cross-talk in mediating this effect (21). Recent studies support the upregulations genes Early growth response-1 (EGR-1) as well as EGR-2 and EGR-3 in GECs and PCs under HG conditions, genes that mediate expression of mesangial matrix proteins, supporting the GEC-PC-MC cross-talk in early DKD (22).

In the later phases of DKD, structural alterations culminate in glomerular and tubulointerstitial fibrosis. Several functional disturbances in the glomeruli that occur in the late DKD and result in renal fibrosis are mediated by an intricate and continuous cross-talk between all the major cells in the kidney. HG-treated GECs secrete exosomes that deliver circular RNA (circRNA) and microRNA (miRNA) to MC, which then produce ECM proteins and initiate renal fibrosis. Loss of glucocorticoid receptor (GR) in the podocytes leads to abnormal activation of the Wnt signaling cascade and affects the integrity and function of GECs, leading to functional decline in late DKD (23). Using ligand analysis, recent studies confirmed closer interactions between fibroblasts with all major cell types in the nephrons from DKD subjects compared to diabetic subjects with no kidney disease or non-diabetic subjects, an observation linked to the interplay of chemokines from fibroblasts with various renal cells (24). These findings establish the close cell-to-cell interactions between various renal cells and renal fibrosis. Figure 1 summarizes the major communication amongst the major renal cell types.

The following section describes specific examples of cross-talk between different glomerular cells in DKD.

Mesangial cells participate in glomerular cell-to-cell cross-talk through vascular endothelial growth factor (VEGF) signaling pathways. Overexpression of VEGF-A in PCs has been shown to affect MC markers, such as desmin, α-SMA, PDGFR-β, and mesangial expansion (25). Furthermore, VEGF-A from PCs has been shown to affect MC differentiation and proliferation (26). Another significant cross-talk that occurs between PCs and MCs involves ET-1-mediated effects. Experimental studies indicate that inhibition of ET-1 receptors or deletion of ET-1 receptors A and B in PCs inhibits MC proliferation and mesangial expansion through inhibition of Wnt-β catenin and NF-κB signaling mediated by ET-1 receptors (27).

Endoplasmic reticulum stress (ERS) has been shown to contribute to the development and progression of DKD. In vitro studies have shown that elutes from MC cultures in HG inhibited ER-associated degradation (ERAD)-related proteins such as Derlin-1 and Derlin-2 in PC, leading to ERS, which amplified podocyte loss, albuminuria, and DKD progression (28).

Additional evidence of PC-MC cross-talk comes from the cell culture studies where HG-exposed MCs were shown to secrete copious exosomes containing TGF-β1, which upregulate TGF-β1 receptor expression on PC. These effects promote PC apoptosis through the TGF-β1-PI3K/Akt signaling pathway (29).

Vascular endothelial growth factor A (VEGF-A), an angiogenic factor produced by podocytes, affects the proliferation and permeability of glomerular capillaries acting through VEGF receptors (VEGFR) 1 and 2 expressed on the surface of GECs. Initially, there is an increased production of VEGF from PCs and increased expression of VEGFR-1 and-2 in GECs in early DKD. These changes result in increased production of NO in the glomerulus through increased eNOS expression and contribute to hyperfiltration and microalbuminuria (30). The increased VEGF-A in early DKD from PCs acting on GECs promotes neo-angiogenesis, which delays fibrogenic processes in the initial stages of DKD. However, as the disease advances, there is a decline in both VEGF-A from PCs and VEGFR-1 and-2 expression in GECs, leading to decreased glomerular eNOS and NO levels contributing to renal fibrosis (6).

Activation of the TGF-β1 receptor on PCs leads to the synthesis of Endothelin-1 (ET-1) from pre-ET-1 through the Smad signaling pathway and acting through ET-1 receptor A located on GECs results in mitochondrial oxidative stress in GECs (31). This illustrates another example of PC-GEC cross-talk leading to podocyte-mediated endothelial cell injury in DKD.

Mitochondrial oxidative stress has also been incriminated in the progression of DKD. Experimental studies examining the effects of HG on GECs demonstrated evidence of significant mitochondrial oxidative stress with increased production of mitochondrial superoxide and 8 hydroxy deoxyguanosine (8 OHdG) and decreased eNOS activity with consequent endothelial dysfunction. Supernatants from such culture studies, when transferred to PCs in culture, promoted PC apoptosis, an effect that was abolished by the elimination of mitochondrial superoxide by TEMPO (32). These observations imply a significant interaction between GECs and PCs, mediated by mitochondrial oxidative stress.

Another instance of PC-GEC cross-talk is illustrated by the effects of NO derived from GECs through the action of eNOS, which affects the structural and functional integrity of PCs since eNOS deficiency results in PC apoptosis (33). Exosomes secreted from HG-treated GECs containing TGF-β mRNA are endocytosed by PCs, which triggers EMT through activation of the Wnt-β catenin signaling pathway (34). There is additional evidence from animal studies about GEC-PC cross-talk involving TGF-β signaling in DKD mediated by bone morphogenic protein-activin membrane-bound inhibitor (BAMBI). BAMBI negatively regulates TGF-β signaling in both GECs and PCs and, therefore, in GEC-BAMBI−/− diabetic mice, podocyte injury and loss were observed, indicating a complex interplay between these cells mediated by TGF-β/Smad signaling pathways (35).

Nitric oxide (NO) has been shown to play a significant role in the onset and advancement of DKD. Nitric oxide is produced from L-arginine, which is the sole substrate for NO generation, a reaction catalyzed by nitric oxide synthases (NOS). All cells in the entire nephron are capable of producing NO, although in variable capacities. The NO production in GECs and podocytes and tubular epithelial cells (TECs) is generally mediated by constitutive NOS (cNOS or eNOS), while in MCs, the reaction is mediated by inducible NOS (iNOS), which requires inflammatory cytokines such as λ-Interferon or lipopolysaccharide or TNF-α to stimulate MCs (30). In the early phases of DKD, there is increased intrarenal NO primarily from iNOS mediated from MCs (with modest contribution from eNOS activation), which leads to afferent glomerular arteriolar vasodilation. Together with angiotensin-mediated efferent arteriolar constriction, the intraglomerular pressure increases, leading to hyperfiltration and microalbuminuria. With progressive DKD, the mitochondrial oxidative stress with increased ROS generation and NO quenching by AGE decreases NO availability, leading to endothelial dysfunction in GECs.

In vitro studies also supported a fine cross-talk between GECs and MCs through NO-dependent mechanisms. In co-cultures involving GECs and MCs, incubation with bradykinin increased intracellular cyclic-GMP (cGMP) in MCs in a NO-dependent pathway. As bradykinin stimulates eNOS present in GECs, the NO released as a result of such eNOS activation leads to cGMP generation in MCs (36). These observations confirm the NO-mediated cross-talk between GECs and MCs.

An important example of GEC-MC cross-talk involves the TGF-β signaling pathway. HG-treated GECs secrete exosomes containing TGF-β mRNA, which are then endocytosed by MCs and, through Smad signaling, result in MC proliferation, mesangial expansion, and secretion of ECM proteins (18). Exosomes containing circular RNA from HG-treated GECs, when taken up by MCs, induced the expression of proteins that caused mesenchymal transdifferentiation (37). Another evidence of GEC-MC cross-talk also involves TGF-β signaling. Inhibition of MC expression of integrin αvβ8 in experimental diabetes resulted in decreased binding of latent TGF-β and releasing excess TGF-β to reach GECs, resulting in GEC apoptosis (38).

There are several examples of PC-TEC cross-talk in DKD. A major instance of such interaction involves bone morphogenic proteins (BMPs) that belong to the TGF-β superfamily group. While TGF-β is a fibrogenic factor, BMPs counterbalance the TGF-β effect. Gremlin is an antagonist of BMP that is overexpressed in TECs, MCs, and PCs when exposed to HG. Animal studies have shown that in transgenic diabetic mice for overexpressing tubule-specific Gremlin, PC damage was exaggerated with severe foot process effacement and podocyte loss (39). These observations suggest tubular overexpression of Gremlin resulted in a cross-talk with PCs, leading to major podocytopathy.

Another example of cross-talk between TECs and PCs involves Bcl2 interacting mediator of cell death (Bim), an apoptotic mediator. Culturing proximal TECs (PTECs) in HG increased Bim expression. Xu et al. showed that when PCs and PTECs were co-cultured, increased Bim expression in PTECs resulted in significant damage to PCs due to increased synaptophysin and F-actin expression. Furthermore, if the PTECs were transfected with Lenti-Bim shRNA and treated with HG, the changes in cocultured in PCs described earlier disappeared, confirming that the cross-talk between PTECs and PCs is mediated by Bim (40).

Hasegawa et al. showed in streptozotocin (STZ)-treated transgenic mice expressing proximal tubule-specific Sirtuin 1 (Sirt1, a nicotinamide adenine dinucleotide-dependent deacetylase, which counters the effects of NF-κB, TGF-β, and p53 signaling) reduced albuminuria and showed better renoprotection compared with control STZ mice (41). Furthermore, such transgenic mice also demonstrated decreased Claudin-1 expression in PCs and parietal epithelial cells of glomeruli, minimizing the epithelial cell damage. Nicotinamide mononucleotide (NMN) upregulated Sirt1 expression in TECs (42) and diminished the Claudin-1-mediated effects on PC damage, thereby reducing albuminuria. The authors concluded that these observations support the interaction of TECs and PCs in the regulation of Claudin-1 expression and effects in PCs mediated by Sirt1.

There is limited literature investigating the cross-talk between TECs and MCs in the context of DKD. Previous experimental evidence supported the role of endothelial cell-myofibroblast transdifferentiation (MFT) in renal fibrosis (43). Recent studies involving co-cultures of PTECs (HK-2 Cells) with MCs in HG media have shown that exosomes from TECs expressing miR-92a-1-5p promoted MFT in MCs (44). The role of miR-92a-1-5p in this cross-talk and the role in DKD was confirmed by the observation of high levels of this exosomal miRNA in the urine of patients with advanced DKD.

A major mediator of cross-talk between TECs and GECs is the Angiopoietin/Tie signaling system (45). Angiopoietin 1 (Ang 1) is predominantly distributed in the TECs and has salutary effects on the renal endothelial cell integrity and function, while Angiopoietin 2 (Ang 2) and Tie (a co-receptor for Ang 1 and 2) are expressed in the GECs and have detrimental effects on the function and integrity of capillaries. Immunohistochemical studies have suggested that as the DKD progresses, Ang 1 expression decreases (after an initial increase) steadily while Ang 2 increases. Thus, the endothelial preservation by the interaction of Ang 1 from TECs on GECs decreases while increasing the Ang 2/Ang 1 ratio promotes progressive endothelial injury with advancing DKD (46).

Fibroblasts are proposed to be the primary sources of myofibroblasts instigating renal fibrosis in DKD. Fibroblast-to-myofibroblast transition (FMT) is the process of activated fibroblasts differentiating into myofibroblasts. Fibroblasts are stromal cells that help repair damaged tissues. They are activated by mechanical stress and paracrine signaling molecules.

In the context of DKD, renal cellular injury following the metabolic insults results in producers of factors required for promoting regeneration and tissue healing, such as TGF-β, platelet-derived growth factor (PDGF), hedgehog, and Wnt ligands. However, persistent injury causes sustained production of these ligands, resulting in paracrine fibrogenic effects (56).

The most prominent and best-studied fibroblast-activating signal is the cytokine TGF-β. Injured epithelia, macrophages, fibroblasts, endothelial cells, and mesangial cells are all potent sources of TGF-β. Active TGF-β1 binds its receptors and activates the canonical TGF-β/Smad signaling pathway in fibroblasts, resulting in the upregulation of its target genes, including α-SMA, ECM molecules, and integrin receptors. Meng et al. reviewed the various cell-type and context-dependent roles played by TGF-β in renal fibrosis (57). Wu et al. recently showed that macrophages undergoing MFT increase the expression of TGF-β, and the conditioned media from these cells activates fibroblasts-to-myofibroblast transition (58). Some integrin receptors, like integrins-αvβ5 and-αvβ6, participate in supplying more of the activated TGF-β. They bind to latent TGF-β, making their activating proteolytic cleavage sites available, thus releasing more of the activated TGF-β into the microenvironment (59–61). This results in a feed-forward loop whereby an activated fibroblast induces FMT in its vicinity. However, other integrin receptors, e.g., integrin-αvβ8 expressed by mesangial cells, have been suggested to protect their neighboring GECs by sequestering TGF-β in its latent form (38). Another connection between TGF-β and integrin signaling is via Yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ). YAP and TAZ are transcription co-activators and, therefore, control gene expression. Stiff ECM promotes the nuclear localization of YAP/TAZ and where they interact with the activated Smad complex, resulting in α-SMA transcription (61).

PDGFs are another set of repair/fibrogenic molecules long known to be growth factors for fibroblasts and promoters of renal MFT. In particular, platelet-derived growth factor-BB (PDGF-BB) induces renal tubulointerstitial cell proliferation, myofibroblast formation, and tubulointerstitial fibrosis in a dose-dependent manner (62). Another growth factor signaling utilized by injured renal cells to activate fibroblasts is the Hedgehog pathway. Upon binding the secreted Hedgehog ligands, the membrane receptor Patched releases the protein smoothened (Smo), which facilitates the transcription of Gli targets. Reporter experiments in mice show that while the ligands are primarily expressed in tubular epithelial cells, the receptor Patches and targets of Gli are exclusively expressed in perivascular fibroblasts and pericytes, suggesting an epithelial-fibroblast paracrine cross-talk via hedgehog signaling (63).

Another group of intercellular communicators driving the MFT of fibroblasts are Wnts. Injury-induced tubular secretion of profibrotic factors has been suggested to be a major contributor to renal fibrosis following acute kidney injury. In assessing the identity of the factors and the necessity of tubular injury, Maarouf et al. found that Wnt1 produced by proximal tubules was sufficient to cause interstitial myofibroblast activation, proliferation, and increased ECM production without injury or, remarkably, any signs of inflammation (64). Constitutive activation of canonical Wnt/β-catenin signaling in murine interstitial pericytes and fibroblasts exhibits spontaneous myofibroblast differentiation even in the absence of injury. Activated myofibroblasts strongly express Wnt4 during renal fibrosis, suggesting autocrine activation in addition to paracrine signaling. However, Wnt4 from interstitial myofibroblasts is not necessary for MFT, probably because of the compensation of other Wnt ligands and Wnt4 from other cells, such as the collecting ductal cells (65).

In addition to the affected secreted factors, signaling pathways, and transcription factors, non-coding RNAs are being recognized to play regulatory roles in the changing renal microenvironment of DKD. In an article published earlier this year, Xing and Rodeo reviewed the emerging roles of non-coding RNAs, including microRNAs, long non-coding RNAs, and circular RNAs, in FMT and fibrotic diseases (66). These include both pro-fibrotic and antifibrotic regulatory RNAs, opening up new therapeutic possibilities. Not only do non-coding RNAs regulate the cellular environment, they are also directly responsible for intercellular cross-communication. For example, high-glucose stimulated proximal TECs secrete exosomes enriched with microRNA (miR)-92a-1-5p. Glomerular MCs take up these exosomes. miR-92a-1-5p then induces ER stress and myofibroblast transdifferentiation of these MCs, resulting in DKD progression (44). Similarly, glucose-stimulated GECs produce more exosomes enriched in circular RNAs, which activate MFT in glomerular MCs (37).

FMT is perhaps the primary contributor of myofibroblasts in fibrotic diseases, but the process is also vital to tissue repair and healing. Targeting this process for fibrosis prevention is going to need more research. For example, TGF-β inhibitors have not worked very well in the clinical setting yet, probably because of TGF-β’s pleiotropic nature and its importance in other systems like immunity, repair, and regeneration. Therefore, understanding the context-dependent differences in fibrosis versus repair/regeneration in the players and pathways involved in FMT is necessary before targeting this process for therapeutic interventions.

An intricate interplay of intercellular communication within renal cells plays a pivotal role in DKD pathophysiology, including in the epithelial-to-mesenchymal transition (EMT) of renal epithelial cells. Even under normal conditions, communication between podocytes, glomerular endothelial cells (GECs), mesangial cells (MCs), and tubular epithelial cells (TECs) is vital for maintaining the renal architecture. The prolonged diabetic milieu changes intercellular communication, leading to glomerulosclerosis, proteinuria, and eventually renal fibrosis via EMT, amongst other cellular changes. EMT within the glomerulus is characterized by podocytes losing epithelial markers and gaining mesenchymal traits, resulting in a compromised glomerular filtration barrier and fibrogenesis. In the extra-glomerular compartment, EMT of proximal TECs (PTECs), distal TECs, and collecting duct epithelial cells contribute to DKD progression (Figure 3). Recent studies have revealed the signaling molecules, such as coding and non-coding RNAs and transforming growth factor-β (TGF-β), that mediate the cross-talk driving EMT in DKD. These complex interactions involve direct cell-to-cell contact, autocrine, and paracrine mechanisms, such as the secretion of exosomes carrying pro-fibrotic signals to neighboring cells.

TGF-β is a multifunctional cytokine that regulates many cellular functions, including cell growth, differentiation, and apoptosis. TGF-β1, the most widely expressed isoform, drives fibrosis in almost all forms of chronic kidney disease (CKD) by activating both canonical (Smad-based) and non-canonical (non-Smad-based) signaling pathways (Figure 3B) (67). In addition to triggering pre-existing myofibroblast activation, TGF-β1 also expands the myofibroblast pool by inducing EMT of renal epithelial cells and as well as an endothelial-to-mesenchymal transition (EndoMT) of glomerular endothelial cells (16, 67). Wu et al. grew GECs under high glucose stress and showed that these cells produced more exosomes than GECs grown under normal glucose levels; the exosomes were also enriched in TGF-β mRNA. Podocytes internalized these exosomes, triggering EMT in them as indicated by the simultaneous loss of epithelial markers (nephrin, ZO-1, WT-1) and gain of mesenchymal markers (α-SMA, desmin, FSP-1), hallmarks of the podocyte EMT (Figure 3B). Canonical Wnt/β-catenin signaling was shown to be activated by the exosomes (34). Thus, paracrine signaling from GECs in the diabetic milieu might trigger podocyte EMT, leading to more myofibroblasts and furthering fibrosis. While not EMT per se, exosomes secreted by high glucose-treated GECs enriched in TGF-β1 mRNA and circRNAs also trigger MC activation and myofibroblast transdifferentiation, aiding fibrosis (18, 37). High-glucose-induced MCs secrete exosomes enriched in the TGF-β1 protein, and podocytes cultured with these exosomes showed several signs of injury, including a reduction in the expression of nephrin and WT-1, suggesting EMT; however, mesenchymal markers were not tested in the study (29).

TGF-β stressed GECs overexpress miR-200c, which decreases VEGF-A expression and secretion in cultured human podocytes, impairing glomerular homeostasis (68). ZEB1 and ZEB2 are known targets of miR-200c and essential transcription factors playing a role in EMT. Moreover, ZEB1 was shown to be downregulated in podocytes by miR200c overexpression in GECs (68), suggesting cross-talk from GECs protecting podocytes from EMT.

Podocytes exposed to the supernatant of MCs cultured in high glucose conditions show a suppression of the endoplasmic reticulum (ER)-associated degradation (ERAD) (28). Membrane-bound or secretory proteins pass through the ER, where their fidelity is monitored via the ER quality control machinery. ERAD is a complex, conserved process where unfolded/misfolded proteins are recognized, polyubiquitinated, translocated to the cytoplasm, and degraded by the 26S proteasome. Suppression of ERAD causes accumulation of misfolded proteins, leading to ER stress and activation of the unfolded protein response (UPR) to recover protein homeostasis. ER stress induces EMT in human lens retinal epithelial cells (69). UPR signaling is exploited in cancer to promote EMT (70). Whether the ERAD suppression induced by communication from activated MCs ultimately leads to EMT of the podocytes remains a likely but unconfirmed scenario in DKD.

EMT of PTECs is an important contributor to DKD pathogenesis. The glomerular barrier is impermeable to proteins or immune complexes in healthy individuals. Glomerular filtration barrier damage leading to albuminuria is an early event in DKD, even utilized for its diagnosis. It is, therefore, conceivable that the PTEC lumen is exposed to all EMT-promoting and-repressing signals that podocytes are exposed to from the other glomerular cells. Tubular compartments of kidneys of a diabetic animal model of DKD show increased expression of the TGF-β1 type II receptor and the activation of the Smad signaling pathway (71), suggesting that GECs and MCs under hyperglycemic stress can induce EMT in PTECs in addition to podocytes via TGF-β. With increasing kidney injury, excess Angiotensin II and TGF-β induce EMT in PTECs (72).

Understanding the dynamics of the intra-renal intercellular cross-talk is crucial for developing therapies that can target the maladaptive communication that leads to EMT and halt the progression of DKD. Technological advances enable researchers to dissect these cellular dialogues with greater precision, offering hope for new interventions to preserve renal function and improve outcomes for patients with diabetes.

Endothelial-to-mesenchymal transition (EndoMT) also plays a pivotal role in the pathophysiology of DKD. EndoMT is a process where endothelial cells lose endothelial traits and acquire mesenchymal and fibroblast-like properties (Figure 4A), contributing to the fibrosis observed in DKD (16). GECs maintain the glomerular filtration barrier along with podocytes. EndoMT of GECs induces EMT of the adjacent podocytes, resulting in barrier dysfunction (34). The increased barrier porosity allows the passage of proteins and signaling molecules through the barrier, resulting in tubular cell injury and albuminuria. Additionally, EndoMT of GECs results in an increased pool of myofibroblasts with up to 30% of renal interstitial myofibroblasts in a DKD animal model reported to be of endothelial origin (16, 73). All these processes culminate in progressive fibrosis. This pathogenic cellular transformation of GECs is influenced by the dynamic cross-talk amongst various cell types within the renal corpuscle, including podocytes, MCs, and PTECs. Researchers are beginning to discover the crucial players of this cross-talk (Figure 4B).

TGF-β is the most common driver of most cases of EndoMT, including EndoMT in DKD (16). GECs growing in high glucose conditions undergo EndoMT as evidenced by a decrease in endothelial markers (CD31 and VE-cadherin) while acquiring mesenchymal markers (α-SMA and FSP) (34). As mentioned before, these cells also produce exosomes enriched in TGF-β mRNA. The EndoMT triggered in high-glucose-stressed GECs could result from autocrine signaling via the TGF-β from the released exosomes. MCs under hyperglycemic conditions secrete TGF-β1-enriched exosomes capable of inducing EMT in podocytes (29). Considering the proximity of MCs, podocytes, and GECs, it is plausible that these exosomes can induce EndoMT of GECs via TGF-β1-dependent mechanisms. Podocytes overexpress TGF-β1 in response to excess intraglomerular passage of proteins (74). Tubular compartments of the kidney of diabetic animals show increased expression of TGF-β1 (71). Thus, injured podocytes, activated MCs, and TECs are possible inducers of GEC EndoMT.

Pathways other than TGF-β also induce and regulate EndoMT (16). For instance, NOD2, a pattern recognition receptor, promotes EndoMT in GECs in response to high glucose by activating mitogen-activated protein kinase (MAPK) signaling (75). STAT5A activation increases ELTD1, which induces EndoMT in GECs (76, 77). MAPK and Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling are immune signaling pathways affected in DKD (78). Endothelial cells undergoing EndoMT release a lot of immune-modulatory chemokines and cytokines (77). All renal cells express pattern recognition receptors and chemokine and cytokine receptors. Thus, GECs undergoing EndoMT can communicate with the other glomerular cells via immune molecules. Sirtuin1 levels in PTECs are reduced even before albuminuria, and increasing PTEC-Sirtuin1 levels protects against glomerular injury and albuminuria in diabetes by maintaining nicotinamide mononucleotide (NMN) concentrations around glomeruli (Figure 1) (41). NMN protects endothelial cells from the damaging signals of IL1β, TNF-α, and angiotensin II (79), all known triggers of EndoMT (80–82). Moreover, NMN has also been shown to be protective against EMT. Thus, it is quite likely that PTECs might interact with and protect GECs from EndoMT.

Thus, almost all major renal cell types (MCs, GECs, podocytes, and PTECs) exposed to a diabetic milieu release signaling molecules capable of inducing or protecting against EndoMT in the adjacent endothelial cells and EMT in the neighboring epithelial cells. The anatomical proximity of these cells allows for a complex communication network mediated by various signaling pathways and molecules, such as the TGF-β/Smad pathway, JAK/STAT pathway, angiopoietins, growth factors, and cytokines. These interactions can lead to changes in cell behavior, resulting in the pathological changes observed in DKD, such as glomerulosclerosis, proteinuria, and fibrosis.

Pericytes are stromal-derived cells that wrap around capillary walls and connect intimately with the adjacent endothelial cells. They are crucial for angiogenesis, vascular stability, and vessel integrity. Unlike retinal pericytes, a renal pericyte can connect with multiple endothelial cells. Some renal pericytes span from the peritubular capillary to the tubule, with processes touching the tubular basement membrane, giving these cells the capability of communicating directly with both endothelial cells and tubular epithelia. In response to kidney injury, pericytes detach from capillary walls, migrate into the interstitial space, proliferate, and transdifferentiate into myofibroblasts, aiding interstitial scar formation and fibrosis (83). While the sources and their proportion of contribution to scar-forming myofibroblasts is a matter of debate, pericytes, along with perivascular fibroblasts, are generally recognized as the primary source of collagen-producing myofibroblasts in kidney fibrosis (53, 84).

Bidirectional communication between pericytes and endothelial cells is essential for this detachment and migration. Blocking either PDGF receptor-β signaling in pericytes or VEGF receptor 2 signaling in endothelial cells attenuates both fibrosis and capillary loss during progressive kidney injury in mice. Blockade of either receptor-mediated signaling pathway prevents pericyte detachment and proliferation (85, 86). In addition to detachment and proliferation, activated PDGF receptor signaling in pericytes also initiates their transdifferentiation into α-SMA and collagen 1-expressing myofibroblasts. Following injury, PGDF expression increases in injured tubules, endothelium, and macrophages in murine models of obstructive and post-ischemic kidney fibrosis. Inhibiting the PDGF-PDGF receptor signaling decreases pericyte differentiation and, therefore, fibrosis (86). As with the other sources, TGF-β signaling also initiates pericyte-to-myofibroblast transition (PMT). Both TGF-β and PDGF signaling are regulated by core fucosylation in initiating pericyte transition (87). Activated PI3K-Akt–mTOR pathway mediates pericyte-to-myofibroblast transition (PMT) by enhancing glycolysis. While inhibiting this pathway reduced PMT of TGF-β-treated pericytes, overexpression of hexokinase II rescued PMT (88). Wnt1 and Wnt4 from various renal sources (proliferating, medullary, and interstitial myofibroblasts, pericytes, and tubular epithelial cells) are also capable of driving myofibroblast transdifferentiation of pericytes (64, 65).

Following pericyte detachment, microvascular endothelial cells are devoid of the protective signaling from pericytes that is essential for microvascular stability. These endothelial cells become prone to injury and dysfunction. Peritubular capillaries are destabilized, leading to microvascular rarefaction (83). Thus, pericytes play a dual role in aiding fibrosis in DKD: (i) pericyte detachment from capillaries destabilizes the microvascular integrity, leading to vascular regression, which results in hypoxic tissues, furthering tissue damage and (ii) detached pericytes proliferate and transdifferentiate into scar-forming myofibroblasts in the renal interstitium directly driving fibrosis.

Macrophage accumulation correlates well with renal fibrosis progression. In addition to their pro-inflammatory role, macrophages can drive fibrosis directly by contributing to the myofibroblast pool. Circulating monocytes originating in the bone marrow differentiate into macrophages that accumulate in the kidneys. The macrophages can undergo myofibroblast transdifferentiation in the process termed macrophage-to-myofibroblast transition (MMT). Macrophage-derived myofibroblasts contribute to kidney fibrosis, as has been evidenced by the findings of co-expression of macrophage and myofibroblast markers, including CD68 or F4/80 (macrophage) and α-SMA (myofibroblast), in a substantial proportion of fibroblast-like cells both in human and experimental fibrotic kidney disease (89). More recently, experiments mapping cell fate in murine models exhibit myeloid-derived myofibroblasts (52). Thus, MMT is an important producer of myofibroblasts in the fibrotic kidney.

Several factors in the renal environment of DKD trigger MMT in macrophages, including hypoxia, hyperglycemia, hyperlipidemia, and an inflammatory milieu. Like the other cell types undergoing MFT, MMT is also controlled by TGF-β/Smad3 signaling with TGF-β1 as the primary inducer. All cells that overproduce TGF-β in DKD (mesangial cells, glomerular endothelial cells, proximal tubular epithelial cells, and infiltrating immune cells like macrophages) are, therefore, potentially signaling macrophages towards MMT. Other major pathways implicated in MMT are adiponectin/AMPK, JAK/STAT, and Wnt signaling pathways. Serum adiponectin levels are elevated in DKD patients. While adipocytes are the primary producers, endothelial cells, inflammatory cells, and epithelial cells have been reported to produce adiponectin. In a murine obstruction model of tubulointerstitial fibrosis, renal interstitial cells showed elevated adiponectin (90). Adiponectin propels MMT in bone marrow-derived monocytes by activating AMPK (90). In cultured mouse monocytes, IL-4 or IL-13 activated STAT6 and induced MMT, as evidenced by the expression of α-SMA and extracellular matrix proteins (fibronectin and collagen I), while in vivo, STAT6 was seen activated in interstitial cells of the obstructed kidney. JAK3 inhibitors or STAT6-deficiency showed less severe fibrosis (91). Both canonical and non-canonical Wnt signaling is elevated in DKD. Because Wnt ligands are ubiquitously expressed, their exact cellular sources involved in DKD remain undefined, but injured tubular epithelia, fibroblasts, and macrophages are known producers. Macrophage-derived Wnt ligands promote β-catenin activation in the tubular epithelium and MFT (92). Tubule-derived Wnt1 induces myofibroblast activation and proliferation (64). Wnt drives renal MMT via canonical Wnt signaling; β-catenin with T-cell factor (TCF) increases MMT, and diverting β-catenin from TCF to Foxo1 inhibits the fibrotic effect of TGF-β and enhances its anti-inflammatory action (93). In a three-way dialogue between macrophages, fibroblasts, and tubular cells, activated Wnt signaling seemingly promotes MMT, FMT, and EMT, respectively.

Exacerbating the situation further, myofibroblast-derived exosomes enhance MMT and kidney fibrosis (94). Deciphering the contents of these exosomes and the myofibroblast-macrophage communication signals would be the next vital step.