Crosstalk between tubular epithelial cells and glomerular endothelial cells in diabetic kidney disease

Abstract In recent years, although the development of clinical therapy for diabetic kidney disease (DKD) has made great progress, the progression of DKD still cannot be controlled. Therefore, further study of the pathogenesis of DKD and improvements in DKD treatment are crucial for prognosis. Traditional studies have shown that podocyte injury plays an important role in this process. Recently, it has been found that glomerulotubular balance and tubuloglomerular feedback (TGF) may be involved in the progression of DKD. Glomerulotubular balance is the specific gravity absorption of the glomerular ultrafiltrate by the proximal tubules, which absorbs only 65% to 70% of the ultrafiltrate. This ensures that the urine volume will not change much regardless of whether the glomerular filtration rate (GFR) increases or decreases. TGF is one of the significant mechanisms of renal blood flow and self‐regulation of GFR, but how they participate in the development of DKD in the pathological state and the specific mechanism is not clear. Injury to tubular epithelial cells (TECs) is the key link in DKD. Additionally, injury to glomerular endothelial cells (GECs) plays a key role in the early occurrence and development of DKD. However, TECs and GECs are close to each other in anatomical position and can crosstalk with each other, which may affect the development of DKD. Therefore, the purpose of this review was to summarize the current knowledge on the crosstalk between TECs and GECs in the pathogenesis of DKD and to highlight specific clinical and potential therapeutic strategies.


Abstract
In recent years, although the development of clinical therapy for diabetic kidney disease (DKD) has made great progress, the progression of DKD still cannot be controlled. Therefore, further study of the pathogenesis of DKD and improvements in DKD treatment are crucial for prognosis. Traditional studies have shown that podocyte injury plays an important role in this process. Recently, it has been found that glomerulotubular balance and tubuloglomerular feedback (TGF) may be involved in the progression of DKD. Glomerulotubular balance is the specific gravity absorption of the glomerular ultrafiltrate by the proximal tubules, which absorbs only 65% to 70% of the ultrafiltrate. This ensures that the urine volume will not change much regardless of whether the glomerular filtration rate (GFR) increases or decreases. TGF is one of the significant mechanisms of renal blood flow and self-regulation of GFR, but how they participate in the development of DKD in the pathological state and the specific mechanism is not clear. Injury to tubular epithelial cells (TECs) is the key link in DKD. Additionally, injury to glomerular endothelial cells (GECs) plays a key role in the early occurrence and development of DKD. However, TECs and GECs are close to each other in anatomical position and can crosstalk with each other, which may affect the development of DKD. Therefore, the purpose of this review was to summarize the current knowledge on the crosstalk between TECs and GECs in the pathogenesis of DKD and to highlight specific clinical and potential therapeutic strategies. energy; and are sensitive to damage. 4 In the diabetic environment, tubular epithelial cells (TECs) are easily affected by metabolic disorders, inflammatory states and changes in urine composition and haemodynamics, resulting in oxidative stress and secretion of a variety of cytokines, leading to interstitial inflammation and fibrosis.
Recent studies have shown that glomerulotubular balance and tubuloglomerular feedback (TGF) affect the progression of DKD.
Based on this, clinical application of novel hypoglycaemic drugs such as daglitazine, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, can increase urinary glucose excretion, control blood sugar and reduce glomerular injury, thereby delaying the deterioration of renal function in DKD. Nektaria found that there are many common signalling pathways between TECs and GECs, in which crosstalk plays a vast role (Figure 1). 5 During the occurrence of DKD, abnormal secretion of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and inflammatory factors and hypoxia promotes injury to GECs. Moreover, injured GECs secrete hepatocyte growth factor (HGF), insulin-like growth factor binding proteins (IGFBPs), extracellular vesicles (EVs) and Kruppel-like factor (KLF), and autophagy can also act on TECs, causing changes in the structure and function of TECs to different degrees. Therefore, the purpose of this review was to briefly summarize the current knowledge on the pathogenesis of DKD, with specific comments on the crosstalk between GECs and TECs and potential therapeutic interventions.

| TEC INJ URY AND DK D
Compared with GEC lesions, TECs are more closely related to the deterioration of renal function. 6 There are different degrees of renal tubular injury in the early stage of DKD. It has been reported that only 1% of diabetic microalbuminuria patients have typical glomerular structural damage, while 1/3 of patients have no or very slight glomerular injury, 7 but renal tubular injury is serious.
The manifestations are thickening of the renal tubular basement membrane, tubular inflammatory lesions, renal tubular atrophy, increased apoptosis, interstitial fibrosis and thinning of peritubular capillaries. 8 It has been found that massive proteinuria in DKD patients causes inflammatory responses, oxidative stress, activation of transforming growth factor-β (TGF-β) and the renin-angiotensin system (RAS) and accumulation of advanced glycation end products (AGEs), 9

| G EC INJ URY AND DK D
GECs are a fundamental part of the renal filtration barrier, which is in direct contact with the blood circulation and is easily affected or damaged by circulating substances such as blood glucose, lipids and inflammatory factors. In the case of high glucose, metabolites and other stimuli activate a variety of signalling pathways (such as RAS, AGEs, polyol pathway and the protein kinase C pathway) and induce intrinsic renal cells to produce a variety of growth factors, cytokines, reactive oxygen species (ROS) and endothelial nitric oxide synthase (eNOS), in addition to mitochondrial DNA damage, and inflammatory responses, resulting in GEC dysfunction. 12 Our previous studies found that high glucose mediated IL to induce the endothelial-mesenchymal transition (EndMT) of GECs. 13

| THE ROLE OF CROSS TALK B E T WEEN TEC S AND G EC S IN DK D
Because of the relationship between the location of TECs and GECs, their abnormal crosstalk plays a key role in the pathogenesis of DKD.
Alterations in the GEC surface layer, including its major component glycocalyx, are a leading cause of microalbuminuria observed in early DKD. In addition, recent studies suggest that GECs contribute to DKD by paracrine communication with other glomerular cells, such as TECs. 17 Emerging evidence suggests that the glomerular filtration barrier and tubulointerstitial compartment are a composite, dynamic entity where any injury of one cell type spreads to other cell types and leads to the dysfunction of the whole apparatus. Gene and protein expression profiling show that glomerular changes in diabetes involve many metabolic and signalling pathways that may occur in individual glomerular cells or through crosstalk between them. 18 In DKD, TECs induce cascading inflammatory responses by releasing cytokines, miRNAs and extracellular vesicles through autocrine or paracrine mechanisms under conditions of high glucose and proteinuria. 19 The glomerular vascular network undergoes apoptosis, necrosis and transdifferentiation with the stimulation of inflammatory factors, and the structure and function of GECs are destroyed.
Damaged GECs reduce the blood supply to the renal tubules, leading to exacerbated TEC damage. In the future, more efforts need to be made to build a kidney cell map and understand the crosstalk between cells. 20

| The inflammatory response of TECs contributes to the injury of GECs
DKD is considered to be an inflammatory disease caused by disorders of glucose and lipid metabolism. 21 Our previous study found that proteinuria causes inflammation of TECs in a CKD mouse model. 22 High concentrations of urinary albumin in DKD patients activate TECs to produce proinflammatory factors such as CRP, IL, TNF-α, NF-κB and ROS, which can lead to GEC injury, apoptosis and EndMT. Inflammation leads to lipid peroxidation, membrane structure destruction, membrane protein function inhibition and membrane transport system dysfunction, such as sodium pump, calcium pump and G protein-coupled receptor dysfunction, which induces GEC apoptosis. In our in vitro experiment, we found that

| Hypoxia-induced responses in TECs promote injury to GECs
High glucose can cause hypoxia. Chronic hypoxia is associated with the occurrence and development of DKD. Expression of hypoxia inducible factor-1α (HIF-1α) in TECs is increased, and HIF-1α enters the nucleus and binds with the HIF-1β subunit at the hypoxia response element (HRE) to form the dimer complex HIF-1, which activates the downstream inflammatory response to promote GEC proliferation and EndMT, inducing autophagy and programmed cell death. 29 Hypoxia increases the expression of Runx1 in GECs. Under hypoxia, transcription of the inflammatory molecules IL-1β, ICAM and TNF-α increases. The stability of the cell membrane decreases, and the apoptosis pathway is activated. Inhibiting Runx1 significantly decreases the hypoxia-induced expression of the inflammatory molecules IL-1β, ICAM and TNF-α, and injury to the GEC membrane and the apoptosis pathway are inhibited.
High mobility group box-1 (HMGB1), a member of the HMG-box family, is a highly rich and widely expressed protein that is highly expressed by TECs and stimulates inflammatory signalling pathways such as the inflammasome and NF-kB pathways under anoxic conditions. HMGB1 induces the release of proinflammatory cytokines and affects the function of GECs. 30 These results suggest that hypoxia leads to GEC damage in a manner that is dependent on TEC secretion of HIF-1 and the HMGB1 pathway.

| TGF-mediated crosstalk between TECs and GECs in DKD
TGF is a main mechanism for regulating renal microcirculation and renal haemodynamics. Macula densa (MD), which is composed of TECs, regulates the contraction and relaxation of glomerular arterioles and changes the GFR. SGLT2 expressed by proximal TECs determines the reabsorption of renal blood glucose. 31 In DKD patients, more glucose molecules enter the renal tubules, which results in increased SGLT2 expression, a large number of Na+ and glucose molecules transporting back to the blood through SGLT2, and decreased Na+ flowing through the MD. In this condition, TGF is weakened by releasing nitric oxide (NO), RAS and so on. 32 The glomerular arterioles become dilated, and the GFR increases.

| Kruppel-like factor (KLF)-mediated EMT in GECs promotes injury to TECs
KLF is a shear stress-induced transcription factor that has endothelial protective effects. 35 Glomerular injury and proteinuria in GEC-specific KLF2 heterozygous knockout diabetic mice were significantly worse than those of wild-type diabetic mice. The low expression of KLF2 in GECs differentially regulates the expression of key angiogenic markers. Moreover, the decrease in endothelial-specific KLF2 also leads to the obvious injury of TECs in diabetic mice, indicating that KLF2 may be involved in GEC/TEC interactions. 36 Studies have shown that KLFs negatively regulate the proliferation of TECs by inducing G1 arrest in TECs. 37 In addition, it has been found that Dnmt1 is involved in the TGF-β1mediated methylation of the KLF4 promoter in vivo and in vitro.

| Hepatocyte growth factor (HGF/c-MET) inhibits EMT in TECs by affecting GECs
HGF is a pluripotent factor found in mature hepatocytes that can promote cell proliferation, division, differentiation and migration. 39

| Insulin-like growth factor binding proteins (IGFBPs) mediate crosstalk between TECs and GECs in DKD
IGFs are a group of peptides with a growth-promoting effect. IGF-1 mRNA and protein are distributed in GECs. Imbalance in the IGF sys-

| Autophagy maintains balance between GECs and TECs
Autophagy is an important mechanism for maintaining environmental stability in glomeruli and renal tubules, which is necessary in human health and diseases. Bone morphogenetic protein

| Glomerulotubular balance maintains balance between GECs and TECs
The glomerulotubular balance is a phenomenon in which the reabsorption of water and solutes by the proximal tubules changes with the GFR. By sensing capillary blood pressure and colloidal osmotic pressure, the filtered glomerular filtration fluid determines the amount of renal tubular reabsorption, maintaining ultrafiltration homeostasis. 47 The results of the EMPA-REG OUTCOME TM clinical study 30 showed that after the initial 4 weeks of treatment, the estimated GFR (eGFR) decreased for a short time in the englicine group. However, during the following long-term treatment, eGFR remained stable in the englinide group and decreased progressively in the placebo group, indicating that englinide improves glomerular hyperfiltration and plays a longterm renal protective role. In addition, Neal et al 34 found that kaglitazine treatment reduced the risk of renal endpoint events by 40%.

| CON CLUS ION
At present, DKD is the main cause of ESRD, and its incidence remains high. Tubular feedback and TGF play a key role in maintaining the normal structure and function of renal tubules and glomeruli. Recently, it has been found that there are many common signalling pathways between TECs and GECs, and the crosstalk between them plays an important role in the occurrence and development of DKD. Improving the injury to TECs and GECs and maintaining normal crosstalk between them may become a new strategy for the prevention and treatment of DKD in the future. Further research efforts should be aimed at demonstrating that prevention of progression of the crosstalk between TECs and GECs is possible and leads to improved outcomes.

ACK N OWLED G EM ENTS
This study was funded by grants from the National Natural Science

CO N FLI C T S O F I NTE R E S T
The authors have no conflicts of interest to declare.

AUTH O R CO NTR I B UTI O N
All authors contributed equally to manuscript writing.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.