Interstitial Fibrosis In Diabetic Nephropathy Research Articles

Inhibition of S100A8/A9 ameliorates renal interstitial fibrosis in diabetic nephropathy

BackgroundRenal interstitial fibrosis (RIF) is one of the main features of diabetic nephropathy (DN), but the molecular mechanisms mediating RIF in DN has yet been fully understood. S100A8 and S100A9 are the proteins associated with immune and inflammation response. Here we reported the expression of S100A8 and S100A9 were significantly increased on tubular epithelial cells in diabetic kidneys through a proteomic analysis. MethodsWe detected the expression of S100A8/A9 in diabetic kidneys by using immunoblotting, real-time PCR and immunostaining. RNA silencing and overexpression were performed by using S100A8/A9 expression/knockdown lentivirus to investigate the connection between S100A8/A9 and epithelial to mesenchymal transition (EMT) process. We also identify the expression of TLR4/NFκB pathway-related molecules in the case mentioned above. Afterwards a CO-IP assay was used to verify that compound AB38b ameliorates the EMT by interfering S100A8/A9 expression. ResultsThe expression of S100A8 and S100A9 were significantly increased on tubular epithelial cells in diabetic kidneys. S100A8/A9 knocking-down alleviate and over-expression promote the renal interstitial fibrosis of diabetic mice. Mechanically, high levels of S100A8/A9 expression in tubular epithelial cells during diabetic condition activated the TLR4/NF-κB signal pathway which promoted the EMT process and finally led to RIF progression. S100A8/A9 knockdown ameliorated RIF of diabetic mice. Further experiments revealed that compound AB38b inhibited the EMT progression of tubular epithelial cells induced by S100A8/A9 through interfering the expressions of S100A8/A9. ConclusionsOur study suggest that abnormal expression of S100A8/A9 in the disease condition promotes EMT process and RIF through TLR4/NF-κB signal pathway. Using small molecular inhibitor AB38b to inhibit the abnormal expressions of S100A8/A9 might be a novel therapeutic strategy in treating DN.

A meta-analysis of the effects of endothelial nitric oxide synthase 4ba polymorphism on renal interstitial fibrosis in diabetic nephropathy.

This study aimed to investigate the relationship between endothelial nitric oxide synthase (eNOS) 4b/a gene polymorphism and renal interstitial fibrosis in patients with diabetic nephropathy (DN) via a meta-analysis, in the hope of providing guidance for the genotypic detection of DN. The Boolean logic search method was adopted. A literature search of PubMed, Medline, CNKI, and other databases from inception to June 2020 was performed using the search terms "eNOS 4b/a", "diabetes", "renal interstitial fibrosis", and "kidney disease". Literatures with a DN group, a non-nephropathy diabetic group, and a normal control group were screened. Review Manager software was employed to perform the meta-analysis. A total of 13 articles were included in the meta-analysis, the majority of which had a low risk of bias (i.e., medium and high quality). The results of the meta-analysis indicated that the genotypic distribution frequency of eNOS4bb in patients with DN was significantly lower than that in the non-nephropathy diabetic group (Z=3.19, P=0.001). The genotypic distribution frequency of eNOS4aa was significantly higher in non-nephropathy diabetic patients than in the normal control group (Z=2.57, P=0.01). The genotypic distribution frequency of eNOS4ba was not statistically different between patients with DN and non-nephropathy diabetic patients (Z=1.45, P=0.15). The genotypic distribution frequency of eNOS4bb in DN patients was significantly lower than that in the normal control group (Z=3.03, P=0.002); however, the genotypic distribution frequency of eNOS4ba was significantly greater than that of the normal controls (Z=2.36, P=0.02), as was that of eNOS4aa (Z=2.34, P=0.02). Genotypic polymorphism of eNOS 4b/a was closely related to DN. Moreover, the genotypic distribution frequency of eNOS4bb in DN renal interstitial patients was lower than that in non-nephropathy diabetic patients and normal controls.

MiRNA‑342 suppresses renal interstitial fibrosis in diabetic nephropathy by targeting SOX6.

Diabetic kidney disease (DKD) is one of the major microvascular complications in patients with type 1 and/or type 2 diabetes, the first cause of end-stage renal disease (ESRD) in several countries and regions. However, the pathogenesis of DKD and the mechanisms through which it leads to ESRD remain unknown. Thus, in this study, we aimed to elucidate some of these mechanisms. The expression of microRNA (miRNA or miR)-342-3p and SRY-box 6 (SOX6) in the renal tissues of mice with DKD and mouse renal mesangial cells (MCs) was determined by RT-qPCR and western blot analysis. The diabetic kidney environment was established using high-glucose medium. SOX6 was verified as a target gene of miR-342-3p by dual-luciferase activity assay. In addition, western blot analysis was employed to determine the changes in the levels of several biomarkers of fibrosis [transforming growth factor (TGF)-β1, fibronectin (FN), collagen IV (referred to as C-IV) and phosphatase and tensin homolog (PTEN)]. Compared with THE control mice, the expression of miR-342-3p in the kidney tissues of mice with DKD was down-regulated, whereas that of SOX6 was upregulated. The same phenomenon was observed in the MCs cultured in high-glucose medium. Subsequently, miR-342-3p inhibited SOX6 expression, promoted cell proliferation and inhibited the apoptosis of MCs. Moreover, the overexpression of miR-342-3p suppressed high glucose-induced renal interstitial fibrosis. In addition, it was found that miR-342-3p inhibited SOX6 expression by binding to the 3′-UTR of SOX6. On the whole, the findings of this study demonstrate that miR-342-3p suppresses the progression of DKD by inducing the degradation of SOX6. Thus, the miR-342-3p/SOX6 axis may serve as a novel therapeutic target in the treatment of DKD.

Silencing of long noncoding RNA XIST protects against renal interstitial fibrosis in diabetic nephropathy via microRNA-93-5p-mediated inhibition of CDKN1A.

Long noncoding RNAs (lncRNAs) have been reported to play an important role in diabetic nephropathy (DN). However, the molecular mechanism involved in this process remains poorly understood. Thus, the present study aimed to explore the function and molecular mechanism of dysregulated lncRNA X-inactive specific transcript (XIST) in DN. DN mouse models were established by streptozotocin treatment, and human renal tubular epithelial HK-2 cells were exposed to high glucose to produce an in vitro model. XIST was highly expressed in renal tissues of patients with DN, mice with DN, and high glucose-exposed HK-2 cells. To identify the interaction among XIST, miR-93-5p, and cyclin-dependent kinase inhibitor 1A (CDKN1A) and to analyze the functional significance of their interaction in renal interstitial fibrosis, we altered endogenous expression of XIST and miR-93-5p and CDKN1A. Dual-luciferase reporter assay results suggested that XIST was highly expressed in the kidney tissue of DN mice and high glucose-exposed HK-2 cells. XIST was identified to be a lncRNA that could bind to miR-93-5p, and CDKN1A was a target of miR-93-5p. Downregulated expression of XIST led to an increase in miR-93-5p expression, thereby decreasing CDKN1A and suppressing renal interstitial fibrosis in DN. Consistently, XIST knockdown reduced the expression of fibrosis markers (fibronectin, collagen type IV, and transforming growth factor-β1). Restoration of CDKN1A or decreasing miR-93-5p yielded a reversed effect on renal interstitial fibrosis. In conclusion, our study demonstrated that silenced XIST inducing miR-93-5p-dependent CDKN1A inhibition was beneficial for preventing renal interstitial fibrosis in DN, which may provide a future strategy to prevent the progression of DN.

CXCL6 Promotes Renal Interstitial Fibrosis in Diabetic Nephropathy by Activating JAK/STAT3 Signaling Pathway.

In this study the role of CXCL6 in diabetic nephropathy (DN) was investigated. It was found to be overexpression in DN patients and DN rat model. And the expression of fibrosis-related cytokines was consistent with the expression of CXCL6. High glucose significantly increased the proliferation of rat renal fibroblasts NRK-49F cell and the expression of CXCL6. Knockdown of CXCL6 ameliorated the pro-proliferation effect of high glucose and decreased the expression of fibrosis-related cytokines, while CXCL6 overexpression exhibited the opposite phenomenon. Gene set enrichment analysis, Western blot and ELISA showed that Janus kinase-signal transducer and activator of transcription (JAK-STAT) and CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION signaling pathways were correlative with CXCL6. This data indicates that CXCL6 may promote fibrosis-related factors to accelerate the development of DN renal interstitial fibrosis by activating JAK/STAT3 signaling pathway. CXCL6 is promising to be a potential novel therapeutic target and candidate biomarker for JAK/STAT3 signaling for the treatment of DN.

Effect of zinc deficiency on mouse renal interstitial fibrosis in diabetic nephropathy.

There is emerging evidence that tubulointerstitial fibrosis is the final common pathway of the majority of chronic progressive renal diseases, including diabetic nephropathy (DN). Zinc, an essential dietary element, has been suggested to be important for a number of protein functions during fibrosis invivo and invitro. However, the effect of zinc deficiency (ZnD) on renal interstitial fibrosis in DN remains unclear. The present study investigated the effect and the underlying mechanisms of ZnD on renal interstitial fibrosis during DN using an streptozotocin‑induced model of diabetes with immunofluorescence staining and western blot analysis. The present study identified that dietary zinc restriction significantly decreased zinc concentrations in the plasma and mouse kidney. ZnD enhanced albuminuria and extracellular matrix protein expression, associated with diabetic renal interstitial fibrosis by activation of renal interstitial fibroblasts and regulation of the expression of fibrosis‑associated factors, which may be mediated by the activation of fibroblasts via the TGF‑β/Smad signaling pathway. The data indicates that ZnD serves an important role in the pathogenic mechanisms of renal interstitial fibrosis during the development of DN.

Resveratrol inhibits renal interstitial fibrosis in diabetic nephropathy by regulating AMPK/NOX4/ROS pathway.

Resveratrol inhibits high glucose-induced NRK cell activation by decreasing NOX4-derived ROS. Resveratrol inhibits high glucose-induced NOX4 expression in NRK cells via activation of AMPK. ROS-activated ERK1/2 signaling is involved in high glucose-induced NRK cell activation. Resveratrol attenuated renal fibrosis in db/db mice via regulation of AMPK/NOX4/ROS signaling.

Resveratrol Inhibits Renal Interstitial Fibrosis in Diabetic Nephropathy by Regulating AMPK/NOX4/ROS Signaling

Resveratrol Inhibits Renal Interstitial Fibrosis in Diabetic Nephropathy by Regulating AMPK/NOX4/ROS Signaling

TLR4 activation promotes podocyte injury and interstitial fibrosis in diabetic nephropathy.

Toll like receptor (TLR) 4 has been reported to promote inflammation in diabetic nephropathy. However the role of TLR4 in the complicated pathophysiology of diabetic nephropathy is not understood. In this study, we report elevated expression of TLR4, its endogenous ligands and downstream cytokines, chemokines and fibrogenic genes in diabetic nephropathy in WT mice with streptozotocin (STZ) diabetes. Subsequently, we demonstrated that TLR4−/− mice were protected against the development of diabetic nephropathy, exhibiting less albuminuria, inflammation, glomerular hypertrophy and hypercellularity, podocyte and tubular injury as compared to diabetic wild-type controls. Marked reductions in interstitial collagen deposition, myofibroblast activation (α-SMA) and expression of fibrogenic genes (TGF-β and fibronectin) were also evident in TLR4 deficient mice. Consistent with our in vivo results, high glucose directly promoted TLR4 activation in podocytes and tubular epithelial cells in vitro, resulting in NF-κB activation and consequent inflammatory and fibrogenic responses. Our data indicate that TLR4 activation may promote inflammation, podocyte and tubular epithelial cell injury and interstitial fibrosis, suggesting TLR4 is a potential therapeutic target for diabetic nephropathy.

Endothelin-1 mediated high glucose-induced epithelial–mesenchymal transition in renal tubular cells

ObjectiveThe pathogenesis of interstitial fibrosis in diabetic nephropathy (DN) is an intractable problem without good therapy. Emerging evidence suggests that epithelial–mesenchymal transition (EMT) is an important mechanism for tubular epithelial cells undergoing profibrotic change in DN. Endothelin-1 (ET-1) is an important cytokine which can cause fibrogenesis and is reportedly involved in DN. However, the role of ET-1 in EMT in DN is unknown. The present study was designed to investigate the role of ET-1 in high glucose-induced EMT and the signaling pathway mediating the effect of ET-1 in renal tubular cells. MethodTubular epithelial cells (NRK52E) were treated with normal glucose (d-glucose 5.6mmol/L, NG), high glucose (30mmol/L, HG), high osmotic (d-glucose 5.6mmol/L+d-mannitol 24.4mmol/L), HG+ETA antagonist BQ123 (2μg/ml), ET-1, ET-1+ hypoxia inducible factor (HIF)-1α siRNA, CoCl2 (100μmol/L), CoCl2+HIF-1α siRNA or CoCl2+BQ123. The supernatant level of ET-1 was measured by ELISA and the expression of vimentin, E-cadherin and HIF-1α was detected by RT-PCR and western blot. ResultThe ET-1 level increased markedly in the supernatant of NRK52E incubated with HG. In NRK52E induced with HG or ET-1, the expression of vimentin was upregulated, whereas the expression of E-cadherin was downregulated. BQ123 attenuated HG- and CoCl2-induced EMT while HIF-1α siRNA did not affect ET-1 induced EMT. ConclusionsHigh glucose induced ET-1 production that mediated the EMT induced by high glucose in renal tubular epithelial cells, and HIF-1α acted as the upstream signal to regulate ET-1.

Effects of mycophenolate mofetil on the expression of monocyte chemoattractant protein-1 and fibronectin in high glucose cultured human mesangial cells.

The effects of high glucose on the expression of monocyte chemoattractant protein-1 (MCP-1) and the main component of the extracellular matrix, fibronectin (FN), were explored in human mesangial cells (HMCs), along with the intervention effects of mycophenolate mofetil (MMF) on these indicators. Cultured HMCs were divided into five groups: 1) normal control group (5 mM glucose); 2) high glucose group (30 mM glucose); 3) mannitol osmotic pressure control group (5 mM glucose + 25 mM mannitol); 4) high glucose + MMF-10 group (30 mM glucose + 10 μg/mL MMF); 5) high glucose + MMF-100 group (30 mM glucose + 100 μg/mL MMF). At 24, 48, and 72 h, reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay methods were used to detect the effects of MMF on MCP-1 mRNA and protein and FN expression in HMCs under high glucose conditions. MCP-1 mRNA and protein expressions and FN secretion significantly increased in HMCs of the high glucose group compared with the normal control group (P < 0.01), with the highest expression observed at 48 h. MMF could reduce the MCP-1 mRNA and protein and FN expression levels (P < 0.01), and the inhibition occurred in a dose- and time-dependent manner (P < 0.05). In conclusion, MMF could inhibit MCP-1 expression and the secretion of FN, indicating that it may delay the progression of glomerulosclerosis and interstitial fibrosis in diabetic nephropathy to ultimately achieve protective effects on the kidney.

A new mechanism of action of sulodexide in diabetic nephropathy: inhibits heparanase-1 and prevents FGF-2-induced renal epithelial-mesenchymal transition

BackgroundEpithelial-mesenchymal transition of tubular cells is a widely recognized mechanism that sustains interstitial fibrosis in diabetic nephropathy (DN). The signaling of FGF-2, a growth factor involved in this mechanism, is regulated by glycosaminoglycans. Heparanase-1, an endoglycosidase that cleaves heparan sulfate, is implicated in the pathogenesis of diabetic nephropathy and is necessary to FGF-2 for the induction of tubular cells transition. Well known Heparanase-1 inhibitors are heparin(s) and sulodexide, a low-molecular weight heparin – dermatan sulphate blend, which is effective in the treatment of DN.MethodsWe have investigated the inhibition by sulodexide and its components of Heparanase-1 by an ELISA assay. We have analyzed its effect on the epithelial-mesenchymal transition of tubular cells by real time gene expression analysis, zymography and migration assay.ResultsResults show that sulodexide is an effective heparanase-1 inhibitor, exclusively in virtue to the heparin component, with an IC50 of 5 μg/ml. In FGF-2 treated tubular cells, sulodexide also prevents the over-expression of the mesenchymal markers αSMA, vimentin and fibronectin and the motility increase, i.e. the epithelial-mesenchymal transition of tubular cells. Moreover, sulodexide prevents FGF-2 induced heparanase-1 and MMP9 increase switching off the autocrine loop that FGF-2 activates to support its signal.ConclusionsThe findings highlight the capacity of sulodexide to inhibit heparanase-1 and to control tubular fibrosis triggered by epithelial-mesenchymal transition. In conclusion, these sulodexide activities support the value of this agent in controlling the progression of nephropathy to renal failure.

IFN-γ suppresses the high glucose-induced increase in TGF-β1 and CTGF synthesis in mesangial cells

Mesangial cells are the main source of renal interstitial fibrosis in diabetic nephropathy (DN). Interferon-γ (IFN-γ) is a key cytokine that may play a potential therapeutic role in reducing fibrosis. Here, we focus on the effects of IFN-γ on human mesangial cells (HMCs) treated with high glucose. This study shows that IFN-γ phosphorylates STAT1, suppresses HMC proliferation, and down-regulates mRNA and protein levels of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) in HMCs treated with high glucose. The regulation of P-STAT1 could change HMC proliferation and the expression of fibrotic cytokines TGF-β1 and CTGF in HMCs. These data indicate that IFN-γ could activate STAT1 to suppress the increase in TGF-β1 and CTGF synthesis in HMCs induced by high glucose. This paper may lead to new therapeutic treatments of DN.

Effect of aldosterone and its antagonist spironolactone on epithelial-mesenchymal transition of normal rat kidney epithelial cells in high glucose

To determine the effect of aldosterone and its antagonist, spironolactone on epithelial-mesenchymal transition (EMT) of normal rat kidney epithelial cells (NRK-52E) in a high glucose milieu,and to explore the mechanism of renoprotection in diabetic nephropathy (DN ) in rats involving aldosterone and spironolacton. NRK-52E cells were simultaneously cultured in the serum-free Dulbecco's modification of Eagle's medium Dulbecco (DMEM) for 12 hours. Then the low glucose (LG) group was cultured in LG (1000 mg/L) DMEM:The high glucose (HG) group was cultured in high glucose (4,500 mg/L) DMEM. The aldosterone (Aldo) groups were cultured in high glucose DMEM with the addition of 10,50 and 100 nmol/L aldosterone respectively. The SP group was cultured in high glucose (4,500 mg/L) DMEM plus 10(-7)mol/L spironolactone. Immunohistochemistry, RT-PCR and Western blot were used to detect E-cadherin and alpha smooth muscle actin(alpha-SMA) mRNA expression. RT-PCR showed that compared with the LG Group, E-cadherin mRNA expression in the HG group was significantly lower, and alpha-SMA mRNA expression was significantly increased(P<0.05). E-cadherin mRNA expression in the 50 nmol/L Aldo group and 100 nmol/L Aldo group was significantly lower than that in the HG group, while the expression of alpha-SMA mRNA was significantly increased in the HG group(P<0.05), with a dose-dependent relationship with aldosterone(r=-0.70,P<0.05;r=0.67, P<0.05). E-cadherin mRNA in the SP group was significantly higher,while alpha-SMA mRNA expression was lower than that in the HG group(P<0.01). Immunohistochemistry and Western blot showed that compared with the LG group, E-cadherin protein expression was significantly reduced, and alpha-SMA expression was significantly increased in the HG group(P<0.01). In the 10 nmol/L Aldo, 50 nmol/L Aldo, and the 100 nmol/L Aldo groups, E-cadherin protein expression was significantly lower, and alpha-SMA protein expression was significantly higher than that in the HG group(P<0.05), with a dose-dependent relationship with aldosterone(r=-0.83,P<0.05;r=0.81, P<0.05). In the SP group, E-cadherin protein expression was higher, and alpha-SMA protein level was lower than that in the HG group(P<0.05). Aldosterone can promote EMT of tubular epithelial cells in a high sugar milieu, leading to renal interstitial fibrosis in Diabetic nephropathy. Spironolactone can inhibit high glucose-induced renal tubular epithelial cells EMT, which may be an important mechanism for the inhibition of renal interstitial fibrosis.

Correlations of tissue macrophages and cytoskeletal protein expression with renal fibrosis in patients with diabetes mellitus

In clinicopathological studies of cellular remodeling in the progression of diabetic nephropathy, it has not been well described whether tissue macrophage numbers and the expression of two cytoskeletal proteins--alpha-smooth muscle actin (alphaSMA) and vimentin--correlate with the disease severity. Renal biopsy specimens from 23 patients with noninsulin-dependent diabetes mellitus (NIDDM) were examined by immunoperoxidase methods for CD68+ macrophages and alphaSMA and vimentin staining in paraffin-embedded samples. alphaSMA staining was evaluated in mesangial and interstitial myofibroblastic cells, and vimentin staining was evaluated in podocytes and mesangial and tubular cells. Glomerular macrophage numbers were not correlated with any clinicopathological scores. However, the interstitial macrophage score was significantly correlated with serum creatinine (sCr) and strongly correlated with the interstitial fibrosis score. Both alphaSMA and vimentin were detectable in the mesangium, without significant correlation with each other. A positive correlation was observed between mesangial alphaSMA and urinary (u-) protein levels. In contrast, an inverse correlation was observed between levels of mesangial vimentin and u-protein. Mesangial alphaSMA, but not vimentin, showed a significant correlation with glomerular sclerosis. Podocytic vimentin levels tended to decrease in patients with higher sCr levels. The severity of interstitial peritubular alphaSMA was correlated strongly with interstitial macrophage proliferation and significantly with the interstitial fibrosis score. The expression of mesangial alphaSMA may play a role in the progression of glomerular damage, while, on the other hand, newly acquired mesangial vimentin seems to be attenuated by heavy proteinuria. In addition, it was suggested that peritubular alphaSMA-positive myofibroblastic cells, in collaboration with interstitial macrophages, contribute to the progression of interstitial fibrosis in diabetic nephropathy.

Diabetic nephropathy: the central role of renal proximal tubular cells in tubulointerstitial injury.

Diabetic nephropathy is now the commonest cause of end stage renal disease and accounts for 30-40% of all patients requiring renal replacement therapy. Furthermore, the incidence of diabetic nephropathy continues to increase, in part due to the improved survival of type 2 diabetic patients as the cardiovascular mortality in this group declines (Ritz and Stefanski, 1996). Clinically incipient nephropathy is first manifest by the onset of persistent microalbuminuria, after which, overt diabetic nephropathy is heralded by the appearance of persistent proteinuria. Subsequently, there is a progressive decline in glomerular filtration rate (GFR) resulting, within 5 years, in end stage renal disease in 50% of patients (Hasslacher et al., 1989). The pathology of the renal lesions are similar in type I and II diabetes (Taft et al., 1994), although it has been suggested that there is more heterogeneity in type II diabetes (Chihara et al., 1986). Studies analysing structural-functional relationships have demonstrated that the development of proteinuria correlates with the degree of mesangial expansion (Mauer et al., 1984; White and Bilous, 2000). Although diabetic nephropathy was traditionally considered a primarily glomerular disease, it is now widely accepted that the rate of deterioration of function correlates best with the degree of renal tubulointerstitial fibrosis (Mauer et al., 1984, Bohle et al., 1991). This suggests that although in the majority of patients the primary event is a condition manifest by glomerular changes resulting in proteinuria, the long-term outcome is determined by events in the renal interstitium. With the increasing awareness of the importance of these pathological interstitial changes, interest has focused on the role of cells, such as the epithelial cells of the proximal tubule (PTC) or the interstitial myofibroblast, in the initiation of fibrosis. The aim of the present review is to analyse the available data supporting the role for the PTC in orchestrating renal interstitial fibrosis in diabetic nephropathy as a result of glucose-dependent alterations in PTC function. The potential for subsequent effects on PTC-fibroblast cross-talk will also be considered.

Role of glomerular ultrafiltration of growth factors in progressive interstitial fibrosis in diabetic nephropathy

The present in vivo and in vivo experiments were performed to test the hypothesis that in rats with glomerular proteinuria, the bioactive growth factors transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF) are ultrafiltered into tubular fluid, can interact with respective receptors in apical tubular cell membranes, increase the expression and basolateral secretion of C-C-chemokines, which interact with cells in the renal interstitium and indirectly cause myofibroblasts to increase the expression of extracellular matrix proteins. HGF and TGF-beta were measured by Western blot and bioassay in glomerular ultrafiltrate that was collected by nephron micropuncture from rats with diabetic nephropathy and control rats. Proximal tubular and collecting duct cells were incubated with diluted proximal tubular fluid or recombinant human HGF (rhHGF) or rhTGF-beta and expression of C-C-chemokines was measured by RT-PCR and ELISA. Interactions of tubular cell chemokines with macrophages and indirectly with myofibroblasts were also examined using cell culture models. In rats with glomerular proteinuria due to diabetic nephropathy mature, bioactive HGF as well as active and latent TGF-beta were detected in early proximal tubular fluid. Specific HGF- and TGF-beta type II receptors were expressed in apical tubular membranes more in diabetic compared to control rats. Incubation of cultured mouse proximal tubular cells (mPTC) or medullary collecting duct cells (mIMCD-3) with diabetic rat proximal tubular fluid increased MCP-1 and RANTES mRNA levels as well as secreted peptide up to threefold. In contrast, high glucose (450 mg/dL), bovine serum albumin (BSA) or rat albumin (each at 100 micrograms/mL) or 10 nmol/L insulin-like growth factor-I (IGF-I; which was also present in glomerular ultrafiltrate in rats with diabetic nephropathy) did not affect expression of these chemokines. Recombinant human TGF-beta as well as rhHGF each increased MCP-1 and RANTES mRNA as well as peptide levels several-fold. In cultured macrophages MCP-1 raised the secretion of TGF-beta, which in turn increased the expression of collagen type I and III as well as fibronectin in renal interstitial myofibroblasts about 2.5 to 4-fold. Proteinuria-induced progressive renal interstitial fibrosis may be caused by glomerular ultrafiltration of high molecular weight bioactive growth factors, HGF and TGF-beta, which "activate" tubular cells through apical membranes. These apical signals are translated into basolateral events that are recognized by cells in the interstitium, such as the basolateral secretion of the C-C-chemokines MCP-1 and RANTES, which may (via macrophages) stimulate interstitial myofibroblasts, and thus lead to accumulation of extracellular matrix proteins and progressive interstitial fibrosis.