PRR promotes hypertensive renal injury by activating Wnt/ß-catenin signaling and inflammation infiltration in mice.

Hypertension stands out as a substantial independent risk factor in the progression of chronic kidney disease; however, the exact pathological mechanisms remain elusive. Our preliminary studies find that Wnt/ß-catenin control renin-angiotensin system (RAS) expression, thus playing an important role in the pathogenesis of hypertension and renal fibrosis. As an integral component of the RAS, the (pro)renin receptor (PRR) plays a crucial role in the activation of the RAS and hypertension. Recent studies suggest a reciprocal relationship between PRR and Wnt/ß-catenin signaling, potentially contributing to hypertensive renal fibrosis development. To assess the role of PRR in mediating hypertensive nephropathy, we manipulated this signaling by over expression of PRR ligand or blockade of PRR by siPRR. In vivo, PRR induction promoted hypertension, proteinuria, renal fibrosis, inflammatory response and ß-catenin activation in Ang II induced hypertension mice. Conversely, blockade of PRR inhibited Ang II mediated hypertension, renal fibrosis and inflammation. In vitro, PRR over expression renal tubular cells exacerbated the Ang II induced fibrotic response and inflammation. Moreover, PRR was upregulated in hypertensive nephropathy patients, and correlated with renal function and renal fibrosis. These results indicate that PRR interact with Wnt/ß-catenin signaling promote the progression of hypertensive nephropathy. PRR could be served as a biomarker for the diagnosis and treatment of hypertensive renal fibrosis.

Identification of Novel Genes and Associated Drugs in Advanced Clear Cell Renal Cell Carcinoma by Bioinformatic Methods.

The current work screened differentially expressed genes (DEGs) related to advanced clear cell renal cell carcinoma (ccRCC) and found potential biomarkers and drugs for advanced ccRCC. After analyzing GSE53757 and GSE66271, we identified DEGs and performed the functional annotation, pathway enrichment, validation, survival analysis, and candidate drug analysis. We obtained 861 common DEGs from datasets between advanced ccRCC tissues and normal kidney tissues. Besides, we performed functional analysis under ontological conditions and carried out pathway analysis. The five most stable core gene groups and top 10 genes were screened using the Cytoscape software. We performed functional and pathway analyses again and found that the core genes were similar to total DEGs. After verification, the expression trends of the 10 hub genes did not change. Survival analysis showed high expressions of TOP2A, BIRC5, BUB1, MELK, RRM2, and TPX2 genes, suggesting that they might participate in cancer occurrence, migration, and relapse of ccRCC. The gene-drug analysis showed that gallium nitrate, cladribine, and amonafide were strongly associated with RRM2 and TOP2A. We found that RRM2 and TOP2A might be predictive biomarkers and novel targeted therapy for advanced ccRCC. These drugs (gallium nitrate, cladribine, and amonafide) might be used for treating advanced ccRCC.

Pentosan polysulfate ameliorates fibrosis and inflammation markers in SV40 MES13 cells by suppressing activation of PI3K/AKT pathway via miR-446a-3p.

BACKGROUND: Renal fibrosis is a common outcome of various renal damage, including diabetic nephropathy (DN), the leading cause of end-stage renal disease. Currently, there are no effective therapies for renal fibrosis. The present study aimed to determine whether pentosan polysulphate sodium (PPS), a FDA approved medication for interstitial cystitis, protects diabetic renal fibrosis. METHODS: Cell viability and apoptosis were evaluated in mouse mesangial cells (SV40-MES13) after incubating with the advanced glycation end products (AGEs), which play important roles in the pathogenesis of DN. Western blot and ELISA were performed to determine the expression of transforming growth factor-beta1 (TGF-ß1) and fibronectin (FN), two biomarkers of renal fibrosis, as well as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), two biomarkers of inflammation. The miRNA-mRNA regulatory network involved in the phosphatidylinositol 3-kinase (PI3K)/Ser and Thr Kinase (AKT) signalling was investigated by miRNA deep sequencing and validated by RT-PCR and miRNA transfection. RESULTS: AGEs significantly inhibited cell proliferation and promoted cell apoptosis, which was associated with the overexpression of TGF-ß1, FN, IL-6, and TNFα. PPS almost completely reversed AGEs-induced biomarkers of fibrosis and inflammation, and significantly altered the miRNA expression profile in AGEs-treated cells. Notably, the PI3K/AKT signalling was one of the most significantly enriched pathways targeted by PPS-related differentially expressed miRNAs. PPS significantly up-regulated miR-466a-3p, which was shown to target PIK3CA, and mediated the inhibitory effect of PPS on AGEs-induced activation of PI3K/AKT pathway. CONCLUSIONS: The treatment of PPS protected against AGEs-induced toxicity in SV40 MES13 cells via miR-466a-3p-mediated inhibition of PI3K/AKT pathway.

Lipidomics Reveals Serum Specific Lipid Alterations in Diabetic Nephropathy.

In diabetes mellitus (DM), disorders of glucose and lipid metabolism are significant causes of the onset and progression of diabetic nephropathy (DN). However, the exact roles of specific lipid molecules in the pathogenesis of DN remain unclear. This study recruited 577 participants, including healthy controls (HCs), type-2 DM (2-DM) patients, and DN patients, from the clinic. Serum samples were collected under fasting conditions. Liquid chromatography-mass spectrometry-based lipidomics methods were used to explore the lipid changes in the serum and identify potential lipid biomarkers for the diagnosis of DN. Lipidomics revealed that the combination of lysophosphatidylethanolamine (LPE) (16:0) and triacylglycerol (TAG) 54:2-FA18:1 was a biomarker panel for predicting DN. The receiver operating characteristic analysis showed that the panel had a sensitivity of 89.1% and 73.4% with a specificity of 88.1% and 76.7% for discriminating patients with DN from HCs and 2-DM patients. Then, we divided the DN patients in the validation cohort into microalbuminuria (diabetic nephropathy at an early stage, DNE) and macroalbuminuria (diabetic nephropathy at an advanced stage, DNA) groups and found that LPE(16:0), phosphatidylethanolamine (PE) (16:0/20:2), and TAG54:2-FA18:1 were tightly associated with the stages of DN. The sensitivity of the biomarker panel to distinguish between patients with DNE and 2-DM, DNA, and DNE patients was 65.6% and 85.9%, and the specificity was 76.7% and 75.0%, respectively. Our experiment showed that the combination of LPE(16:0), PE(16:0/20:2), and TAG54:2-FA18:1 exhibits excellent performance in the diagnosis of DN.

Serum integrative omics reveals the landscape of human diabetic kidney disease.

OBJECTIVE: Diabetic kidney disease (DKD) is the most common microvascular complication of type 2 diabetes mellitus (2-DM). Currently, urine and kidney biopsy specimens are the major clinical resources for DKD diagnosis. Our study proposes to evaluate the diagnostic value of blood in monitoring the onset of DKD and distinguishing its status in the clinic. METHODS: This study recruited 1,513 participants including healthy adults and patients diagnosed with 2-DM, early-stage DKD (DKD-E), and advanced-stage DKD (DKD-A) from 4 independent medical centers. One discovery and four testing cohorts were established. Sera were collected and subjected to training proteomics and large-scale metabolomics. RESULTS: Deep profiling of serum proteomes and metabolomes revealed several insights. First, the training proteomics revealed that the combination of α2-macroglobulin, cathepsin D, and CD324 could serve as a surrogate protein biomarker for monitoring DKD progression. Second, metabolomics demonstrated that galactose metabolism and glycerolipid metabolism are the major disturbed metabolic pathways in DKD, and serum metabolite glycerol-3-galactoside could be used as an independent marker to predict DKD. Third, integrating proteomics and metabolomics increased the diagnostic and predictive stability and accuracy for distinguishing DKD status. CONCLUSIONS: Serum integrative omics provide stable and accurate biomarkers for early warning and diagnosis of DKD. Our study provides a rich and open-access data resource for optimizing DKD management.

Exogenous Wnt1 Prevents Acute Kidney Injury and Its Subsequent Progression to Chronic Kidney Disease.

Studies suggest that Wnt/ß-catenin agonists are beneficial in the treatment of acute kidney injury (AKI); however, it remains elusive about its role in the prevention of AKI and its progression to chronic kidney disease (CKD). In this study, renal Wnt/ß-catenin signaling was either activated by overexpression of exogenous Wnt1 or inhibited by administration with ICG-001, a small molecule inhibitor of ß-catenin signaling, before mice were subjected to ischemia/reperfusion injury (IRI) to induce AKI and subsequent CKD. Our results showed that in vivo expression of exogenous Wnt1 before IR protected mice against AKI, and impeded the progression of AKI to CKD in mice, as evidenced by both blood biochemical and kidney histological analyses. In contrast, pre-treatment of ICG-001 before IR had no effect on renal Wnt/ß-catenin signaling or the progression of AKI to CKD. Mechanistically, in vivo expression of exogenous Wnt1 before IR suppressed the expression of proapoptotic proteins in AKI mice, and reduced inflammatory responses in both AKI and CKD mice. Additionally, exogenous Wnt1 inhibited apoptosis of tubular cells induced by hypoxia-reoxygenation (H/R) treatment in vitro. To conclude, the present study provides evidences to support the preventive effect of Wnt/ß-catenin activation on IR-related AKI and its subsequent progression to CKD.

PC-1 NF suppresses high glucose-stimulated inflammation and extracellular matrix accumulation in glomerular mesangial cells via the Wnt/ß-catenin signaling.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease worldwide with high morbidity and mortality. Glomerular mesangial cell (MC) proliferation, inflammatory cell infiltration and extracellular matrix (ECM) accumulation are the main pathological characteristics of DN. A previous study revealed that polycystin-1 N-terminal fragment (PC-1 NF) fusion protein could inhibit ECM accumulation in a mesangial proliferative glomerulonephritis model. However, the role of PC-1 NF fusion protein in DN remains unknown. The results of the present study indicated that PC-1 NF fusion protein significantly abolished high glucose (HG)-induced glomerular MC viability over three time points measured (24, 48 and 72 h). In addition, PC-1 NF suppressed the levels of monocyte chemotactic peptide-1 and tumor necrosis factor α, as well as the expression of fibronectin and collagen IV, in HG-stimulated MCs. Furthermore, PC-1 NF fusion protein efficiently inhibited the activation of Wnt/ß-catenin signaling pathway in HG-stimulated MCs. Taken together, these data indicated that PC-1 NF fusion protein inhibited HG-induced MC proliferation, inflammation, and ECM expression via the modulation of the Wnt signaling pathway. The present study indicated that PC-1 NF fusion protein may be a potential agent in treating DN.

Early activation of fibroblasts is required for kidney repair and regeneration after injury.

Acute kidney injury (AKI) is a devastating condition with high morbidity and mortality. AKI is characterized by tubular injury, inflammation, and vascular impairment. However, the role of interstitial fibroblasts in the pathogenesis of AKI is largely unknown. Here, we show that fibroblasts were activated, as defined by vimentin expression, at 1 h after AKI triggered by ischemia-reperfusion injury (IRI). They rapidly entered the cell cycle with Ki-67-positive staining, which started at 1 h and peaked at 12 h after IRI, whereas tubular cell proliferation peaked at 3 d. The trigger for such an early activation of fibroblasts was identified as sonic hedgehog (Shh), which was rapidly induced in renal tubules and could target interstitial fibroblasts. Tubule-specific knockout of Shh in mice inhibited fibroblast activation and aggravated kidney injury and functional decline after IRI. Likewise, pharmacologic inhibition of Shh signaling with cyclopamine also hindered fibroblast activation and exacerbated kidney damage. These studies uncover that tubule-derived Shh triggers the early activation of fibroblasts, which is required for kidney repair and regeneration. Our findings for the first time illustrate a previously unrecognized importance of interstitial fibroblasts in conferring renal protection in AKI.-Zhou, D., Fu, H., Liu, S., Zhang, L., Xiao, L., Bastacky, S. I., Liu, Y. Early activation of fibroblasts is required for kidney repair and regeneration after injury.

Wnt/ß-catenin regulates blood pressure and kidney injury in rats.

Activation of the renin-angiotensin system (RAS) plays a pivotal role in mediating hypertension, chronic kidney and cardiovascular diseases. As Wnt/ß-catenin regulates multiple RAS genes, we speculated that this developmental signaling pathway might also participate in blood pressure (BP) regulation. To test this, we utilized two rat models of experimental hypertension: chronic angiotensin II infusion and remnant kidney after 5/6 nephrectomy. Inhibition of Wnt/ß-catenin by ICG-001 blunted angiotensin II-induced hypertension. Interestingly, angiotensin II was able to induce the expression of multiple Wnt genes in vivo and in vitro, thereby creating a vicious cycle between Wnt/ß-catenin and RAS activation. In the remnant kidney model, renal ß-catenin was upregulated, and delayed administration of ICG-001 also blunted BP elevation and abolished the induction of angiotensinogen, renin, angiotensin-converting enzyme and angiotensin II type 1 receptor. ICG-001 also reduced albuminuria, serum creatinine and blood urea nitrogen, and inhibited renal expression of fibronectin, collagen I and plasminogen activator inhibitor-1, and suppressed the infiltration of CD3+ T cells and CD68+ monocytes/macrophages. In vitro, incubation with losartan prevented Wnt/ß-catenin-mediated fibronectin, α-smooth muscle actin and Snail1 expression, suggesting that the fibrogenic action of Wnt/ß-catenin is dependent on RAS activation. Taken together, these results suggest an intrinsic linkage of Wnt/ß-catenin signaling with BP regulation. Our studies also demonstrate that hyperactive Wnt/ß-catenin can drive hypertension and kidney damage via RAS activation.

Annexin A2 and FTH1 are potential biomarkers for lupus nephritis.

Lupus nephritis (LN) occurs in ~50% of patients with systemic lupus erythematosus and is a major cause of morbidity and mortality of the affected individuals. Therefore, identification of novel and predictive biomarkers for the early diagnosis and progression of LN is required. The present study included 10 patients with LN whose diagnoses were confirmed by renal biopsy and 5 healthy participants as control subjects. Sera were collected both from patients with LN and healthy controls. Subsequently, mesangial cells were treated with these sera for 24 h. Differential proteins between groups were detected by two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis. 2D-DIGE maps of cellar proteins were obtained for LN and normal control groups. A total of 45 proteins were characterized, and 2 low-abundance proteins were identified. Compared with the normal human sera group, expression level of Annexin A2 was elevated in patients with LN, while the expression of the ferritin heavy chain (FTH1) decreased in the LN group; the analysis was carried out by DeCyder version 7.0 automatically. The results of the present study suggest that Annexin A2 and FTH1 contributed to the progression of LN and could serve as potential biomarkers for this disease.

Fibroblast-Specific ß-Catenin Signaling Dictates the Outcome of AKI.

AKI is a devastating condition with high morbidity and mortality. The pathologic features of AKI are characterized by tubular injury, inflammation, and vascular impairment. Whether fibroblasts in the renal interstitium have a role in the pathogenesis of AKI is unknown. In this study, we investigated the role of fibroblast-specific ß-catenin signaling in dictating the outcome of AKI, using conditional knockout mice in which ß-catenin was specifically ablated in fibroblasts (Gli1-ß-cat-/-). After ischemia-reperfusion injury (IRI), Gli1-ß-cat-/- mice had lower serum creatinine levels and less morphologic injury than Gli1-ß-cat+/+ littermate controls. Moreover, we detected fewer apoptotic cells, as well as decreased cytochrome C release; reduced expression of Bax, FasL, and p53; and increased phosphorylation of Akt, in the Gli1-ß-cat-/- kidneys. Gli1-ß-cat-/- kidneys also exhibited upregulated expression of proliferating cell nuclear antigen and Ki-67, which are markers of cell proliferation. Furthermore, Gli1-ß-cat-/- kidneys displayed suppressed NF-κB signaling and cytokine expression and reduced infiltration of inflammatory cells. Notably, loss of ß-catenin in fibroblasts induced renal expression of hepatocyte growth factor (HGF) and augmented the tyrosine phosphorylation of c-met receptor after IRI. In vitro, treatment with Wnt ligands or ectopic expression of active ß-catenin inhibited HGF mRNA and protein expression and repressed HGF promoter activity. Collectively, these results suggest that fibroblast-specific ß-catenin signaling can control tubular injury and repair in AKI by modulating HGF expression. Our studies uncover a previously unrecognized role for interstitial fibroblasts in the pathogenesis of AKI.

Tubule-Derived Wnts Are Required for Fibroblast Activation and Kidney Fibrosis.

Cell-cell communication via Wnt ligands is necessary in regulating embryonic development and has been implicated in CKD. Because Wnt ligands are ubiquitously expressed, the exact cellular source of the Wnts involved in CKD remains undefined. To address this issue, we generated two conditional knockout mouse lines in which Wntless (Wls), a dedicated cargo receptor that is obligatory for Wnt secretion, was selectively ablated in tubular epithelial cells or interstitial fibroblasts. Blockade of Wnt secretion by genetic deletion of Wls in renal tubules markedly inhibited myofibroblast activation and reduced renal fibrosis after unilateral ureteral obstruction. This effect associated with decreased activation of ß-catenin and downstream gene expression and preserved tubular epithelial integrity. In contrast, fibroblast-specific deletion of Wls exhibited little effect on the severity of renal fibrosis after obstructive or ischemia-reperfusion injury. In vitro, incubation of normal rat kidney fibroblasts with tubule-derived Wnts promoted fibroblast proliferation and activation. Furthermore, compared with kidney specimens from patients without CKD, biopsy specimens from patients with CKD also displayed increased expression of multiple Wnt proteins, predominantly in renal tubular epithelium. These results illustrate that tubule-derived Wnts have an essential role in promoting fibroblast activation and kidney fibrosis via epithelial-mesenchymal communication.

Matrix Metalloproteinase-7 Is a Urinary Biomarker and Pathogenic Mediator of Kidney Fibrosis.

Matrix metalloproteinase-7 (MMP-7), a secreted zinc- and calcium-dependent endopeptidase, is a transcriptional target of canonical Wnt/ß-catenin signaling. Because Wnt/ß-catenin is activated in diseased kidney, we hypothesized that urinary MMP-7 level may be used as a noninvasive surrogate biomarker for fibrotic lesions. To test this hypothesis, we conducted a cross-sectional study, measuring urinary MMP-7 levels in a cohort of 102 patients with CKD. Compared with normal subjects, patients with various kidney disorders had markedly elevated urinary levels of MMP-7. Furthermore, urinary MMP-7 levels closely correlated with renal fibrosis scores in patients. In mice, knockout of MMP-7 ameliorated the fibrotic lesions and expression of matrix genes induced by obstructive injury. Genetic ablation of MMP-7 also preserved E-cadherin protein expression and substantially reduced the expression of total and dephosphorylated ß-catenin and the de novo expression of vimentin and fibroblast-specific protein 1 in renal tubules of obstructed kidneys. In vitro, MMP-7 proteolytically degraded E-cadherin in proximal tubular cells, leading to ß-catenin liberation and nuclear translocation and induction of ß-catenin target genes by a mechanism independent of Wnt ligands. Finally, pharmacologic inhibition of MMP-7 immediately after obstructive injury reduced renal fibrosis in vivo These results suggest that MMP-7 not only can serve as a noninvasive biomarker but also is an important pathogenic mediator of kidney fibrosis.

Sustained Activation of Wnt/ß-Catenin Signaling Drives AKI to CKD Progression.

AKI is increasingly recognized as a major risk factor for progression to CKD. However, the factors governing AKI to CKD progression are poorly understood. In this study, we investigated this issue using moderate (20 minutes) and severe (30 minutes) ischemia/reperfusion injury (IRI) in mice. Moderate IRI led to acute kidney failure and transient Wnt/ß-catenin activation, which was followed by the restoration of kidney morphology and function. However, severe IRI resulted in sustained and exaggerated Wnt/ß-catenin activation, which was accompanied by development of renal fibrotic lesions characterized by interstitial myofibroblast activation and excessive extracellular matrix deposition. To assess the role of sustained Wnt/ß-catenin signaling in mediating AKI to CKD progression, we manipulated this signaling by overexpression of Wnt ligand or pharmacologic inhibition of ß-catenin. In vivo, overexpression of Wnt1 at 5 days after IRI induced ß-catenin activation and accelerated AKI to CKD progression. Conversely, blockade of Wnt/ß-catenin by small molecule inhibitor ICG-001 at this point hindered AKI to CKD progression. In vitro, Wnt ligands induced renal interstitial fibroblast activation and promoted fibronectin expression. However, activated fibroblasts readily reverted to a quiescent phenotype after Wnt ligands were removed, suggesting that fibroblast activation requires persistent Wnt signaling. These results indicate that sustained, but not transient, activation of Wnt/ß-catenin signaling has a decisive role in driving AKI to CKD progression.

Extracellular Superoxide Dismutase Protects against Proteinuric Kidney Disease.

Extracellular superoxide dismutase (EC-SOD), also known as SOD3, is an antioxidant expressed at high levels in normal adult kidneys. Because oxidative stress contributes to a variety of kidney injuries, we hypothesized that EC-SOD may be protective in CKD progression. To study this hypothesis, we used a murine model of ADR nephropathy characterized by albuminuria and renal dysfunction. We found that levels of EC-SOD diminished throughout the course of disease progression and were associated with increased levels of NADPH oxidase and oxidative stress markers. EC-SOD null mice were sensitized to ADR injury, as evidenced by increases in albuminuria, serum creatinine, histologic damage, and oxidative stress. The absence of EC-SOD led to increased levels of NADPH oxidase and an increase in ß-catenin signaling, which has been shown to be pathologic in a variety of kidney injuries. Exposure of EC-SOD null mice to either chronic angiotensin II infusion or to daily albumin injections also caused increased proteinuria. In contrast, EC-SOD null mice subjected to nonproteinuric CKD induced by unilateral ureteral obstruction exhibited no differences compared with wild-type mice. Finally, we also found a decrease in EC-SOD in human CKD biopsy samples, similar to our findings in mice. Therefore, we conclude that EC-SOD is protective in CKDs characterized by proteinuria.

Sonic hedgehog is a novel tubule-derived growth factor for interstitial fibroblasts after kidney injury.

Tubular epithelium constitutes the majority of the renal parenchyma and is the primary target of various kidney injuries. However, how the injured tubules drive interstitial fibroblast activation and proliferation remains poorly understood. Here, we investigated the role of sonic hedgehog (Shh), a secreted extracellular signaling protein, in fibroblast proliferation. Shh was induced in renal tubular epithelia in animal models of CKD induced by ischemia/reperfusion injury (IRI), adriamycin, or renal mass ablation, and in renal tubules of kidney biopsy specimens from CKD patients with different etiologies. Using Gli1-CreER(T2) reporter mice, we identified interstitial fibroblasts as the principal targets of renal Shh signaling in vivo. In vitro, incubation with Shh promoted normal rat kidney fibroblast proliferation, which was assessed by cell counting, MTT assay, and BrdU incorporation assay, and stimulated the induction of numerous proliferation-related genes. However, Shh had no effect on the proliferation of renal tubular epithelial cells. In vivo, overexpression of Shh promoted fibroblast expansion and aggravated kidney fibrotic lesions after IRI. Correspondingly, blockade of Shh signaling by cyclopamine, a small molecule inhibitor of Smoothened, inhibited fibroblast proliferation, reduced myofibroblast accumulation, and attenuated renal fibrosis. These studies identify Shh as a novel, specific, and potent tubule-derived growth factor that promotes interstitial fibroblast proliferation and activation. Our data also suggest that blockade of Shh signaling is a plausible strategy for therapeutic intervention of renal fibrosis.