Evidence of a Role for Fibroblast Transient Receptor Potential Canonical 3 Ca2+ Channel in Renal Fibrosis

Transient receptor potential canonical (TRPC) Ca²⁺-permeant channels, especially TRPC3, are increasingly implicated in cardiorenal diseases. We studied the possible role of fibroblast TRPC3 in the development of renal fibrosis. In vitro, a macromolecular complex formed by TRPC1/TRPC3/TRPC6 existed in isolated cultured rat renal fibroblasts. However, specific blockade of TRPC3 with the pharmacologic inhibitor pyr3 was sufficient to inhibit both angiotensin II- and 1-oleoyl-2-acetyl-sn-glycerol–induced Ca²⁺ entry in these cells, which was detected by fura-2 Ca²⁺ imaging. TRPC3 blockade or Ca²⁺ removal inhibited fibroblast proliferation and myofibroblast differentiation by suppressing the phosphorylation of extracellular signal-regulated kinase (ERK1/2). In addition, pyr3 inhibited fibrosis and inflammation-associated markers in a noncytotoxic manner. Furthermore, TRPC3 knockdown by siRNA confirmed these pharmacologic findings. In adult male Wistar rats or wild-type mice subjected to unilateral ureteral obstruction, TRPC3 expression increased in the fibroblasts of obstructed kidneys and was associated with increased Ca²⁺ entry, ERK1/2 phosphorylation, and fibroblast proliferation. Both TRPC3 blockade in rats and TRPC3 knockout in mice inhibited ERK1/2 phosphorylation and fibroblast activation as well as myofibroblast differentiation and extracellular matrix remodeling in obstructed kidneys, thus ameliorating tubulointerstitial damage and renal fibrosis. In conclusion, TRPC3 channels are present in renal fibroblasts and control fibroblast proliferation, differentiation, and activation through Ca²⁺-mediated ERK signaling. TRPC3 channels might constitute important therapeutic targets for improving renal remodeling in kidney disease.     

Inhibition of Thrombin-Activated Fibrinolysis Inhibitor Increases Survival in Experimental Kidney Fibrosis

Uncontrolled diabetes, inflammation, and hypertension are key contributors to progressive renal fibrosis and subsequent loss of renal function. Reduced fibrinolysis appears to be a feature of ESRD, but its contribution to the fibrotic program has not been extensively studied. Here, we show that in patients with CKD, the activity levels of serum thrombin-activated fibrinolysis inhibitor and plasmin strongly correlated with the degree of renal function impairment. We made similar observations in rats after subtotal nephrectomy and tested whether pharmacologic inhibition of thrombin-activated fibrinolysis inhibitor with UK-396082 could reduce renal fibrosis and improve renal function. Compared with untreated animals, UK-396082–treated animals had reduced glomerular and tubulointerstitial fibrosis after subtotal nephrectomy. Renal function, as measured by an increase in creatinine clearance, was maintained and the rate of increase in proteinuria was reduced in UK-396082–treated animals. Furthermore, cumulative survival improved from 16% to 80% with inhibition of thrombin-activated fibrinolysis inhibitor. Taken together, these data support the importance of the fibrinolytic axis in regulating renal fibrosis and point to a potentially important therapeutic role for suppression of thrombin-activated fibrinolysis inhibitor activity.     

EGF Receptor Deletion in Podocytes Attenuates Diabetic Nephropathy

The generation of reactive oxygen species (ROS), particularly superoxide, by damaged or dysfunctional mitochondria has been postulated to be an initiating event in the development of diabetes complications. The glomerulus is a primary site of diabetic injury, and podocyte injury is a classic hallmark of diabetic glomerular lesions. In streptozotocin-induced type 1 diabetes, podocyte-specific EGF receptor (EGFR) knockout mice (EGFR^podKO) and their wild-type (WT) littermates had similar levels of hyperglycemia and polyuria, but EGFR^podKO mice had significantly less albuminuria and less podocyte loss compared with WT diabetic mice. Furthermore, EGFR^podKO diabetic mice had less TGF-β1 expression, Smad2/3 phosphorylation, and glomerular fibronectin deposition. Immunoblotting of isolated glomerular lysates revealed that the upregulation of cleaved caspase 3 and downregulation of Bcl2 in WT diabetic mice were attenuated in EGFR^podKO diabetic mice. Administration of the SOD mimetic mito-tempol or the NADPH oxidase inhibitor apocynin attenuated the upregulation of p-c-Src, p-EGFR, p-ERK1/2, p-Smad2/3, and TGF-β1 expression and prevented the alteration of cleaved caspase 3 and Bcl2 expression in glomeruli of WT diabetic mice. High-glucose treatment of cultured mouse podocytes induced similar alterations in the production of ROS; phosphorylation of c-Src, EGFR, and Smad2/3; and expression of TGF-β1, cleaved caspase 3, and Bcl2. These alterations were inhibited by treatment with mito-tempol or apocynin or by inhibiting EGFR expression or activity. Thus, results of our studies utilizing mice with podocyte-specific EGFR deletion demonstrate that EGFR activation has a major role in activating pathways that mediate podocyte injury and loss in diabetic nephropathy.     

Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation

BackgroundAKI is characterized by abrupt and reversible kidney dysfunction, and incomplete recovery leads to chronic kidney injury. Previous studies by us and others have indicated that macrophage infiltration and polarization play key roles in recovery from AKI. The role in AKI recovery played by IFN regulatory factor 4 (IRF4), a mediator of polarization of macrophages to the M2 phenotype, is unclear.MethodsWe used mice with myeloid or macrophage cell–specific deletion of Irf4 (MΦ Irf4 ^−/−) to evaluate Irf4’s role in renal macrophage polarization and development of fibrosis after severe AKI.ResultsSurprisingly, although macrophage Irf4 deletion had a minimal effect on early renal functional recovery from AKI, it resulted in decreased renal fibrosis 4 weeks after severe AKI, in association with less-activated macrophages. Macrophage Irf4 deletion also protected against renal fibrosis in unilateral ureteral obstruction. Bone marrow–derived monocytes (BMDMs) from MΦ Irf4 ^−/− mice had diminished chemotactic responses to macrophage chemoattractants, with decreased activation of AKT and PI3 kinase and increased PTEN expression. PI3K and AKT inhibitors markedly decreased chemotaxis in wild-type BMDMs, and in a cultured macrophage cell line. There was significant inhibition of homing of labeled Irf4 ^−/− BMDMs to postischemic kidneys. Renal macrophage infiltration in response to AKI was markedly decreased in MΦ Irf4 ^−/− mice or in wild-type mice with inhibition of AKT activity.ConclusionsDeletion of Irf4 from myeloid cells protected against development of tubulointerstitial fibrosis after severe ischemic renal injury in mice, due primarily to inhibition of AKT-mediated monocyte recruitment to the injured kidney and reduced activation and subsequent polarization into a profibrotic M2 phenotype.     

Disappearance of Tophi in Familial Juvenile Hyperuricemic Nephropathy after Kidney Transplantation

A 40-year-old man who had been on hemodialysis for 25 months due to familial juvenile hyperuricemic nephropathy (FJHN) received a kidney transplant. Biopsy of his native kidney had shown tubulo-interstitial nephropathy. Genetic analysis confirmed abnormal uromodulin expression due to a mutation in the exon 4 of the UMOD gene. He had multiple tophi on the day of transplantation, including some on his fingers. He received immunosuppressive treatment including polyclonal antilymphocyte antibodies, mycophenolate mofetil, steroids and cyclosporine and achieved excellent renal function, with serum creatinine at 13 mg/L on day 10 posttransplantation and 9.4 mg/L at 6 months. His uric acid excretion rate increased from 4.4% at day 2 posttransplantation to 7.7% 6 months after transplantation. The number and sizes of the tophi were reduced 3 months posttransplantation, and nearly disappeared at month 6. Serum uric acid level decreased slowly from 650 mumol/L before transplantation to 300 mumol/L. Reduction of tophi was probably due to the absence of the mutated UMOD gene in the transplanted kidney.     

Therapeutic Targets in the Treatment of Allograft Fibrosis

The dramatic improvements in short-term graft survival and acute rejection rates could only have been dreamed of 20 years ago. Late graft loss following kidney transplantation is now the critical issue of this decade. Frequently, graft loss is associated with the development of tubular atrophy and interstitial fibrosis within the kidney (i.e. chronic allograft nephropathy; CAN). Major treatment strategies in this disorder are non-specific and the focus of intervention has been on limiting injurious events. Following graft injury is a fibrogenesis phase featuring both proliferative and infiltrative responses mediated by chemokines, cytokines and growth factors. In particular, TGFβ has been strongly implicated in the pathogenesis of chronic injury and epithelial-mesenchymal transformation (EMT) may be part of this process. The cascade of events results in matrix accumulation, due to either increased production and/or reduced degradation of matrix. Recent investigations into the pathogenesis of tissue fibrosis have suggested a number of new strategies to ameliorate matrix synthesis. While the majority of therapies have focused on TGFβ, this may not be an ideal maneuver in transplant settings and alternative targets identified in other fibrotic diseases will be discussed. Attacking graft fibrosis should be a new focus in organ transplantation.     

Mineralocorticoid Receptor Antagonist for Renal Protection

The renin–angiotensin system (RAS) plays an important role in the pathophysiology of cardiovascular and renal diseases. In chronic kidney disease (CKD), blockade of RAS by angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) has been shown to reduce proteinuria and retard the progression of renal function deterioration. However, aldosterone, another key hormone of the RAS, is not directly targeted by ACEI or ARB. Hyperaldosteronism, apart from promoting sodium and fluid retention, causes inflammation and fibrosis in the heart and kidney. Studies have shown that although plasma aldosterone level shows an initial decrease following ACEI or ARB treatment, it returns to pretreatment level or even increases paradoxically after prolonged treatment. This “aldosterone breakthrough” forms the basis of adding mineralocorticoid receptor (MR) antagonist on top of ACEI or ARB for renal protection. New insights into the pathophysiological role of aldosterone in CKD further expands its potential indications, and there was a growing body of evidence in the past 10 years, which showed a substantial antiproteinuric effect and possibly a considerable renoprotective effect of MR antagonist. Since aldosterone does not act on the efferent glomerular arteriole and has no effect on intraglomerular hemodynamics, the very fact that MR antagonist ameliorates proteinuria sheds light on the physiology of glomerular permeability barrier. This review summarizes the data regarding the theoretical benefit as well as clinical use of MR antagonist in renal diseases.     

Kidney tissue elastography and interstitial fibrosis observed in kidney biopsy

IntroductionKidney interstitial fibrosis is an important risk factor for the progression of chronic kidney disease. Kidney elastography is a noninvasive imaging modality that might be used to assess tissue fibrosis. In this study, we aimed to investigate the relationship between tissue stiffness detected in kidney elastography and interstitial fibrosis observed in kidney biopsy.Materials and methodsPatients who were hospitalized in a tertiary care university hospital with a kidney biopsy indication were included in this study. In all patients, the transverse and sagittal elastography measurements were made using a sonoelastography device before the biopsy. The total histological score was calculated.ResultsFifty-seven native kidney patients with proteinuria were included in the study. Patients were divided into two groups according to the presence (n = 6) and absence of fibrosis (n = 51) as detected by kidney biopsy. A significant correlation was found between the presence of fibrosis detected by biopsy and elastography outcomes (p = .046, r = .192). A significant correlation was found between the urea and creatinine levels and transverse elastography measurements (p = .036, r = .240). No correlation was observed between the transverse elastography measurements and total histological score consisting of glomerular, vascular, and tubular scores (r = .006, p = .967)ConclusionThe findings of our study suggest a significant relationship between the elastography measurements and interstitial fibrosis. Because of the high negative predictive value (91%), we suggest that elastography should mainly be used as an exclusion test for the presence of fibrosis. We also believe that elastography may be useful to evaluate the fibrosis status in kidney diseases.     

Inhibition of PAX2 Gene Expression by siRNA (Polyethylenimine) in Experimental Model of Obstructive Nephropathy

Objective: It was found that PAX2 (Paired Box 2) re-expression in renal tubular epithelial cells correlated with renal interstitial fibrosis of rats with obstructive nephropathy. The purpose of the present study was to identify whether RNA interference (RNAi) induced by polyethylenimine (jetPEI) could inhibit PAX2 gene re-expression and impact renal interstitial fibrosis of rats with obstructive nephropathy. Methods: Four pairs of small interfering RNA (siRNA) interference sequences and a negative control were designed and synthesized according to the whole PAX2 gene mRNA sequence. siRNA was then transfected into renal capsule in a rodent model of unilateral ureteral obstruction (UUO), using in vivo jetPEI as delivery vector. The expression of PAX2 mRNA and protein in renal tissue was determined by real-time quantitative polymerase chain reaction (PCR) and Western blot, respectively. A carboxyfluorescein (FAM)-labeled negative control was also synthesized at the same time to identify transfection in vivo. Renal interstitial fibrosis was observed under light microscope after PAX2 gene silenced. Results: Green fluorescence was observed around renal tubule epithelial cells by fluorescence microscopy 3 days after the procedure validating the success of transfection. While the PAX2 gene expression was inhibited by all siRNA interference sequences, the strongest inhibition was observed with siRNA3. The PAX2 mRNA and protein were inhibited by 55% and 81%, respectively. Renal tubular damage and renal interstitial fibrosis were remitted obviously after PAX2 gene silenced. Conclusion: siRNA was able to inhibit the expression of PAX2 in tubular epithelial cells in the UUO model. The siRNA may thus provide therapeutic potential for retarding the pathological progression of UUO.     

Anti‐Phospholipase A2 Receptor Antibodies in Recurrent Membranous Nephropathy

About 70% of patients with primary membranous nephropathy (MN) have circulating anti‐phospholipase A₂ receptor (PLA₂R) antibodies that correlate with disease activity, but their predictive value in post‐transplant (Tx) recurrent MN is uncertain. We evaluated 26 patients, 18 with recurrent MN and 8 without recurrence, with serial post‐Tx serum samples and renal biopsies to determine if patients with pre‐Tx anti‐PLA₂R are at increased risk of recurrence as compared to seronegative patients and to determine if post‐Tx changes in anti‐PLA₂R correspond to the clinical course. In the recurrent group, 10/17 patients had anti‐PLA₂R at the time of Tx versus 2/7 patients in the nonrecurrent group. The positive predictive value of pre‐Tx anti‐PLA₂R for recurrence was 83%, while the negative predictive value was 42%. Persistence or reappearance of post‐Tx anti‐PLA₂R was associated with increasing proteinuria and resistant disease in 6/18 cases; little or no proteinuria occurred in cases with pre‐Tx anti‐PLA₂R and biopsy evidence of recurrence in which the antibodies resolved with standard immunosuppression. Some cases with positive pre‐Tx anti‐PLA₂R were seronegative at the time of recurrence. In conclusion, patients with positive pre‐Tx anti‐PLA₂R should be monitored closely for recurrent MN. Persistence or reappearance of antibody post‐Tx may indicate a more resistant disease.     

Potential Therapeutic Targets for Cisplatin-Induced Kidney Injury: Lessons from Other Models of AKI and Fibrosis

The effectiveness of cisplatin, a mainstay in the treatment of many solid organ cancers, is hindered by dose-limiting nephrotoxicity. Cisplatin causes AKI in 30% of patients. Patients who do not develop AKI by clinical standards during treatment are still at risk for long-term decline in kidney function and the development of CKD. The connection between AKI and CKD has become increasingly studied, with renal fibrosis a hallmark of CKD development. To prevent both the short- and long-term effects of cisplatin, researchers must use models that reflect both types of pathology. Although a lot is known about cisplatin-induced AKI, very little is known about the mechanisms by which repeated low levels of cisplatin lead to fibrosis development. In this review, strategies used in various rodent models to prevent kidney injury, its progression to fibrosis, or both, are examined to gain mechanistic insights and identify potential therapeutic targets for cisplatin-induced kidney pathologies. Reviewing the results from these models highlights the diverse and highly complex role of cell death, cell senescence, endoplasmic reticulum stress, autophagy, and immune cell activation in acute and chronic kidney injuries. The use of several models of kidney injury is needed for development of agents that will prevent all aspects of cisplatin-induced kidney injury.     

慢性肾脏病3期糖尿病肾病与非糖尿病肾病患者贫血特点及影响因素分析

目的：评估慢性肾脏病3期（CKD3）糖尿病肾病（ON）与非糖尿病肾病患者之间贫血特点的差异及影响因素。方法：选取2012年2月～2013年5月在上海市第六人民医院肾内科住院的CKD3患者,其中糖尿病肾病42例。非糖尿病肾病患者49例,检测患者红细胞计数（RBC）、血红蛋白（Hb）、总蛋白（TP）、白蛋白（mb）、促红细胞生长素（EPO）、铁蛋白、总铁结合力、血清铁、叶酸、维生素B12、肌酐、尿素氮、钙、磷、甲状旁腺素、25羟维生素D3等指标进行对比分析。结果：42例CKD3期糖尿病肾病患者总的贫血发生率为67％,49例非糖肾组患者总的贫血发生率为39％。糖尿病肾病组红细胞计数、血红蛋白、白蛋白、血清铁均明显低于非糖肾组,组间差异具有统计学意义（P〈0.05）,DN组促红细胞生长素水平高于非DN组（P〈0.05）。结论：CKD3糖尿病肾病患者的贫血程度较非糖肾组严重,其贫血的发生与低蛋白血症、糖尿病肾外因素及铁代谢异常密切相关。