Pirfenidone Is Renoprotective in Diabetic Kidney Disease

Although several interventions slow the progression of diabetic nephropathy, current therapies do not halt progression completely. Recent preclinical studies suggested that pirfenidone (PFD) prevents fibrosis in various diseases, but the mechanisms underlying its antifibrotic action are incompletely understood. Here, we evaluated the role of PFD in regulation of the extracellular matrix. In mouse mesangial cells, PFD decreased TGF-β promoter activity, reduced TGF-β protein secretion, and inhibited TGF-β–induced Smad2-phosphorylation, 3TP-lux promoter activity, and generation of reactive oxygen species. To explore the therapeutic potential of PFD, we administered PFD to 17-wk-old db/db mice for 4 wk. PFD treatment significantly reduced mesangial matrix expansion and expression of renal matrix genes but did not affect albuminuria. Using liquid chromatography with subsequent electrospray ionization tandem mass spectrometry, we identified 21 proteins unique to PFD-treated diabetic kidneys. Analysis of gene ontology and protein–protein interactions of these proteins suggested that PFD may regulate RNA processing. Immunoblotting demonstrated that PFD promotes dosage-dependent dephosphorylation of eukaryotic initiation factor, potentially inhibiting translation of mRNA. In conclusion, PFD is renoprotective in diabetic kidney disease and may exert its antifibrotic effects, in part, via inhibiting RNA processing.Diabetic nephropathy (DN) is the single major cause of the emerging epidemic of ESRD in the United States,¹ accounting for nearly 50% of all new cases.² Characteristic morphologic lesions of DN include glomerular hypertrophy, thickening of the basement membrane, and mesangial expansion.³ Several interventions, such as tight glycemic control and antihypertensive therapy, especially angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers,^4–9 have been shown to slow the progression of established disease. Nevertheless, DN remains a major long-term complication of both types 1 and 2 diabetes,^10,11 because treatment commenced after the manifestation of overt clinical nephropathy often does not arrest progression to ESRD.¹¹ The annual medical cost for treatment of patients with diabetes ESRD is expected to be $18 to 30 billion (US) during the next decade.^12–15 It is therefore imperative to identify novel drug-therapeutic regimens that can ideally arrest further progression of the disease after manifestation of nephropathy.Pirfenidone (PFD; 5-methyl-1-phenyl-2-(1H)-pyridone) is a low molecular weight synthetic molecule that exerts dramatic antifibrotic properties in cell culture and various animal models of fibrosis.^16,17 PFD has emerged as a promising oral treatment with few adverse effects in open-label clinical studies. A study of hemodialysis patients with a history of sclerosing peritonitis demonstrated that it may not be necessary to adjust dosages of PFD for renal impairment and that the drug is well tolerated even in ESRD.¹⁸ In an open-label study wherein PFD was administered to patients with advanced refractory focal sclerosis, there was a good safety profile in patients with impaired renal function and heavy proteinuria, and PFD slowed the rate of decline of renal function by 25%.¹⁹ In a Phase III trial for patients with idiopathic pulmonary fibrosis in Japan, PFD was reported to promote stabilization and improvement of lung function.²⁰ Of note, there have been no reports that PFD may worsen renal blood flow, lower BP, affect glycemic parameters, or cause hyperkalemia, thereby making this treatment approach truly unique as compared with presently available renin-angiotensin-aldosterone antagonists. Thus, the combined experience with PFD in patients and in animal models of progressive kidney disease suggests that the compound is safe and may provide stabilization of renal function.To determine whether PFD is potentially beneficial in diabetic kidney disease, we studied the effects of PFD in cell culture experiments and in the db/db mouse model of diabetic kidney disease. In cell culture studies, PFD inhibited TGF-β production and TGF-β signaling and reduced TGF-β–induced reactive oxygen species (ROS) production. In the db/db mouse, PFD promoted resolution of mesangial matrix when administered after the onset of nephropathy. For identification of novel pathways of PFD relevant to DN, proteomic studies of the whole kidneys followed by bioinformatic analyses revealed RNA processing as a novel mechanism of PFD action. In support of a role of PFD to affect mRNA translation, PFD was found to regulate the activity of eukaryotic initiation factor (eIF4E), a key mRNA cap-structure binding protein, in mesangial cells in culture.     

Cyclodextrin Protects Podocytes in Diabetic Kidney Disease

Diabetic kidney disease (DKD) remains the most common cause of end-stage kidney disease despite multifactorial intervention. We demonstrated that increased cholesterol in association with downregulation of ATP-binding cassette transporter ABCA1 occurs in normal human podocytes exposed to the sera of patients with type 1 diabetes and albuminuria (DKD⁺) when compared with diabetic patients with normoalbuminuria (DKD⁻) and similar duration of diabetes and lipid profile. Glomerular downregulation of ABCA1 was confirmed in biopsies from patients with early DKD (n = 70) when compared with normal living donors (n = 32). Induction of cholesterol efflux with cyclodextrin (CD) but not inhibition of cholesterol synthesis with simvastatin prevented podocyte injury observed in vitro after exposure to patient sera. Subcutaneous administration of CD to diabetic BTBR (black and tan, brachiuric) ob/ob mice was safe and reduced albuminuria, mesangial expansion, kidney weight, and cortical cholesterol content. This was followed by an improvement of fasting insulin, blood glucose, body weight, and glucose tolerance in vivo and improved glucose-stimulated insulin release in human islets in vitro. Our data suggest that impaired reverse cholesterol transport characterizes clinical and experimental DKD and negatively influences podocyte function. Treatment with CD is safe and effective in preserving podocyte function in vitro and in vivo and may improve the metabolic control of diabetes.Diabetic kidney disease (DKD) is responsible for nearly half of the incidents of end-stage kidney disease in the U.S. (1), yet our current understanding of the pathophysiological processes responsible for DKD has led to limited improvements in patient outcomes. Multifactorial intervention reduces the rate of progression of DKD but does not prevent end-stage kidney disease in type 1 (T1D) or type 2 diabetes (T2D) (2,3). A key factor for this translation gap is the current lack of adequate mechanistic insight into DKD in humans.The kidney glomerulus is a highly specialized structure that ensures the selective ultrafiltration of plasma so that essential proteins are retained in the blood (4). Podocytes are glomerular epithelial cells that contribute to the glomerular filtration barrier through a tight regulation of actin cytoskeleton remodeling (4). Currently, the diagnosis of DKD relies on the detection of microalbuminuria (5). However, a growing body of evidence suggests that key histological lesions precede the development of albuminuria (6,7); among them, decreased podocyte number (podocytopenia) has been described as an independent predictor of DKD progression (8–12). Although we have previously shown that podocyte insulin resistance and susceptibility to apoptosis is already present at the time of onset of microalbuminuria in experimental models of DKD, the cause of podocyte injury in early DKD remains unknown (13).We used a previously established cell-based assay in which differentiated human podocytes are exposed to 4% patient sera for 24 h (14) to identify new pathways and targets in DKD. Podocytes exposed to the sera of patients with DKD showed increased cholesterol accumulation in association with downregulation of ATP-binding cassette transporter 1 (ABCA1) expression that was independent of circulating cholesterol.ABCA1 is a major regulator of cellular cholesterol homeostasis by mediating efflux to lipid-poor apolipoprotein acceptors in the bloodstream (15). ABCA1 genetic variants are strongly associated with the risk of coronary artery disease (16). Furthermore, the capacity of patient sera to induce ABCA1-mediated cholesterol efflux in macrophages is impaired in patients with T2D and incipient or overt nephropathy (17). Excessive cholesterol accumulation has been described in glomeruli of rodent models of T1D and T2D (18–20) and may contribute to DKD development and progression. Finally, inactivating mutations of ABCA1 result in Tangier disease, which causes premature atherosclerosis and proteinuria (21).Although interventions that increase ABCA1 expression (such as liver X receptor agonists) may be beneficial in DKD, they have a relatively high incidence of adverse events (22) as well as intrinsic lipogenic effects (23). We used β-cyclodextrins, cyclic oligosaccharides consisting of seven β(1-4)-glucopyranose rings, to remove cholesterol from differentiated human podocytes in vitro and from diabetic animals in vivo. The exact mechanism by which cyclodextrins (CDs) remove cholesterol from cells is not completely understood, but the formation of cholesterol/CD inclusion complexes at the membrane surface plays an important role in this process (24).We hypothesized that 2-hydroxypropyl-β-cyclodextrin, which was recently approved by the U.S. Food and Drug Administration (FDA) for the cure of Niemann-Pick disorder (25,26), would be an effective way to sequester cholesterol and to protect podocytes from cholesterol-dependent damage in DKD in vivo and in vitro.     

Cardiovascular Disease Reduction in the Outpatient Kidney Transplant Clinic

Cardiovascular disease (CVD) is an important cause of death in kidney transplant recipients. Future CVD mortality was estimated by a risk calculator in all patients (n = 439) with a functioning transplant (>6 months), followed at our center. In addition to CURRENT mortality rates, an OPTIMAL rate (adding anti-hypertensive and lipid-lowering therapy in uncontrolled patients) and an HISTORIC rate (higher systolic blood pressures and the absence of statin use in our population 5 years ago) were also calculated. Overall, the predicted CURRENT CVD mortality rates are 0.82 (95% CI 0.81-0.83) of HISTORIC rates. Predicted OPTIMAL CVD mortality rates are 0.90 (95% CI 0.87-0.92) of CURRENT rates. To achieve OPTIMAL rates, a 27% increase in blood pressure and lipid-lowering drug use is required. There were few contraindications to these medications, implying that physician prescribing was the major barrier to minimizing risk. Despite OPTIMAL rates, the transplant population's relative risk is 2.3 (median, 95% CI 2.1-2.5) times higher than that in the general population. Therefore, targeted therapy to reduce CVD risk can have substantial benefit, but CVD mortality may continue to exceed that in the general population.     

Early Prediction of Cardiovascular Disease in Kidney Transplant Recipients

Cardiovascular disease (CVD) is frequent after kidney transplantation (KT). This study investigated CVD prediction in KT by information available before KT or within 6 months after KT. The study cohort consisted of 629 patients with KT in 2005–10 and with adult age at KT. The end point was incidence up to 2015 of CVD (coronary heart disease, cerebrovascular disease, peripheral artery disease). Graft failure, non-CVD death with functioning graft, and loss to follow-up were considered competing events. CVD prediction was investigated for 34 variables by means of competing-risks regression. Follow-up range was 0.28–10.00 years (mean ± SD, 7.30 ± 3.10). First incident event was CVD in 103 patients and competing events in 146 patients. In the multivariable model for pre-KT variables only, CVD predictors were male sex (hazard ratio [HR], 1.68; 95% confidence interval [CI], 1.06–2.66), diabetic nephropathy (HR, 6.63; 95% CI, 1.81–24.35), pre-KT dialysis for ≥5 years (HR, 1.52; 95% CI, 1.02–2.27), pre-KT CVD (HR, 4.87; 95% CI, 2.84–8.35), and age at KT ≥45 years (HR, 2.98; 95% CI, 1.83–4.87). In the model for pre-KT and post-KT variables together, the sole post-KT CVD predictor was estimated glomerular filtration rate <60 mL/min at the 6-month visit (HR, 1.75; 95% CI, 1.11–2.77). Diabetic nephropathy, pre-KT dialysis, pre-KT CVD, and age at KT predicted 91.2% of incident CVD. Early available information effectively predicted CVD in KT independently from competing events.