Dual sEH/COX-2 Inhibition Using PTUPB—A Promising Approach to Antiangiogenesis-Induced Nephrotoxicity

Kidney injury from antiangiogenic chemotherapy is a significant clinical challenge, and we currently lack the ability to effectively treat it with pharmacological agents. Thus, we set out to investigate whether simultaneous soluble epoxide hydrolase (sEH) and cyclooxygenase-2 (COX-2) inhibition using a dual sEH/COX-2 inhibitor PTUPB could be an effective strategy for treating antiangiogenic therapy-induced kidney damage. We used a multikinase inhibitor, sorafenib, which is known to cause serious renal side effects. The drug was administered to male Sprague–Dawley rats that were on a high-salt diet. Sorafenib was administered over the course of 56 days. The study included three experimental groups; 1) control group (naïve rats), 2) sorafenib group [rats treated with sorafenib only (20 mg/kg/day p.o.)], and 3) sorafenib + PTUPB group (rats treated with sorafenib only for the initial 28 days and subsequently coadministered PTUPB (10 mg/kg/day i.p.) from days 28 through 56). Blood pressure was measured every 2 weeks. After 28 days, sorafenib-treated rats developed hypertension (161 ± 4 mmHg). Over the remainder of the study, sorafenib treatment resulted in a further elevation in blood pressure through day 56 (200 ± 7 mmHg). PTUPB treatment attenuated the sorafenib-induced blood pressure elevation and by day 56, blood pressure was 159 ± 4 mmHg. Urine was collected every 2 weeks for biochemical analysis. After 28 days, sorafenib rats developed pronounced proteinuria (9.7 ± 0.2 P/C), which intensified significantly (35.8 ± 3.5 P/C) by the end of day 56 compared with control (2.6 ± 0.4 P/C). PTUPB mitigated sorafenib-induced proteinuria, and by day 56, it reduced proteinuria by 73%. Plasma and kidney tissues were collected on day 56. Kidney histopathology revealed intratubular cast formation, interstitial fibrosis, glomerular injury, and glomerular nephrin loss at day 56 in sorafenib-treated rats. PTUPB treatment reduced histological features by 30%–70% compared with the sorafenib-treated group and restored glomerular nephrin levels. Furthermore, PTUPB also acted on the glomerular permeability barrier by decreasing angiotensin-II-induced glomerular permeability to albumin. Finally, PTUPB improved in vitro the viability of human mesangial cells. Collectively, our data demonstrate the potential of using PTUPB or dual sEH/COX-2 inhibition as a therapeutic strategy against sorafenib-induced glomerular nephrotoxicity.

Integrated Metabolomics and Network Pharmacology to Establish the Action Mechanism of Qingrekasen Granule for Treating Nephrotic Syndrome

Nephrotic syndrome (NS) is a clinical syndrome resulting from abnormal glomerular permeability, mainly manifesting as edema and proteinuria. Qingrekasen granule (QRKSG), a Chinese Uyghur folk medicine, is a single-flavor preparation made from chicory (Cichorium intybus L.), widely used in treating dysuria and edema. Chicory, the main component in QRKSG, effectively treats edema and protects kidneys. However, the active components in QRKSG and its underlying mechanism for treating NS remain unclear. This study explored the specific mechanism and composition of QRKSG on an NS rat model using integrated metabolomics and network pharmacology. First, metabolomics explored the relevant metabolic pathways impacted by QRKSG in the treatment of NS. Secondly, network pharmacology further explored the possible metabolite targets. Afterward, a comprehensive network was constructed using the results from the network pharmacology and metabolomics analysis. Finally, the interactions between the active components and targets were predicted by molecular docking, and the differential expression levels of the target protein were verified by Western blotting. The metabolomics results showed “D-Glutamine and D-glutamate metabolism” and “Alanine, aspartate, and glutamate metabolism” as the main targeted metabolic pathways for treating NS in rats. AKT1, BCL2L1, CASP3, and MTOR were the core QRKSG targets in the treatment of NS. Molecular docking revealed that these core targets have a strong affinity for flavonoids, terpenoids, and phenolic acids. Moreover, the expression levels of p-PI3K, p-AKT1, p-mTOR, and CASP3 in the QRKSG group significantly decreased, while BCL2L1 increased compared to the model group. These findings established the underlying mechanism of QRKSG, such as promoting autophagy and anti-apoptosis through the expression of AKT1, CASP3, BCL2L1, and mTOR to protect podocytes and maintain renal tubular function.

Merokok dan Gangguan Fungsi Ginjal

Smoking increases the production of angiotensin II as an effect of renin secretion stimulated by the efferent sympathetic system through beta-1 adrenergic stimulation of the juxtaglomerular apparatus. Angiotensin II will cause tubular and glomerular injuries through the mechanism of pressure-induced renal injury and ischemia-induced renal injury as a secondary result of intrarenal vasoconstriction and decreased renal blood flow. In addition, there is secondary tubular injury due to angiotensin-induced proteinuria. Angiotensin II activates renal fibroblasts to undergo differentiation into myofibroblasts, stimulates TGF-ß profibrotic cytokines, induces oxidative stress, stimulates chemokines and osteopontin which can cause local inflammation, and stimulates mesangial cell proliferation and hypertrophy. Glomerular capillary hypertension causes an increase in glomerular permeability resulting in an increase in albumin filtration which will further trigger kidney damage through various pathways, including induction of tubular chemokine expression and activation of complement leading to infiltration of inflammatory cells in the interstitium and trigger fibrogenesis. This phenomenon involves endothelial cells and glomerular podocytes and will trigger exacerbation of proteinuria and glomerulosclerosis with the end result in the formation of kidney scar tissue and a decrease in glomerular filtration rate (GFR).Keywords: smoking; renal function; TGF-ß; glomerular filtration rate (GFR) Abstrak: Merokok akan meningkatkan produksi angiotensin II sebagai efek dari sekresi renin yang distimulasi oleh sistim simpatik eferen melalui stimulasi beta-1 adrenergik pada aparatus jukstaglomerular. Angiotensin II akan menyebabkan cedera tubulus dan glomerulus melalui mekanisme pressure-induced renal injury dan ischemia-induced renal injury sebagai akibat sekunder dari vasokonstriksi intrarenal dan penurunan aliran darah ginjal. Selain itu terjadi cedera tubulus sekunder dari proteinuria yang diinduksi angiotensin. Angiotensin II akan mengaktifkan fibroblas ginjal berdiferensiasi menjadi miofibroblas, menstimulasi sitokin profibrotik TGF-ß, menginduksi stres oksidatif, menstimulasi kemokin dan osteopontin yang dapat menyebabkan inflamasi local, dan menstimulasi proliferasi dan hipertrofi sel mesangial. Hipertensi kapiler glomerulus akan menyebabkan peningkatan permeabilitas glomerulus sehingga terjadi peningkatan filtrasi albumin yang selanjutnya memicu kerusakan ginjal melalui berbagai jalur, diantaranya induksi ekspresi kemokin tubulus dan aktivasi komplemen yang akan mengarah pada infiltrasi sel-sel inflamasi pada interstisium dan memicu fibrogenesis. Fenomena ini melibatkan sel endotel dan podosit glomerulus dan akan mencetuskan eksaserbasi proteinuria dan glomerulosklerosis dengan hasil akhir berupa terbentuknya jaringan parut ginjal dan penurunan laju filtrasi glomerulus (LFG).Kata kunci: merokok; fungsi ginjal; TGF-ß; laju filtrasi glomerulus (LFG)

Podocyte Sphingolipid Signaling in Nephrotic Syndrome.

Podocytes play a vital role in the pathogenesis of nephrotic syndrome (NS), which is clinically characterized by heavy proteinuria, hypoalbuminemia, hyperlipidemia, and peripheral edema. The pathogenesis of NS has evolved through several hypotheses ranging from immune dysregulation theory and increased glomerular permeability theory to the current concept of podocytopathy. Podocytopathy is characterized by dysfunction or depletion of podocytes, which may be caused by unknown permeability factor, genetic disorders, drugs, infections, systemic disorders, and hyperfiltration. Over the last two decades, numerous studies have been done to explore the molecular mechanisms of podocyte injuries or NS and to develop the novel therapeutic strategies targeting podocytopathy for treatment of NS. Recent studies have shown that normal sphingolipid metabolism is essential for structural and functional integrity of podocytes. As a basic component of the plasma membrane, sphingolipids not only support the assembly of signaling molecules and interaction of receptors and effectors, but also mediate various cellular activities, such as apoptosis, proliferation, stress responses, necrosis, inflammation, autophagy, senescence, and differentiation. This review briefly summarizes current evidence demonstrating the regulation of sphingolipid metabolism in podocytes and the canonical or noncanonical roles of podocyte sphingolipid signaling in the pathogenesis of NS and associated therapeutic strategies.

Autoimmunity in Focal Segmental Glomerulosclerosis: A Long-Standing Yet Elusive Association

Focal segmental glomerulosclerosis (FSGS) is a histological term that describes a pathologic renal entity affecting both adults and children, with a wide array of possible underlying etiologies. Podocyte damage with scarring, the hallmark of this condition, leads to altered permeability of the glomerular barrier, which may result in massive proteinuria and relentless renal function deterioration. A definite cause of focal segmental glomerulosclerosis can be confirmed in a minority of cases, while most forms have been traditionally labeled as primary or idiopathic. Despite this definition, increasing evidence indicates that primary forms are a heterogenous group rather than a single disease entity: several circulating factors that may affect glomerular permeability have been proposed as potential culprits, and both humoral and cellular immunity have been implicated in the pathogenesis of the disease. Consistently, immunosuppressive drugs are considered as the cornerstone of treatment for primary focal segmental glomerulosclerosis, but response to these agents and long-term outcomes are highly variable. In this review we provide a summary of historical and recent advances on the pathogenesis of primary focal segmental glomerulosclerosis, focusing on implications for its differential diagnosis and treatment.

Non-purine selective xanthine oxidase inhibitor ameliorates glomerular endothelial injury in InsAkita diabetic mice

Endothelial dysfunction represents a predominant early feature of diabetes, rendering patients with diabetes prone to renal complications, e.g., proteinuria. Recent studies have indicated a possible role for xanthine oxidase (XO) in the pathogenesis of vascular dysfunctions associated with diabetes. In the present study, we investigated the contribution of XO activation on the progression of diabetic nephropathy in a mouse model using selective XO inhibitors. Male Ins2Akita heterozygous mice were used with wild-type mice as controls. Akita mice were treated with topiroxostat (Topi) or vehicle for 4 wk. Serum uric acid levels were significantly reduced in Akita + Topi mice compared with Akita + vehicle mice. The Akita + Topi group had a significant reduction in urinary albumin excretion compared with the Akita + vehicle group. Mesangial expansion, glomerular collagen type IV deposition, and glomerular endothelial injury (assessed by lectin staining and transmission electron microscopy) were considerably reduced in the Akita + topi group compared with the Akita + vehicle group. Furthermore, glomerular permeability was significantly higher in the Akita + vehicle group compared with the wild-type group. These changes were reduced with the administration of Topi. We conclude that XO inhibitors preserve glomerular endothelial functions and rescue compromised glomerular permeability, suggesting that XO activation plays a vital role in the pathogenesis of diabetic nephropathy.

Effect of angiotensin II on diabetic glomerular hyperpermeability: an in vivo permeability study in rats

Angiotensin II drives the pathogenesis of diabetic kidney disease, and its systemic administration induces glomerular hyperpermeability in normal rats. However, the response of diabetic glomerular permeability to angiotensin II is largely unknown. In the present study, we investigated the impact of extended systemic administration of angiotensin II on the glomerular permeability of streptozotocin (STZ)-induced late diabetes in rats. We examined the changes in the glomerular permeability after subcutaneous infusion of angiotensin II at 200 ng·kg−1·min−1 for 7 days in male Wistar diabetic rats with 3 mo of STZ-induced diabetes (i.e., blood glucose of ∼20 mmol/L). We also compared these changes with the effects on nondiabetic rats. The sieving coefficients (θ) for inert polydisperse Ficoll molecules, which had a radius of 10–90 Å (Ficoll70–90 Å), were measured in vivo. The θ for large Ficoll molecules was selectively enhanced after infusion of extended angiotensin II in both diabetic (θ for Ficoll70–90 Å = 0.00244 vs. 0.00079, P < 0.001) and nondiabetic animals (θ for Ficoll70–90 Å = 0.00029 vs. 0.00006, P < 0.001). These changes were compatible with the more than twofold increase in the macromolecular glomerular transport through the large-pore pathways after infusion of angiotensin II in both diabetic and nondiabetic animals. Angiotensin II infusion enhanced the large shunt-like glomerular transport pathway of STZ-induced late diabetes. Such defects can account for the large-molecular-weight IgM-uria that is observed in severe diabetic kidney disease.

The PKGIα/VASP pathway is involved in insulin- and high glucose-dependent regulation of albumin permeability in cultured rat podocytes

Podocytes, the principal component of the glomerular filtration barrier, regulate glomerular permeability to albumin via their contractile properties. Both insulin- and high glucose (HG)-dependent activation of protein kinase G type Iα (PKGIα) cause reorganization of the actin cytoskeleton and podocyte disruption. Vasodilator-stimulated phosphoprotein (VASP) is a substrate for PKGIα and involved in the regulation of actin cytoskeleton dynamics. We investigated the role of the PKGIα/VASP pathway in the regulation of podocyte permeability to albumin. We evaluated changes in high insulin- and/or HG-induced transepithelial albumin flux in cultured rat podocyte monolayers. Expression of PKGIα and downstream proteins was confirmed by western blot and immunofluorescence. We demonstrate that insulin and HG induce changes in the podocyte contractile apparatus via PKGIα-dependent regulation of the VASP phosphorylation state, increase VASP colocalization with PKGIα, and alter the subcellular localization of these proteins in podocytes. Moreover, VASP was implicated in the insulin- and HG-dependent dynamic remodelling of the actin cytoskeleton and, consequently, increased podocyte permeability to albumin under hyperinsulinaemic and hyperglycaemic conditions. These results indicate that insulin- and HG-dependent regulation of albumin permeability is mediated by the PKGIα/VASP pathway in cultured rat podocytes. This molecular mechanism may explain podocytopathy and albuminuria in diabetes.

P0089KEAP1/NRF2 PATHWAY REGULATES GFR BY INCREASING THE GLOMERULAR EFFECTIVE AREA WITHOUT AFFECTING THE AFFERENT/EFFERENT ARTERIOLE RATIO

Background and Aims The Keap1/Nrf2 pathway regulates the expression of a series of cytoprotective, anti-inflammatory and antioxidant genes. The Nrf2 activator, bardoxolone methyl (BARD), has consistently increased estimated GFR (eGFR) in clinical studies in patients with chronic kidney disease. BARD demonstrated improvement of renal function assessed by inulin clearance, the clinical gold standard for measuring GFR, in diabetic kidney disease patients. These findings suggest the Keap1/Nrf2 system is deeply involved in the regulatory mechanisms of GFR. However, the precise mechanisms are not fully elucidated. We pharmacologically and genetically investigated the mechanisms of GFR regulation by Keap1/Nrf2 system using in vivo multiphoton microscope (MPM) imaging techniques. Method C57BL/6 (Cont), Nrf2 knockout (Nrf2-KO), and Nrf2-activated Keap1-knockdown mice (Keap1-KD) were used. The mice were treated the synthetic triterpenoid RTA dh404 (10 mg/kg/day by gavage) which is a Nrf2 activator for rodents, for a week. We successfully developed the technique to evaluate single-nephron GFR (SNGFR) using MPM (Circulation 2019). The glomerular hemodynamics, diameter of the afferent/efferent arterioles and glomerular permeability were also evaluated. The calcium influx into cells in response to ATP and angiotensin II stimulation and the effect on [Ca2+]i by RTAdh404 were evaluated using Fluo 4 and Fura red in cultured mesangial cells and podocytes. Production of reactive oxygen species and nitric oxide (NO) availability were assessed by fluorescent method using CellROX® Deep Red and diaminofluorescein-FM diacetate (DAF-FM DA) upon the exposure to these stimuli. Results SNGFR in Keap1-KD mice was significantly higher than in the control (9.13±0.55 vs 4.40±0.39 nl/min, Figure 1). RTA dh404 increased SNGFR in the control but not in the Nrf2-KO mice (6.00±0.40 vs 4.66±0.35 nl/min, Figure 1). There was no significant change in the ratio of the glomerular afferent/efferent arteriole diameter in all groups. RTA dh404 treatment increased glomerular volume but did not affect the glomerular permeability of albumin and 40kd-dextran. RTA dh404-treatment inhibited calcium influx into cultured podocytes and mesangial cells induced by angiotensin II or ATP, thereby affecting contractile responses. Oxidative stress and NO-bioavailablity were also ameliorated with RTA dh404. Conclusion The Keap1/Nrf2 pathway plays a pivotal role in controlling GFR and presumably underlies the effect of BARD on GFR in patients.