Reactive oxygen species are involved in insulin-dependent regulation of autophagy in primary rat podocytes

Autophagy is an intracellular defense mechanism responsible for the turnover of damaged or non-functional cellular constituents. This process provides cells with energy and essential compounds under unfavorable environmental conditions—such as oxidative stress and hyperglycemia, which are both observed in diabetes. The most common diabetes complication is diabetic nephropathy (DN), which can lead to renal failure. This condition often includes impaired podocyte function. Here we investigated autophagic activity in rat podocytes cultured with a high insulin concentration (300 nM). Autophagy was activated after 60 min of insulin stimulation. Moreover, this effect was abolished following pharmacological (apocynin) or genetic (siRNA) inhibition of NAD(P)H oxidase activity, indicating that insulin-dependent autophagy stimulation involved reactive oxygen species (ROS). We also observed a continuous and time-dependent increase of podocyte albumin permeability in response to insulin, and this process was slightly improved by autophagy inhibition following short-term insulin exposure. Our results suggest that insulin may be a factor affecting the development of diabetic nephropathy.     

Thioredoxin-interacting protein mediates NALP3 inflammasome activation in podocytes during diabetic nephropathy

Numerous studies have shown that the NALP3 inflammasome plays an important role in various immune and inflammatory diseases. However, whether the NALP3 inflammasome is involved in the pathogenesis of diabetic nephropathy (DN) is unclear. In our study, we confirmed that high glucose (HG) concentrations induced NALP3 inflammasome activation both in vivo and in vitro. Blocking NALP3 inflammasome activation by NALP3/ASC shRNA and caspase-1 inhibition prevented IL-1β production and eventually attenuated podocyte and glomerular injury under HG conditions. We also found that thioredoxin (TRX)-interacting protein (TXNIP), which is a pro-oxidative stress and pro-inflammatory factor, activated NALP3 inflammasome by interacting with NALP3 in HG-exposed podocytes. Knocking down TXNIP impeded NALP3 inflammasome activation and alleviated podocyte injury caused by HG. In summary, the NALP3 inflammasome mediates podocyte and glomerular injury in DN, moreover, TXNIP participates in the formation and activation of the NALP3 inflammasome in podocytes during DN, which represents a novel mechanism of podocyte and glomerular injury under diabetic conditions.     

The Ras GTPase-activating-like protein IQGAP1 is downregulated in human diabetic nephropathy and associated with ERK1/2 pathway activation

Podocyte injury may contribute to the pathogenesis of diabetic nephropathy (DN), but the underlying mechanism of hyperglycemia induced podocyte damage is not fully understood. The Ras GTPase-activating-like protein IQGAP1 is associated to the slit diaphragm proteins and the actin cytoskeleton in podocyte. Here, we studied IQGAP1 expression alterations in human DN biopsies and extracellular signal-regulated kinase (ERK)-dependent pathways of IQGAP1 expression in podocyte under high glucose (HG) media. In vivo, analysis of renal biopsies from patients with DN revealed a significant reduction in IQGAP1 expression compared to controls. In vitro, IQGAP1 mRNA and protein expression were observed to decline under HG media at 48 h. But phosphorylation of ERK1/2 was activated under HG media at 24 h and 48 h. However, HG-induced downregulation of IQGAP1 protein was attenuated by specific ERK1/2 activation inhibitor PD98059. Taken together, these results highlight the importance of IQGAP1 in DN, and suggest that IQGAP1 expression in podocyte under HG media is modulated by the ERK1/2 pathway, which may lead to the future development of therapies targeting IQGAP1 dysfunction in podocytes in DN.     

Insulin increases glomerular filtration barrier permeability through dimerization of protein kinase G type Iα subunits

The increase in the permeability of the glomerular barrier filtration to albumin is a well-known feature of diabetic microvasculature and a negative prognostic factor for vascular complications. However, the underlying mechanisms are incompletely understood. We demonstrated recently that superoxide anion generation increases dimerization of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. Here we investigated whether high insulin concentration is involved in PKGI-dependent hyperpermeability of the diabetic glomerular filtration barrier. We assessed changes in insulin-induced glomerular permeability by measuring glomerular capillary permeability to albumin in isolated glomeruli from Wistar and obese and lean Zucker rats and transmembrane albumin flux in cultured rat podocytes. Expression of PKGIα and upstream proteins was confirmed in the podocytes using Western blotting and immunofluorescence. Insulin (300 nM, 5 min) increased NAD(P)H-dependent glomerular albumin permeability in Wistar rats and PKGI-dependent transmembrane albumin flux in cultured podocytes. Podocyte exposure to insulin in non-reducing conditions increased PKGIα interprotein disulfide bond formation, altered the phosphorylation of the PKG target proteins MYPT1 and MLC, and disrupted the actin cytoskeleton. The role of NADPH oxidase (NOX) in insulin-induced reactive oxygen species (ROS) generation and insulin-evoked increases in albumin permeability in podocytes was confirmed with NOX2 and NOX4 siRNA. Glomerular albumin permeability was increased in hyperinsulinemic Zucker obese rats with isolated glomeruli showing increased expression of PKGIα and NOX4. Taken together, these data demonstrate that insulin increases glomerular barrier albumin permeability via a PKGI-dependent mechanism involving NAD(P)H-dependent generation of superoxide anion. These findings reveal a role for insulin in the pathophysiology of diabetic glomerular nephropathy.     

Astragaloside IV ameliorates renal injury in streptozotocin-induced diabetic rats through inhibiting NF-κB-mediated inflammatory genes expression

Accumulating evidence suggests that inflammatory processes are involved in the development of diabetic nephropathy (DN). However, there are no effective interventions for inflammation in the diabetic kidneys. Here, we tested the hypothesis that Astragaloside IV(AS-IV), a novel saponin purified from Astragalus membranaceus (Fisch) Bge, ameliorates DN in streptozotocin (STZ)-induced diabetic rats through anti-inflammatory mechanisms. Diabetes was induced with STZ (65 mg/kg) by intraperitoneal injection in rats. Two weeks after STZ injection, rats were divided into three groups (n = 8/each group), namely, diabetic rats, diabetic rats treated with AS-IV at 5 and 10 mg kg^?1 d^?1, p.o., for 8 weeks. The normal rats were chosen as nondiabetic control group (n = 8). The rats were sacrificed 10 weeks after induction of diabetes. AS-IV ameliorated albuminuria, renal histopathology and podocyte foot process effacement in diabetic rats. Renal NF-κB activity, as wells as protein and mRNA expression were increased in diabetic kidneys, accompanied by an increase in mRNA expression and protein content of TNF-α, MCP-1 and ICAM-1 in kidney tissues. The α₁-chain type IV collagen mRNA was elevated in the kidneys of diabetic rats. All of these abnormalities were partially restored by AS-IV. AS-IV also decreased the serum levels of TNF-α, MCP-1 and ICAM-1 in diabetic rats. These findings suggest that AS-IV, a novel anti-inflammatory agent, attenuated DN in rats through inhibiting NF-κB mediated inflammatory genes expression.     

MDM2 is implicated in high‐glucose‐induced podocyte mitotic catastrophe via Notch1 signalling

Podocyte injury and depletion are essential events involved in the pathogenesis of diabetic nephropathy (DN). As a terminally differentiated cell, podocyte is restricted in ‘post‐mitosis’ state and unable to regenerate. Re‐entering mitotic phase will cause podocyte disastrous death which is defined as mitotic catastrophe (MC). Murine double minute 2 (MDM2), a cell cycle regulator, is widely expressed in renal resident cells including podocytes. Here, we explore whether MDM2 is involved in podocyte MC during hyperglycaemia. We found aberrant mitotic podocytes with multi‐nucleation in DN patients. In vitro, cultured podocytes treated by high glucose (HG) also showed an up‐regulation of mitotic markers and abnormal mitotic status, accompanied by elevated expression of MDM2. HG exposure forced podocytes to enter into S phase and bypass G2/M checkpoint with enhanced expression of Ki67, cyclin B1, Aurora B and p‐H3. Genetic deletion of MDM2 partly reversed HG‐induced mitotic phase re‐entering of podocytes. Moreover, HG‐induced podocyte injury was alleviated by MDM2 knocking down but not by nutlin‐3a, an inhibitor of MDM2‐p53 interaction. Interestingly, knocking down MDM2 or MDM2 overexpression showed inhibition or activation of Notch1 signalling, respectively. In addition, genetic silencing of Notch1 prevented HG‐mediated podocyte MC. In conclusion, high glucose up‐regulates MDM2 expression and leads to podocyte MC. Notch1 signalling is an essential downstream pathway of MDM2 in mediating HG‐induced MC in podocytes.     

Thioredoxin-interacting protein deficiency alleviates phenotypic alterations of podocytes via inhibition of mTOR activation in diabetic nephropathy

Thioredoxin-interacting protein (TXNIP) is induced by high glucose (HG), whereupon it acts to inhibit thioredoxin, thereby promoting oxidative stress. We have found that TXNIP knockdown in human renal tubular cells helped prevent the epithelial-to-mesenchymal transition (EMT). Here, we studied the potential effect of TXNIP on podocyte phenotypic alterations in diabetic nephropathy (DN) in vivo and in vitro. In conditionally immortalized mouse podocytes under HG conditions, knocking down TXNIP disrupted EMT, reactive oxygen species (ROS) production, and mammalian target of rapamycin (mTOR) pathway activation. Further, Raptor short hairpin RNA (shRNA), Rictor shRNA, and mTOR specific inhibitor KU-0063794 were used to assess if the mTOR signal pathway is involved in HG-induced EMT in podocytes. We found that Raptor shRNA, Rictor shRNA, and KU-0063794 could all restrain HG-induced EMT and ROS production in podocytes. In addition, antioxidant Tempol or N-acetylcysteine presented a prohibitive effect on HG-induced EMT in podocytes. Streptozotocin was utilized to render equally diabetic in wild-type (WT) control and TXNIP ^−/− (TKO) mice. Diabetes did not increase levels of 24-hr urinary protein, serum creatinine, blood urea nitrogen, and triglyceride in TXNIP ^−/− mice. Podocyte phenotypic alterations and podocyte loss were detected in WT but not in TKO diabetic mice. Oxidative stress was also suppressed in diabetic TKO mice relative to WT controls. Also, TXNIP deficiency suppresses the activation of mTOR in glomeruli of streptozotocin-induced diabetic mice. Moreover, TXNIP expression, mTOR activation, Nox1, and Nox4 could be detected in renal biopsy tissues of patients with DN. This suggests that decreased TXNIP could ameliorate phenotypic alterations of podocytes via inhibition of mTOR in DN, highlighting TXNIP as a promising therapeutic target.     

Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury

Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca²⁺ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca²⁺-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)–induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca²⁺ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca²⁺ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.     

Nestin protects mouse podocytes against high glucose-induced apoptosis by a Cdk5-dependent mechanism

Podocyte apoptosis contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms that mediate hyperglycemia‐induced podocyte apoptosis remain poorly understood. Recent findings indicate that the disruption of the cytoskeleton is related to the podocyte apoptosis. In the present study, we investigated the involvement of nestin, an important cytoskeleton‐associated class VI intermediate filament (IF) protein, in the high glucose (HG)‐induced podocyte apoptosis. Our data showed that HG decreased the expression level of nestin, either mRNA or protein, in a time‐dependent manner in cultured podocytes. Also, through knockdown of nestin expression by miRNA interference, the HG‐induced podocyte apoptotic rate was significantly increased. The expression of cleaved caspase‐3 was also markedly elevated. Considering that nestin is a substrate of cyclin‐dependent kinase 5 (Cdk5), we further assessed the expression of Cdk5 in HG‐treated podocytes. The results showed that HG stimulation increased the protein and mRNA expression of Cdk5 in a time‐dependent manner in cultured mouse podocytes. The protein activator of Cdk5, p35, was also increased in a time‐dependent manner by HG stimulation, and downregulation of Cdk5 by miRNA interference attenuated the nestin reduction in HG‐treated podocytes; the HG‐induced podocyte apoptosis, the increased cleaved caspase‐3 expression and the Bax/Bcl‐2 ratio were all effectively attenuated. These data suggested that nestin, which is dependent on Cdk5 regulation, plays a cytoprotective role in HG‐induced podocyte apoptosis. J. Cell. Biochem. 113: 3186–3196, 2012. © 2012 Wiley Periodicals, Inc.     

Fatty acids are novel nutrient factors to regulate mTORC1 lysosomal localization and apoptosis in podocytes

Podocyte apoptosis is a potent mechanism of proteinuria in diabetic nephropathy. More detailed mechanistic insight into podocyte apoptosis is needed to better understand the pathogenesis of diabetic nephropathy. An elevated level of serum free fatty acid (FFA), as well as hyperglycemia, is a clinical characteristic in diabetes, although its causal role in podocyte apoptosis remains unclear. This study examined the effect of three types of FFAs, saturated, monounsaturated and polyunsaturated FFAs, on podocyte apoptosis. Palmitate, a saturated FFA, induced endoplasmic reticulum (ER) stress-dependent apoptosis in podocytes. Oleate, a monounsaturated FFA, and eicosapentaenoic acid (EPA), an ω − 3 polyunsaturated FFA did not induce apoptosis; rather, they antagonized palmitate-induced apoptosis. Palmitate activated mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a nutrient-sensing kinase regulating a wide range of cell biology. Furthermore, inhibition of mTORC1 activity by rapamycin or siRNA for Raptor, a component of mTORC1, ameliorated palmitate-induced ER stress and apoptosis in podocytes. Activity of mTORC1 is regulated by upstream kinases and Rag/Ragulator-dependent recruitment of mTOR onto lysosomal membranes. Palmitate activated mTORC1 by enhancing recruitment of mTOR onto lysosomal membranes, which was inhibited by co-incubation with oleate or EPA. Inhibition of mTOR translocation onto lysosomes by transfection with dominant-negative forms of Rag ameliorated palmitate-induced apoptosis. This study suggests that saturated and unsaturated FFAs have opposite effects on podocyte apoptosis by regulating mTORC1 activity via its translocation onto lysosomal membranes, and the results provide a better understanding of the pathogenesis in diabetic nephropathy and a novel role of mTORC1 in cell apoptosis.     

Synthesis and PGE2 production inhibition of s-triazine derivatives as a novel scaffold in RAW 264.7 macrophage cells

We present the synthesis and biological evaluation of a collection of s-triazine derivatives as a novel scaffold of compounds with the capability to inhibit the PGE₂ production in LPS-induced RAW 264.7 macrophage cells. A total of 12 derivatives were synthesized and assayed for PGE₂ reduction at 10 μM concentration. Two compounds (7b and 7i) exhibiting >90% inhibition of PGE₂ production were found to have IC₅₀ values of 5.76 and 5.52 μM, respectively. They were counter screened for inhibition on COX-2 activity in a cell free assay. Specifically, compound 7i (R¹ = 4-Bn-Ph, R² = Cl, R³ = Ph, R⁵ = CO₂Me) was highly active in cells while maintaining little COX-2 inhibition (∼0% at 10 μM). Molecular docking study provides the possibility that compound 7i could inhibit PGE₂ production by blocking the PGH₂ binding site of mPGES-1 instead of COX-2 enzyme. Based on this result, our synthetic efforts will focus on intensive structure–activity relationship (SAR) study of s-triazine scaffold to discovery a potential PGE₂ synthesis inhibitor.     

Release and inhibitory effects of prostaglandin D2 in guinea pig urinary bladder and the role of urothelium

BackgroundWhile studying a urothelium-derived inhibitory factor in guinea pig urinary bladders we observed considerable release of prostanoids, including PGD₂-like activity. The present study was carried out to identify the prostanoids and to study their roles in modulating guinea pig urinary bladder motility.MethodsRelease of PGE₂ and PGD₂ in isolated guinea pig urinary bladder preparations was analyzed by high performance liquid chromatography (HPLC) combined with bioassay on bladder strips. Isolated urothelium-intact (UI) or -denuded (UD) bladder strips were subjected to electrical field stimulation (EFS) and applications of PGE₂ and PGD₂.ResultsA resting release of 95 ± 9 (n = 5) ng g tissue^− 1 h^− 1 PGE₂-like activity and 210 ± 34 (n = 4) ng g tissue^− 1 h^− 1 PGD₂-like activity was found, where PGD₂-like was subject to marked spontaneous inactivation during isolation. Prostanoids release was decreased by 70–90% by the cyclo-oxygenase inhibitor diclofenac in UI preparations. Urothelium removal decreased prostanoids release by more than 90%.PGE₂ increased basal tone and spontaneous contractions, whereas PGD₂ had little or no effect on these. Exogenous PGE₂ enhanced and PGD₂ inhibited contractile responses to EFS, exogenous acetylcholine- and ATP, whereas PGD₂ caused marked dose-dependent inhibition. PGE₂ and PGD₂ effects were more pronounced in diclofenac-treated UD tissues.ConclusionsPGD₂ and PGE₂ are released from guinea pig bladder urothelium and PGD₂ has inhibitory effects on bladder motility, mainly through a postjunctional action on smooth muscle responsiveness.General significanceThe release and inhibitory effects merit further studies in relation to normal biological function as well as overactive bladder syndrome.     

Inhibition of mitochondrial fission protects podocytes from albumin-induced cell damage in diabetic kidney disease

AimsIdentifying the mechanisms that underlie progression from endothelial damage to podocyte damage, which leads to massive proteinuria, is an urgent issue that must be clarified to improve renal outcome in diabetic kidney disease (DKD). We aimed to examine the role of dynamin-related protein 1 (Drp1)-mediated regulation of mitochondrial fission in podocytes in the pathogenesis of massive proteinuria in DKD.MethodsDiabetes- or albuminuria-associated changes in mitochondrial morphology in podocytes were examined by electron microscopy. The effects of albumin and other diabetes-related stimuli, including high glucose (HG), on mitochondrial morphology were examined in cultured podocytes. The role of Drp1 in podocyte damage was examined using diabetic podocyte-specific Drp1-deficient mice treated with neuraminidase, which removes endothelial glycocalyx.ResultsNeuraminidase-induced removal of glomerular endothelial glycocalyx in nondiabetic mice led to microalbuminuria without podocyte damage, accompanied by reduced Drp1 expression and mitochondrial elongation in podocytes. In contrast, streptozotocin-induced diabetes significantly exacerbated neuraminidase-induced podocyte damage and albuminuria, and was accompanied by increased Drp1 expression and enhanced mitochondrial fission in podocytes. Cell culture experiments showed that albumin stimulation decreased Drp1 expression and elongated mitochondria, although HG inhibited albumin-associated changes in mitochondrial dynamics, resulting in apoptosis. Podocyte-specific Drp1-deficiency in mice prevented diabetes-related exacerbation of podocyte damage and neuraminidase-induced development of albuminuria. Endothelial dysfunction-induced albumin exposure is cytotoxic to podocytes. Inhibition of mitochondrial fission in podocytes is a cytoprotective mechanism against albumin stimulation, which is impaired under diabetic condition. Inhibition of mitochondrial fission in podocytes may represent a new therapeutic strategy for massive proteinuria in DKD.     

Exosomal microRNA-16-5p from human urine-derived stem cells ameliorates diabetic nephropathy through protection of podocyte

Diabetic nephropathy (DN) remains one of the severe complications associated with diabetes mellitus. It is worthwhile to uncover the underlying mechanisms of clinical benefits of human urine-derived stem cells (hUSCs) in the treatment of DN. At present, the clinical benefits associated with hUSCs in the treatment of DN remains unclear. Hence, our study aims to investigate protective effect of hUSC exosome along with microRNA-16-5p (miR-16-5p) on podocytes in DN via vascular endothelial growth factor A (VEGFA). Initially, miR-16-5p was predicated to target VEGFA based on data retrieved from several bioinformatics databases. Notably, dual-luciferase report gene assay provided further verification confirming the prediction. Moreover, our results demonstrated that high glucose (HG) stimulation could inhibit miR-16-5p and promote VEGFA in human podocytes (HPDCs). miR-16-5p in hUSCs was transferred through the exosome pathway to HG-treated HPDCs. The viability and apoptosis rate of podocytes after HG treatment together with expression of the related factors were subsequently determined. The results indicated that miR-16-5p secreted by hUSCs could improve podocyte injury induced by HG. In addition, VEGA silencing could also ameliorate HG-induced podocyte injury. Finally, hUSC exosomes containing overexpressed miR-16-5p were injected into diabetic rats via tail vein, followed by qualification of miR-16-5p and observation on the changes of podocytes, which revealed that overexpressed miR-16-5p in hUSCs conferred protective effects on HPDCs in diabetic rats. Taken together, the present study revealed that overexpressed miR-16-5p in hUSC exosomes could protect HPDCs induced by HG and suppress VEGFA expression and podocytic apoptosis, providing fresh insights for novel treatment of DN.     

Wnt/β‐catenin signalling pathway mediates high glucose induced cell injury through activation of TRPC6 in podocytes

Objectives

Diabetic nephropathy is a major complication of diabetes and a frequent cause of end‐stage renal disease and recent studies suggest that podocyte damage may play a role in the pathogenesis of this. At early onset of diabetic nephropathy there is podocyte drop‐out, which is thought to provoke glomerular albuminuria and subsequent glomerular injury; however, the underlying molecular mechanisms of this remain poorly understood. Here we report that we tested the hypothesis that early diabetic podocyte injury is caused, at least in part, by up‐regulation of transient receptor potential cation channel 6 (TRPC6), which is regulated by the canonical Wnt signalling pathway, in mouse podocytes.

Materials and methods

Mechanism of injury initiation in mouse podocytes, by high concentration of D‐glucose (HG, 30 mM), was investigated by MTT, flow cytometry, real‐time quantitative PCR, and western blot analysis.

Results

HG induced apoptosis and reduced viability of differentiated podocytes. It caused time‐dependent up‐regulation of TRPC6 and activation of the canonical Wnt signalling pathway, in mouse podocytes. In these cells, blockade of the Wnt signalling pathway by dickkopf related protein 1 (Dkk1) resulted in effective reduction of TRPC6 up‐regulation and amelioration of podocyte apoptosis. Furthermore, reduction of cell viability induced by HG was attenuated by treatment with Dkk1.

Conclusion

These findings indicate that the Wnt/β‐catenin signalling pathway may potentially be active in pathogenesis of TRPC6‐mediated diabetic podocyte injury.

     

Ubiquitin C-terminal hydrolase L1 deletion ameliorates glomerular injury in mice with ACTN4-associated focal segmental glomerulosclerosis

Renal ubiquitin C-terminal hydrolase L1 (UCHL1) is upregulated in a subset of human glomerulopathies, including focal segmental glomerulosclerosis (FSGS), where it may serve to promote ubiquitin pools for degradation of cytotoxic proteins. In the present study, we tested whether UCHL1 is expressed in podocytes of a mouse model of ACTN4-associated FSGS. Podocyte UCHL1 protein was detected in glomeruli of K256E-ACTN4^pod +/UCHL1+/+ mice. UCHL1+/− mice were intercrossed with K256E-ACTN4^pod + mice and monitored for features of glomerular disease. 10-week-old K256E-ACTN4^pod +/UCHL1−/− mice exhibited significantly ameliorated albuminuria, glomerulosclerosis, tubular pathology and blood pressure. Interestingly, while UCHL1 deletion diminished both tubular and glomerular apoptosis, WT1-positive nuclei were unchanged. Finally, UCHL1 levels correlated positively with poly-ubiquitinated proteins but negatively with K256E-α-actinin-4 levels, implying reduced K256E-α-actinin-4 proteolysis in the absence of UCHL1. Our data suggest that UCHL1 upregulation in ACTN4-associated FSGS fuels the proteasome and that UCHL1 deletion may impair proteolysis and thereby preserve K256E/wt-α-actinin-4 heterodimers, maintaining podocyte cytoskeletal integrity and protecting the glomerular filtration barrier.     

MAD2B promotes podocyte injury through regulating Numb-dependent Notch 1 pathway in diabetic nephropathy

Rationale: Recent studies have demonstrated that the loss of podocyte is a critical event in diabetic nephropathy (DN). Previously, our group have found that the mitotic arrest deficient protein MAD2B was involved in high glucose (HG)-induced podocyte injury by regulating APC/C activity. However, the exact mechanism of MAD2B implicated in podocyte injury is still lacking.Methods: The experiments were conducted by using kidney tissues from streptozotocin (STZ) induced diabetic mice with or without podocyte-specific deletion of MAD2B and the cultured podocytes exposed to different treatments. Glomerular pathological injury was evaluated by periodic acid-Schiff staining and transmission electron microscopy. The endogenous interaction between MAD2B and Numb was discovered by yeast two-hybrid analysis and co-immunoprecipitation assay. The expressions of MAD2B, Numb and related pathway were detected by western blot, immunochemistry and immunofluorescence.Results: The present study revealed that MAD2B was upregulated in diabetic glomeruli and cultured podocytes under hyperglycemic conditions. Podocyte-specific deletion of MAD2B alleviated podocyte injury and renal function deterioration in mice of diabetic nephropathy. Afterwards, MAD2B was found to interact with Numb, which was downregulated in diabetic glomeruli and HG-stimulated cultured podocytes. Interestingly, MAD2B genetic deletion could partly reverse the decline of Numb in podocytes exposed to HG and in diabetic mice, and the expressions of Numb downstream molecules such as NICD and Hes-1 were decreased accordingly. In addition, overexpression of Numb ameliorated HG-induced podocyte injury.Conclusions: The present findings suggest that upregulated MAD2B expression contributes to Numb depletion and activation of Notch 1 signaling pathway, which ultimately leads to podocyte injury during DN progression.     

Prostaglandin E1 (PGE1), but not prostaglandin E2 (PGE2), alters luteal and endometrial luteinizing hormone (LH) occupied and unoccupied LH receptors and mRNA for LH receptors in ovine luteal tissue to prevent luteolysis

Loss of luteal progesterone secretion at the end of the ovine estrous cycle is via uterine PGF₂α secretion. However, uterine PGF₂α secretion is not decreased during early pregnancy in ewes. Instead, the embryo imparts a resistance to PGF₂α. Prostaglandins E (PGE; PGE₁ + PGE₂) are increased in endometrium and uterine venous blood during early pregnancy in ewes to prevent luteolysis. Chronic intrauterine infusion of PGE₁ or PGE₂ prevents spontaneous or IUD, estradiol-17β, or PGF₂α-induced premature luteolysis in nonbred ewes. The objective was to determine whether chronic intrauterine infusion of PGE₁ or PGE₂ affected mRNA for LH receptors, occupied and unoccupied receptors for LH in luteal and caruncular endometrium, and luteal function. Ewes received Vehicle, PGE₁, or PGE₂ every 4 h from days 10 to 16 of the estrous cycle via a cathether installed in the uterine lumen ipsilateral to the luteal-containing ovary.Jugular venous blood was collected daily for analysis of progesterone and uterine venous blood was collected on day-16 for analysis of PGF₂α and PGE. Corpora lutea and caruncular endometrium were collected from day-10 preluteolytic control ewes and day-16 ewes treated with Vehicle, PGE₁ or PGE₂ for analysis of the mRNA for LH receptors and occupied and unoccupied receptors for LH. Luteal weights on day-16 in ewes treated with PGE₁ or PGE₂ and day-10 control ewes were similar (P ≥ 0.05), but were greater (P ≤ 0.05) than in day-16 Vehicle-treated ewes. Progesterone profiles on days 10–16 differed (P ≤ 0.05) among treatment groups: PGE₁ > PGE₂ > Vehicle-treated ewes. Concentrations of PGF₂α and PGE in uterine venous plasma on day-16 were similar (P ≥ 0.05) in the three treatment groups. Luteal mRNA for LH receptors and unoccupied and occupied LH receptors were similar (P ≥ 0.05) in day-10 control ewes and day-16 ewes treated with PGE₂ and were lower (P ≤ 0.05) in day-16 Vehicle-treated ewes. PGE₂ prevented loss (P ≤ 0.05) of day-16 luteal mRNA for LH receptors and occupied and unoccupied LH receptors. Luteal and caruncular tissue mRNA for LH receptors and occupied and unoccupied LH receptors were greater (P ≤ 0.05) on day-16 of PGE₁-treated ewes than any treatment group. mRNA for LH receptors and occupied and unoccupied receptors for LH in caruncules were greater (P ≤ 0.05) in day-16 Vehicle or PGE₂-treated ewes than in day-10 control ewes. It is concluded that PGE₁ and PGE₂ share some common mechanisms to prevent luteolysis; however, only PGE₁ increased luteal and endometrial mRNA for LH receptors and occupied and unoccupied LH receptors. PGE₂ prevents a decrease in luteal mRNA for LH receptors and occupied and unoccupied receptors for LH without altering endometrial mRNA for LH receptors or occupied and unoccupied receptors for LH.     

High glucose enhances TGF-β1 expression in rat bone marrow stem cells via ERK1/2-mediated inhibition of STAT3 signaling

AimsThis study was to investigate the effect of high glucose (HG) on TGF-β1 expression and the underlying mechanisms in bone marrow stem cells.Main methodsRat bone marrow multipotent adult progenitor cells (MAPCs) were cultured in normal (5.5 mM d-glucose) and HG media (25.5 mM d-glucose) for up to 14 days. l-Glucose (20 mM plus 5.5 mM d-glucose) was used as high osmolarity control. TGF-β1 expression was evaluated using quantitative RT-PCR, ELISA, and immunofluorescence staining for its mRNA and protein level in the cells and in the conditioned media. The expression and activation of ERK1/2 and STAT3 were examined in MAPCs cultured in HG media with Western blot.Key findingsMeasurable level of TGF-β1 was detected in the cells cultured in normal media. TGF-β1 expression was substantially increased in MAPCs after 36 h of culture in HG media with over 20-fold increase in the mRNA and 5-fold increase in protein level over control. Interestingly, ERK1/2 phosphorylation was significantly increased in MAPCs cultured in HG media, while in STAT3 (Tyr705), not STAT3 (Ser727), phosphorylation was dramatically decreased. Treatment of cells with the specific MEK1 inhibitor PD98059 or U0126 suppressed ERK1/2 phosphorylation and TGF-β1 expression, and completely restored the level of STAT3 (Tyr705) phosphorylation in MAPCs cultured in HG media. Treatment of the cells with the specific STAT3 phosphorylation inhibitor AG490 significantly blocked STAT3 (Tyr705) phosphorylation and increased TGF-β1 expression without change in ERK1/2 phosphorylation in MPACs.SignificanceHG increased TGF-β1 expression through inhibition of STAT3 (Tyr705) by enhanced ERK1/2 signaling in MAPCs.     

Flufenamic acid is a tool for investigating TRPC6-mediated calcium signalling in human conditionally immortalised podocytes and HEK293 cells

Mutations in the cation channel TRPC6 result in a renal-specific phenotype of familial nephrotic syndrome, affecting intracellular calcium ([Ca²⁺]_i) signalling in the glomerular podocyte. Tools to study native TRPC6 activity are scarce, although there has been recent success with flufenamic acid (FFA). We confirm the specificity of FFA for TRPC6 both in an artificial expression system and in a human conditionally immortalised podocyte cell line (ciPod).Cells were loaded with fura-2AM and changes in intracellular calcium ([Ca²⁺]_i) were calculated. 200 μM FFA induced an increase in [Ca²⁺]_i in HEK293 cells with native TRPC6 expression, which was enhanced by overexpression of TRPC6 and completely blocked in the absence of extracellular calcium. Expressed TRPC7 did not significantly affect the response to FFA whereas expressed TRPC3 reduced it. FFA also induced an increase ciPod in [Ca²⁺]_i, which was inhibited using SKF96365 and 2-APB, but not indomethacin. In ciPod, adenovirus (Ad-v) wild type (WT) TRPC6 increased [Ca²⁺]_i activity to FFA compared to native TRPC6, whereas activity was significantly reduced with Ad-v dominant negative (DN) TRPC6. The niflumic acid (NFA) induced increase in [Ca²⁺]_i in ciPod was not affected by Ad-v TRPC6 DN, and in HEK293 cells was not affected by WT TRPC6.In conclusion, FFA activates TRPC6 [Ca²⁺]_i signalling in both ciPod and HEK293 cells independently of TRPC3 and TRPC7, and independently of properties of the fenamate family.