Glomerular Structural Integrity Research Articles

Activation of TRPC6 by AngⅡ Induces Podocyte Injury and Participates in Proteinuria of Nephrotic Syndrome.

Background: Nephrotic syndrome (NS) is a common glomerular disease, and podocyte injury is the character of primary NS, usually caused by minimal change disease and membranous nephropathy. Podocytopathy is primarily associated with glomerular proteinuria. Losartan, an angiotensin receptor blocker (ARB), is commonly used in the treatment of NS, and the AngiotensinⅡ (AngⅡ)–transient receptor potential ion channel 6 (TRPC6) axis has been reported to act on podocytes to regulate proteinuria in NS. Therefore, the purpose of this study was to explore the relationship in between AngⅡ–TRPC6, podocyte injury, and proteinuria based on the adriamycin (ADR) NS rat model. Method: All male rats were divided into three groups: control group, model group, and ARB group. The rats in the model group were induced by ADR, and the rats in the ARB group received losartan after induction of renal injury for 4 weeks. The changes in parameters related to renal dysfunction, and glomerular and podocyte structural damage, such as AngⅡ, AngⅡ type I receptor (AT1R), TRPC6, CaN, Caspase-3, Nephrin, and Podocin, were analyzed. Furthermore, the kidneys were isolated for study via transmission electron microscopy (TEM), immunohistochemistry, and western blot (WB) after the rats were sacrificed. In vitro, immortalized mouse MPC5 podocytes were used to investigate the regulatory effect of flufenamic acid (Flu) and SAR7334 (SAR) on the AngⅡ-TRPC6 signaling axis. Flow cytometry and WB were conducted to determine the relationship between podocyte injury and AngⅡ-TRPC6. Results: In vivo results showed that NS rats developed massive albuminuria and abnormal renal function, accompanied by abnormally increased levels of AngⅡ, TRPC6, AT1R, and CaN and a decreased expression of actin molecules in podocytes, extensive fusion of foot processes (FP), loss of glomerular structural integrity, collapse of podocyte structure, and skeletal reorganization. In vitro experiments indicated that both AngⅡ and Flu (the specific agonist of TRPC6) stimulated the expressions of TRPC6, AT1R, and Caspase-3 in podocytes. The AngⅡ receptor–blocker losartan and TRPC6-specific inhibitor SAR blocked the overexpression of the aforementioned proteins. In addition, SAR also attenuated the degradation of podocyte structural proteins and inhibited the fluorescence intensity of intracellular calcium (Ca2+) and cell apoptosis. Conclusion: The involvement of AngⅡ in the occurrence of NS proteinuria may be related to podocyte injury induced by activated TRPC6.

Glomerular Damage in Trichloroethylene-Sensitized Mice: Targeting Cathepsin L-Induced Hyperactive mTOR Signaling.

Trichloroethylene (TCE) is a serious health hazard for workers with daily exposure, causing occupational medicamentosa-like dermatitis due to TCE (OMDT) and glomerular damage. Recent studies suggest that mTORC1 signaling is activated in various glomerular disorders; however, the role of mTORC1 signaling in TCE-induced glomerular damage remains to be explored. In the present study, 6 OMDT patients were enrolled and a TCE-sensitized mouse model was established to investigate molecular mechanisms underlying the glomerular damage associated with OMDT. Glomerular damage was assessed by levels of urine nephrin, H&E staining, and renal function test. Ultrastructural change of podocyte was investigated by transmission electron microscopy. The podocyte-related molecules including nephrin, α-actinin-4, and integrin β1 were visualized by immunofluorescence. The activation of mTORC1 signaling was confirmed by Western blot. Glomerular apoptosis was examined by the TUNEL test and Western blotting. Expression and location of cathepsin L (CTSL) were assessed by RT-PCR and immunofluorescence. Our results showed that TCE sensitization caused damage to glomerular structural integrity and also increased the activation of mTORC1 signaling, which was accompanied by podocyte loss, hypertrophy, and glomerular apoptosis. Importantly, we also found that over-expressed CTSL was mainly located in podocyte and CTSL inhibition could partially block the activation of mTORC1 signaling. Thus, our findings suggested a novel mechanism whereby hyperactive mTOR signaling contributes to TCE sensitization–induced and immune-mediated glomerular damage via CTSL activation.

DUAL ACTING COX‐2 AND SOLUBLE EPOXIDE HYDROLASE INHIBITOR ATTENUATES GLOMERULAR INJURY IN FOCAL SEGMENTAL GLOMERULAR SCLEROSIS

Proteinuric glomerular diseases like focal and segmental glomerulosclerosis (FSGS) are the leading cause of chronic and end‐stage kidney diseases. FSGS is characterized by glomerular injury, and, at the clinical level, a classic hallmark of FSGS is proteinuria. Although FSGS is associated with high morbidity and mortality, treatments are limited that emphasize the need for novel therapies. We developed a novel dual modulator, PTUPB that concurrently acts as a soluble epoxide hydrolase (sEH) inhibitor as well as a cyclooxygenase‐2 inhibitor (sEHi/COXi). PTUPB has previously demonstrated robust kidney actions including reduction of glomerular injury in diabetic nephropathy. In the current study, we investigated renal actions of PTUPB in a genetic FSGS model, ROP Os/+ mice. ROP Os/+ mice developed proteinuria and glomerular injury between 16–20 weeks of age (8‐fold higher albuminuria vs. WT). We treated 20 weeks old male wild type (WT) and 20 weeks old ROP Os/+ mice with vehicle or PTUPB (10 mg/kg/d, i.p.; n=4–5/group)for 4 weeks. At the end of the 4 week treatment period urine, blood, and kidney tissue were collected for biochemical and histological analysis. The glomerular injury was assessed from albuminuria, histopathology, mRNA expression of proteins critical for glomerular structural integrity and function. Vehicle treated ROP Os/+ had 10‐fold higher albuminuria compared to WT and PTUPB treatment markedly attenuated albuminuria in ROP Os/+ mice by 70%. The ROP Os/+ mice developed glomerular injury with a 3‐fold higher glomerular injury score compared to WT and PTUPB treatment decreased glomerular injury by 28%. We further demonstrated that there was 20–30% lower renal mRNA expression of nephrin, podocin, and synaptopodin in vehicle‐treated ROP Os/+ mice, and PTUPB treatment improved the increased expression for these glomerular proteins in ROP Os/+ mice. These findings demonstrate that the dual modulator PTUPB that concurrently inhibits sEH and COX‐2 has exciting potential to treat FSGS and related glomerular diseases.Support or Funding InformationDK103616 and Dr. Ralph and Marian Falk Medical Research Trust Bank of America, N.A. to John D. Imig

Use of fucoidan to treat renal diseases: A review of 15 years of clinic studies.

Fucoidan is a sulfated polysaccharide extracted from brown seaweeds. Studies have shown that fucoidan has curative effects on the chronic renal failure, acute kidney injury, and diabetic nephropathy both in vitro and in vivo. Saccharina japonica is the most economically important brown seaweed cultivated in China and is consumed as a marine vegetable in East Asia. Over the past thousand years, Chinese people have traditionally used this plant to cure edema, a symptom of kidney diseases. The fucoidan extracted from Saccharina japonica is composed primarily of fucose and galactose with smaller amounts of other monosaccharides. Structure-activity relationship studies reveal that the molecular weight, monosaccharide compositions, the sulfation degree and the positions of sulfates influences the renoprotective activity. Low molecular weight fucoidan exhibits better activity than fucoidan. Pharmacological studies indicate that fucoidan inhibits renal fibrosis and glomerular sclerosis by reducing the accumulation of extracellular matrix. In addition, fucoidan reduces the inflammatory response and P-selectin expression, maintains the glomerular basement membrane and glomerular structural integrity, improves glomerular filtration function, and protects renal glycosaminoglycans from abnormal degradation. The effective constituent of Haikun Shenxi capsule (HSC) is the fucoidan extracted from Saccharina japonica. HSC was approved for treating renal diseases by the Chinese Food and Drug Administration in 2003. Based on the results of peer-reviewed publications, we will recapitulate the structure, pharmacology, reported clinical cases, clinical efficacy, and future perspectives of HSC. This review will summarize the knowledge of HSC gained in China to stimulate in-depth academic and clinical studies of HSC world widely.

Early and late scanning electron microscopy findings\xa0in diabetic kidney disease

Diabetic nephropathy (DN), the single strongest predictor of mortality in patients with type 2 diabetes, is characterized by initial glomerular hyperfiltration with subsequent progressive renal function loss with or without albuminuria, greatly accelerated with the onset of overt proteinuria. Experimental and clinical studies have convincingly shown that early interventions retard disease progression, while treatment if started late in the disease course seldom modifies the slope of GFR decline. Here we assessed whether the negligible renoprotection afforded by drugs in patients with proteinuric DN could be due to loss of glomerular structural integrity, explored by scanning electron microscopy (SEM). In diabetic patients with early renal disease, glomerular structural integrity was largely preserved. At variance SEM documented that in the late stage of proteinuric DN, glomerular structure was subverted with nearly complete loss of podocytes and lobular transformation of the glomerular basement membrane. In these circumstances one can reasonably imply that any form of treatment, albeit personalized, is unlikely to reach a given cellular or molecular target. These findings should persuade physicians to start the putative renoprotective therapy soon after the diagnosis of diabetes or in an early phase of the disease before structural integrity of the glomerular filter is irreversibly compromised.

Combined melatonin and exendin-4 therapy preserves renal ultrastructural integrity after ischemia-reperfusion injury in the male rat.

We tested whether combined melatonin (Mel) and exendin-4 (Ex4) treatment can better preserve glomerular structural integrity after ischemia-reperfusion (IR) injury compared with either alone. Adult male Sprague Dawley rats (n = 50) were equally divided into sham control (SC), IR, IR-Ex4 (10 μg/kg subcutaneously 30 min after reperfusion and daily for 5 days), IR-Mel (20 mg/kg intraperitoneally at 30 min postreperfusion and 50 mg/kg at 6 and 18 hr), and IR-Ex4-Mel were euthanized at day 14. Serum creatinine level and urine protein-to-creatinine ratio at days 3 and 14 were highest in IR group and lowest in SC, significantly higher in IR-Ex4 and IR-Mel groups than in IR-Ex4-Mel group (all P < 0.001) without significant difference between IR-Ex4 and IR-Mel groups. Changes in podocyte injury score (PIS) and kidney injury score were highest in IR group and lowest in SC, significantly higher in IR-Ex4 and IR-Mel groups than in IR-Ex4-Mel, and significantly higher in IR-Mel group than in IR-Ex4 group (all P < 0.001). Immunohistochemical microscopic findings of the expressions of FSP-1 and WT-1 (two glomerular damage indicators) and KIM-1 and snail (two renal tubular-damaged indicators) showed an identical pattern, whereas the expressions of ZO-1, p-cadherin, podocin, dystroglycan, fibronectin, and synaptopodin (six indices of glomerular integrity) demonstrated an opposite pattern compared to that of PIS among five groups (all P < 0.001). Protein expressions of inflammatory (TNF-α/NF-κB/MMP-9) and oxidative stress (NOX-1, NOX-2, oxidized protein) biomarkers exhibited an identical pattern to that of PIS among five groups (all P < 0.001). Combined melatonin-exednin-4 therapy further protected glomerulus from IR injury.

Spironolactone inhibits podocyte motility via decreasing integrin β1 and increasing integrin β3 in podocytes under high-glucose conditions.

Integrin β1 and β3 expression by podocytes is required to maintain glomerular structural integrity. Previous studies have shown that aldosterone (ALD) is involved in glomerular podocyte injury, and mineralocorticoid receptor (MR) blocker spironolactone effectively reduces proteinuria in patients with diabetic nephropathy. The present study was designed to observe the effects of spironolactone on β1 and β3 integrin expression and podocyte motility under in vitro diabetic conditions. Immortalized mouse podocytes were cultured in media containing normal glucose (NG) levels, high glucose (HG) or HG plus spironolacton. The expression of β1 and β3 integrin in podocytes was detected by reverse transcription quantitative polymerase chain reaction, immunofluorescence and western blot analyses. The effects of spironolacton on podocyte motility was further evaluated using a wound healing assay. HG stimulation markedly decreased mRNA and protein expression of integrin β1, and significantly increased mRNA and protein expression of integrin β3 in cultured podocytes. However, simultaneous treatment with spironolacton (10‑7 mol/l) significantly attenuated HG-mediated increases in integrin β3 and decreases in integrin β1 expression. Furthermore, the migration of podocytes induced by HG was abrogated by concomitant treatment with spironolacton. In conclusion, the present study suggested that HG decreased the expression of integrin β1 in cultured podocytes, accompanied with an increase of integrin β3. Spironolactone inhibited cell motility and stabilized podoctyes treated with HG, probably through partly normalizing the expression of integrin β1 and decreasing the expression of integrin β3.

Down-Regulation of Integrin β1 and Focal Adhesion Kinase in Renal Glomeruli under Various Hemodynamic Conditions

Given that integrin β1 is an important component of the connection to maintain glomerular structural integrity, by binding with multiple extracellular matrix proteins and mediating intracellular signaling. Focal adhesion kinase (FAK) is the most essential intracellular integrator in the integrin β1-FAK signalling pathway. Here, we investigated the changes of the two molecules and visualized the possbile interaction between them under various hemodynamic conditions in podocytes. Mice kidney tissues were prepared using in vivo cryotechnique (IVCT) and then were stained and observed using light microscopy, confocal laser scanning microscopy and immunoelectron microscopy. The expression of these molecules were examined by western blot. Under the normal condition, integrin β1 stained continually and evenly at the membrane, and FAK was located in the cytoplasm and nuclei of the podocytes. There were significant colocalized plaques of two molecules. But under acute hypertensive and cardiac arrest conditions, integrin β1 decreased and stained intermittently. Similarly, FAK decreased and appeared uneven. Additionally, FAK translocated to the nuclei of the podocytes. As a result, the colocalization of integrin β1 and FAK reduced obviously under these conditions. Western blot assay showed a consistent result with the immunostaining. Collectively, the abnormal redistribution and decreased expressions of integrin β1 and FAK are important molecular events in regulating the functions of podocytes under abnormal hemodynamic conditions. IVCT could offer considerable advantages for morphological analysis when researching renal diseases.

The targeted podocyte

The podocyte plays a key role both in maintenance of the glomerular filtration barrier and in glomerular structural integrity. Podocyte injury and loss contribute to proteinuria and progressive sclerosis. Inhibitors of mammalian target of rapamycin (mTOR) have variably decreased or caused proteinuria and sclerosis in human disease and experimental settings. In this issue of the JCI, two interesting studies of podocyte-specific manipulation of the mTOR system shed light on the complexity of this pathway in the podocyte.

β1 integrin expression by podocytes is required to maintain glomerular structural integrity

Integrins are transmembrane heteromeric receptors that mediate interactions between cells and extracellular matrix (ECM). β1, the most abundantly expressed integrin subunit, binds at least 12 α subunits. β1 containing integrins are highly expressed in the glomerulus of the kidney; however their role in glomerular morphogenesis and maintenance of glomerular filtration barrier integrity is poorly understood. To study these questions we selectively deleted β1 integrin in the podocyte by crossing β1flox/flox mice with podocyte specific podocin-cre mice (pod-Cre), which express cre at the time of glomerular capillary formation. We demonstrate that podocyte abnormalities are visualized during glomerulogenesis of the pod-Cre;β1flox/flox mice and proteinuria is present at birth, despite a grossly normal glomerular basement membrane. Following the advent of glomerular filtration there is progressive podocyte loss and the mice develop capillary loop and mesangium degeneration with little evidence of glomerulosclerosis. By 3 weeks of age the mice develop severe end stage renal failure characterized by both tubulointerstitial and glomerular pathology. Thus, expression of β1 containing integrins by the podocyte is critical for maintaining the structural integrity of the glomerulus.