Mild electrical stimulation and heat shock ameliorates progressive proteinuria and renal inflammation in mouse model of Alport syndrome.
ABSTRACT: Alport syndrome is a hereditary glomerulopathy with proteinuria and nephritis caused by defects in genes encoding type IV collagen in the glomerular basement membrane. All male and most female patients develop end-stage renal disease. Effective treatment to stop or decelerate the progression of proteinuria and nephritis is still under investigation. Here we showed that combination treatment of mild electrical stress (MES) and heat stress (HS) ameliorated progressive proteinuria and renal injury in mouse model of Alport syndrome. The expressions of kidney injury marker neutrophil gelatinase-associated lipocalin and pro-inflammatory cytokines interleukin-6, tumor necrosis factor-? and interleukin-1? were suppressed by MES+HS treatment. The anti-proteinuric effect of MES+HS treatment is mediated by podocytic activation of phosphatidylinositol 3-OH kinase (PI3K)-Akt and heat shock protein 72 (Hsp72)-dependent pathways in vitro and in vivo. The anti-inflammatory effect of MES+HS was mediated by glomerular activation of c-jun NH(2)-terminal kinase 1/2 (JNK1/2) and p38-dependent pathways ex vivo. Collectively, our studies show that combination treatment of MES and HS confers anti-proteinuric and anti-inflammatory effects on Alport mice likely through the activation of multiple signaling pathways including PI3K-Akt, Hsp72, JNK1/2, and p38 pathways, providing a novel candidate therapeutic strategy to decelerate the progression of patho-phenotypes in Alport syndrome.
Project description:Nephrotic syndrome (NS) is a renal disorder that is characterized by massive proteinuria, hypoalbuminemia and edema. One of the main causes of NS is focal segmental glomerulosclerosis (FSGS), which has extremely poor prognosis. Although steroids and immunosuppressants are the first line of treatment, some FSGS cases are refractory, prompting the need to find new therapeutic strategies. We have previously demonstrated that an optimized combination treatment of mild electrical stimulation (MES) and heat shock (HS) has several biological benefits including the amelioration of the pathologies of the genetic renal disorder Alport syndrome. Here, we investigated the effect of MES?+?HS on adriamycin (ADR)-induced NS mouse model. MES?+?HS suppressed proteinuria and glomerulosclerosis induced by ADR. The expressions of pro-inflammatory cytokines and pro-fibrotic genes were also significantly downregulated by MES?+?HS. MES?+?HS decreased the expression level of cleaved caspase-3 and the number of TUNEL-positive cells, indicating that MES?+?HS exerted anti-apoptotic effect. Moreover, MES?+?HS activated the Akt signaling and induced the phosphorylation and inhibition of the apoptotic molecule BAD. In in vitro experiment, the Akt inhibitor abolished the MES?+?HS-induced Akt-BAD signaling and anti-apoptotic effect in ADR-treated cells. Collectively, our study suggested that MES?+?HS modulates ADR-induced pathologies and has renoprotective effect against ADR-induced NS via regulation of Akt-BAD axis.
Project description:Induction of heat shock protein (HSP) 72 improves metabolic profiles in diabetic model mice. However, its effect on pancreatic ?-cells is not known. The current study investigated whether HSP72 induction can reduce ?-cell stress signaling and apoptosis and preserve ?-cell mass. MIN6 cells and db/db mice were sham-treated or treated with heat shock (HS) and mild electrical stimulation (MES) (HS+MES) to induce HSP72. Several cellular markers, metabolic parameters, and ?-cell mass were evaluated. HS+MES treatment or HSP72 overexpression increased HSP72 protein levels and decreased tumor necrosis factor (TNF)-?-induced Jun NH(2)-terminal kinase (JNK) phosphorylation, endoplasmic reticulum (ER) stress, and proapoptotic signal in MIN6 cells. In db/db mice, HS+MES treatment for 12 weeks significantly improved insulin sensitivity and glucose homeostasis. Upon glucose challenge, a significant increase in insulin secretion was observed in vivo. Compared with sham treatment, levels of HSP72, insulin, pancreatic duodenal homeobox-1, GLUT2, and insulin receptor substrate-2 were upregulated in the pancreatic islets of HS+MES-treated mice, whereas JNK phosphorylation, nuclear translocation of forkhead box class O-1, and nuclear factor-?B p65 were reduced. Apoptotic signals, ER stress, and oxidative stress markers were attenuated. Thus, HSP72 induction by HS+MES treatment protects ?-cells from apoptosis by attenuating JNK activation and cell stresses. HS+MES combination therapy may preserve pancreatic ?-cell volume to ameliorate glucose homeostasis in diabetes.
Project description:Low-intensity electrical current (or mild electrical stimulation; MES) influences signal transduction and activates phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Because insulin resistance is characterized by a marked reduction in insulin-stimulated PI3K-mediated activation of Akt, we asked whether MES could increase Akt phosphorylation and ameliorate insulin resistance. In addition, it was also previously reported that heat shock protein 72 (Hsp72) alleviates hyperglycemia. Thus, we applied MES in combination with heat shock (HS) to in vitro and in vivo models of insulin resistance. Here we show that 10-min treatment with MES at 5 V (0.1 ms pulse duration) together with HS at 42 degrees C increased the phosphorylation of insulin signaling molecules such as insulin receptor substrate (IRS) and Akt in HepG2 cells maintained in high-glucose medium. MES (12 V)+mild HS treatment of high fat-fed mice also increased the phosphorylation of insulin receptor beta subunit (IRbeta) and Akt in mice liver. In high fat-fed mice and db/db mice, MES+HS treatment for 10 min applied twice a week for 12-15 weeks significantly decreased fasting blood glucose and insulin levels and improved insulin sensitivity. The treated mice showed significantly lower weight of visceral and subcutaneous fat, a markedly improved fatty liver and decreased size of adipocytes. Our findings indicated that the combination of MES and HS alleviated insulin resistance and improved fat metabolism in diabetes mouse models, in part, by enhancing the insulin signaling pathway.
Project description:Proteinuria is an important cause of tubulointerstitial damage. Anti-proteinuric interventions are not always successful, and residual proteinuria often leads to renal failure. This indicates the need for additional treatment modalities by targeting the harmful downstream consequences of proteinuria. We previously showed that proteinuria triggers renal lymphangiogenesis before the onset of interstitial inflammation and fibrosis. However, the interrelationship of these interstitial events in proteinuria is not yet clear. To this end, we specifically blocked lymphangiogenesis (anti-VEGFR3 antibody), monocyte/macrophage influx (clodronate liposomes) or lymphocyte and myofibroblast influx (S1P agonist FTY720) separately in a rat model to investigate the role and the possible interaction of each of these phenomena in tubulointerstitial remodeling in proteinuric nephropathy. Proteinuria was induced in 3-month old male Wistar rats by adriamycin injection. After 6?weeks, when proteinuria has developed, rats were treated for another 6?weeks by anti-VEGFR3 antibody, clodronate liposomes or FTY720 up to week 12. In proteinuric rats, lymphangiogenesis, influx of macrophages, T cells and myofibroblasts, and collagen III deposition and interstitial fibrosis significantly increased at week 12 vs week 6. Anti-VEGFR3 antibody prevented lymphangiogenesis in proteinuric rats, however, without significant effects on inflammatory and fibrotic markers or proteinuria. Clodronate liposomes inhibited macrophage influx and partly reduced myofibroblast expression; however, neither significantly prevented the development of lymphangiogenesis, nor fibrotic markers and proteinuria. FTY720 prevented myofibroblast accumulation, T-cell influx and interstitial fibrosis, and partially reduced macrophage number and proteinuria; however, it did not significantly influence lymphangiogenesis and collagen III deposition. This study showed that proteinuria-induced interstitial fibrosis cannot be halted by blocking lymphangiogenesis or the influx of macrophages. On the other hand, FTY720 treatment did prevent T-cell influx, myofibroblast accumulation and interstitial fibrosis, but not renal lymphangiogenesis and proteinuria. We conclude that tubulointerstitial fibrosis and inflammation are separate from lymphangiogenesis, at least under proteinuric conditions.
Project description:BACKGROUND: The induction of heat shock protein (HSP) 72 by mild electrical stimulation with heat shock (MES + HS), which improves visceral adiposity and insulin resistance in mice, may be beneficial in treating metabolic syndrome (MS) or type 2 diabetes mellitus (T2DM). METHODS: Using open-label crossover trials, 40 subjects with MS or T2DM were randomly assigned using computer-generated random numbers to 12 weeks of therapeutic MES + HS followed by 12 weeks of no treatment, or vice versa. During the intervention period, physical and biochemical markers were measured. FINDINGS: Compared to no treatment, MES + HS treatment was associated with a significant decrease in visceral adiposity (- 7.54 cm(2) (- 8.61%), 95% CI - 8.55 to - 6.53 (p = 0.037) in MS, - 19.73 cm(2) (- 10.89%), 95% CI - 20.97 to - 18.49 (p = 0.003) in T2DM). Fasting plasma glucose levels were decreased by 3.74 mg/dL (- 5.28%: 95% CI - 4.37 to - 3.09 mg/dL, p = 0.029) in MS and by 14.97 mg/dL (10.40%: 95% CI - 15.79 to 14.15 mg/dL, p < 0.001) in T2DM, and insulin levels were also reduced by 10.39% and 25.93%, respectively. HbA1c levels showed a trend toward reduction (- 0.06%) in MS, and was significantly declined by - 0.43% (95% CI - 0.55 to - 0.31%, p = 0.009) in T2DM. HbA1c level of less than 7.0% was achieved in 52.5% of the MES + HS-treated T2DM patients in contrast to 15% of the non-treated period. Several insulin resistance indices, inflammatory cytokines or adipokines, including C-reactive protein, adiponectin, and tumor necrosis factor-?, were all improved in both groups. In isolated monocytes, HSP72 expression was increased and cytokine expression was reduced following MES + HS treatment. Glucose excursions on meal tolerance test were lower after using MES + HS in T2DM. INTERPRETATION: This combination therapy has beneficial impacts on body composition, metabolic abnormalities, and inflammation in subjects with MS or T2DM. Activation of the heat shock response by MES + HS may provide a novel approach for the treatment of lifestyle-related diseases. FUNDING: Funding for this research was provided by MEXT KAKENHI (Grants-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology, Japan).
Project description:Laboratory research and previous study suggest that aliskiren, a direct renin inhibitor, has anti-proteinuric effects. We conducted a randomized crossover study to evaluate the anti-proteinuric effect of aliskiren in patients with immunoglobulin A (IgA) nephropathy.We studied 22 patients with biopsy-proven IgA nephropathy and persistent proteinuria despite angiotensin converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB). Patients were randomized to either oral aliskiren 300 mg/day or placebo for 16 weeks and then crossed over to the other treatment arm after a washout period. Proteinuria, estimated glomerular filtration rate (eGFR), blood pressure, and serum potassium were monitored.After aliskiren treatment, there was a significant reduction in proteinuria in 4 weeks (1.76±0.95 to 1.03±0.69 g:g-Cr, p<0.0001), which remained at a low level throughout the treatment period. There was a significant difference in proteinuria between the aliskiren and placebo groups from 4 to 16 weeks after treatment (p<0.01 for all comparisons). After aliskiren treatment, there were modest but statistically significant reductions in eGFR (57.2±29.1 to 54.8±29.3 ml/min/1.73 m(2), p?=?0.013) and diastolic blood pressure (72.6±12.3 to 66.2±11.2 mmHg, p<0.0001). None of the patient developed severe hyperkalemia (serum potassium ?6.0 mmol/l) during the study period.Aliskiren has anti-proteinuric effect in patients with IgA nephropathy and persistent proteinuria despite ACE inhibitor or ARB. Further studies are needed to confirm the renal protecting effect of direct renin inhibition in chronic proteinuric kidney diseases.ClinicalTrials.gov NCT00870493.
Project description:Focal segmental glomerulosclerosis (FSGS) is a histological lesion with many causes, including inherited genetic defects, with significant proteinuria being the predominant clinical finding at presentation. Mutations in COL4A3 and COL4A4 are known to cause Alport syndrome (AS), thin basement membrane nephropathy, and to result in pathognomonic glomerular basement membrane (GBM) findings. Secondary FSGS is known to develop in classic AS at later stages of the disease. Here, we present seven families with rare or novel variants in COL4A3 or COL4A4 (six with single and one with two heterozygous variants) from a cohort of 70 families with a diagnosis of hereditary FSGS. The predominant clinical finding at diagnosis was proteinuria associated with hematuria. In all seven families, there were individuals with nephrotic-range proteinuria with histologic features of FSGS by light microscopy. In one family, electron microscopy showed thin GBM, but four other families had variable findings inconsistent with classical Alport nephritis. There was no recurrence of disease after kidney transplantation. Families with COL4A3 and COL4A4 variants that segregated with disease represent 10% of our cohort. Thus, COL4A3 and COL4A4 variants should be considered in the interpretation of next-generation sequencing data from such patients. Furthermore, this study illustrates the power of molecular genetic diagnostics in the clarification of renal phenotypes.
Project description:AIM:Type IV collagen nephropathies include Alport Syndrome and thin basement membrane nephropathy (TBMN), which are caused by mutations in COL4A3/A4/A5 genes. Recently, reports of patients with heterozygous mutations in COL4A3/A4 have been increasing. The clinical course of these patients has a wide variety, and they are diagnosed as TBMN, autosomal dominant Alport syndrome (ADAS), or familial focal segmental glomerular sclerosis. However, diagnosis, frequency and clinicopathological manifestation of them remains unclear. We tested COL4A3/A4/A5 genes in patients with hereditary nephritis that was difficult to diagnose clinicopathologically, and investigated who should undergo such testing. METHODS:We performed immunostaining for α5 chain of type IV collagen [α5 (IV)] in 27 patients from 21 families who fitted the following criteria: (i) haematuria and proteinuria (± renal dysfunction); (ii) family history of haematuria, proteinuria, and/or renal dysfunction (autosomal dominant inheritance); (iii) no specific glomerulonephritis; and (iv) thinning, splitting, or lamellation of the glomerular basement membrane (GBM) on electron microscopy. Then we performed genetic testing in 19 patients from 16 families who showed normal α5 (IV) patterns. We conducted a retrospective analysis of their clinicopathological findings. RESULTS:Among 16 families, 69% were detected heterozygous mutations in COL4A3/A4, suggesting the diagnosis of TBMN/ADAS. Twenty-one percent of patients developed end stage renal disease. All patients showed thinning of GBM, which was accompanied by splitting or lamellation in seven patients. CONCLUSION:A considerable fraction of patients with hereditary nephritis that is difficult to diagnose clinicopathologically have TBMN/ADAS. It is important to recognize TBMN/ADAS and perform genetic testing in appropriate patients.
Project description:We assessed the lipopolysaccharide (LPS) or heat shock (HS) induction of heat shock protein-72 (HSP72) in peripheral blood mononuclear cells (PBMCs) of patients with severe sepsis (SS) or trauma-related systemic inflammatory response syndrome (SIRS), compared to healthy individuals (H); we also investigated any pre- or posttreatment modulating glutamine (Gln) effect.SS (11), SIRS (10), and H (19) PBMCs were incubated with 1 μg/mL LPS or 43°HS. Gln 10 mM was either added 1 h before or 1 h after induction or was not added at all. We measured monocyte (m), lymphocyte (l), mRNA HSP72, HSP72 polymorphisms, interleukins (ILs), monocyte chemoattractant protein-1 (MCP-1), and cortisol levels.Baseline lHSP72 was higher in SS (p < 0.03), and mHSP72 in SIRS (p < 0.02), compared to H. Only HS induced l/mHSP72/mRNA HSP72; LPS induced IL-6, IL-8, IL-10, and MCP-1. Induced mRNA was related to l/mHSP72, and was related negatively to cytokines. Intracellular l/mHSP72/HSP72 mRNA was related to serum ILs, not being influenced by cortisol, illness severity, and HSP72 polymorphisms. Gln did not induce mRNA in any group but modified l/mHSP72 after LPS/HS induction unpredictably.HSP72 mRNA and l/mHSP72 are higher among critically ill patients, further induced by HS, not by LPS. HSP72 proteins and HSP72 mRNA are related to serum ILs and are negatively related to supernatant cytokines, not being influenced by HSP72 polymorphisms, cortisol, or illness severity. Gln may depress l/mHSP72 after LPS exposure and enhance them after HS induction, but it may not affect early induced HSP72 mRNA.
Project description:Alport post-transplant nephritis (APTN) is an aggressive form of anti-glomerular basement membrane disease that targets the allograft in transplanted patients with X-linked Alport syndrome. Alloantibodies develop against the NC1 domain of ?5(IV) collagen, which occurs in normal kidneys, including renal allografts, forming distinct ?345(IV) and ?1256(IV) networks. Here, we studied the roles of these networks as antigens inciting alloimmunity and as targets of nephritogenic alloantibodies in APTN. We found that patients with APTN, but not those without nephritis, produce two kinds of alloantibodies against allogeneic collagen IV. Some alloantibodies targeted alloepitopes within ?5NC1 monomers, shared by ?345NC1 and ?1256NC1 hexamers. Other alloantibodies specifically targeted alloepitopes that depended on the quaternary structure of ?345NC1 hexamers. In Col4a5-null mice, immunization with native forms of allogeneic collagen IV exclusively elicited antibodies to quaternary ?345NC1 alloepitopes, whereas alloimmunogens lacking native quaternary structure elicited antibodies to shared ?5NC1 alloepitopes. These results imply that quaternary epitopes within ?345NC1 hexamers may initiate alloimmune responses after transplant in X-linked Alport patients. Thus, ?345NC1 hexamers are the culprit alloantigen and primary target of all alloantibodies mediating APTN, whereas ?1256NC1 hexamers become secondary targets of anti-?5NC1 alloantibodies. Reliable detection of alloantibodies by immunoassays using ?345NC1 hexamers may improve outcomes by facilitating early, accurate diagnosis.