Activation of Piezo1 Inhibits Kidney Cystogenesis.

The disruption of calcium signaling associated with polycystin deficiency has been proposed as the primary event underlying the increased abnormally patterned epithelial cell growth characteristic of Polycystic Kidney Disease. Calcium can be regulated through mechanotransduction, and the mechanosensitive cation channel Piezo1 has been implicated in sensing of intrarenal pressure and in urinary osmoregulation. However, a possible role for PIEZO1 in kidney cystogenesis remains undefined. We hypothesized that cystogenesis in ADPKD reflects altered mechanotransduction, suggesting activation of mechanosensitive cation channels as a therapeutic strategy for ADPKD. Here, we show that Yoda-1 activation of PIEZO1 increases intracellular Ca 2+ and reduces forskolin-induced cAMP levels in mIMCD3 cells. Yoda-1 reduced forskolin-induced IMCD cyst surface area in vitro and in mouse metanephros ex vivo in a dose-dependent manner. Knockout of polycystin-2 dampened the efficacy of PIEZO1 activation in reducing both cAMP levels and cyst surface area in IMCD3 cells. However, collecting duct-specific Piezo1 knockout neither induced cystogenesis in wild-type mice nor affected cystogenesis in the Pkd1 RC/RC model of ADPKD. Our study suggests that polycystin-2 and PIEZO1 play a role in mechanotransduction during cystogenesis in vitro , and ex vivo , but that in vivo cyst expansion may require inactivation or repression of additional suppressors of cystogenesis and/or growth. Our study provides a preliminary proof of concept for PIEZO1 activation as a possible component of combination chemotherapy to retard or halt cystogenesis and/or cyst growth.

Yiwei Xiaoyu granules for treatment of chronic atrophic gastritis with deficiency syndrome of the spleen and stomach.

BACKGROUND: The Correa sequence, initiated by Helicobacter pylori (H. pylori), commonly progresses to gastric cancer through the stage of chronic atrophic gastritis (CAG). Although eradication of H. pylori only reduces the risk of gastric cancer, it does not eliminate the risk for neoplastic progression. Yiwei Xiaoyu granules (YWXY) are a commonly used composite preparation in Chinese clinics. However, the pursuit of excellence in clinical trials and the establishment of standardized animal experiments are still needed to contribute to full understanding and application of traditional Chinese medicine in the treatment of CAG. AIM: To demonstrate the effectiveness of YWXY in patients with CAG and spleen-stomach deficiency syndrome (DSSS), by alleviating histological scores, improving response rates for pathological lesions, and achieving clinical efficacy in relieving DSSS symptoms. METHODS: We designed a double-blind, randomized, controlled trial. The study enrolled seventy-two H. pylori-negative patients (mean age, 52.3 years; 38 men) who were randomly allocated to either the treatment group or control group in a 1:1 ratio, and treated with 15 g YWXY or 0.36 g Weifuchun (WFC) tablet combined with the respective dummy for 24 wk. The pre-randomization phase resulted in the exclusion of 72 patients: 50 participants did not meet the inclusion criteria, 12 participants declined to participate, and 10 participants were excluded for various other reasons. Seven visits were conducted during the study, and histopathological examination with target endoscopic biopsy of narrow-band imaging was requested before the first and seventh visits. We also evaluated endoscopic performance scores, total symptom scores, serum pepsinogen and gastrin-17. RESULTS: Six patients did not complete the trial procedures. Treatment with YWXY improved the Operative Link on Gastric Intestinal Metaplasia Assessment (OLGIM) stage, compared with WFC (P < 0.05). YWXY provided better relief from symptoms of DSSS and better improvement in serum gastric function, compared with WFC (P < 0.05). CONCLUSION: YWXY compared with WFC significantly reduced the risk of mild or moderate atrophic disease, according to OLGIM stage, significantly relieved symptoms of DSSS, and improved serum gastric function.

Biodegradation of Oil by a Newly Isolated Strain Acinetobacter junii WCO-9 and Its Comparative Pan-Genome Analysis.

Waste oil pollution and the treatment of oily waste present a challenge, and the exploitation of microbial resources is a safe and efficient method to resolve these problems. Lipase-producing microorganisms can directly degrade waste oil and promote the degradation of oily waste and, therefore, have very significant research and application value. The isolation of efficient oil-degrading strains is of great practical significance in research into microbial remediation in oil-contaminated environments and for the enrichment of the microbial lipase resource library. In this study, Acinetobacter junii WCO-9, an efficient oil-degrading bacterium, was isolated from an oil-contaminated soil using olive oil as the sole carbon source, and its enzyme activity of ρ-nitrophenyl decanoate (ρ-NPD) decomposition was 3000 U/L. The WCO-9 strain could degrade a variety of edible oils, and its degradation capability was significantly better than that of the control strain, A junii ATCC 17908. Comparative pan-genome and lipid degradation pathway analyses indicated that A. junii isolated from the same environment shared a similar set of core genes and that the species accumulated more specific genes that facilitated resistance to environmental stresses under different environmental conditions. WCO-9 has accumulated a complete set of oil metabolism genes under a long-term oil-contamination environment, and the compact arrangement of abundant lipase and lipase chaperones has further strengthened the ability of the strain to survive in such environments. This is the main reason why WCO-9 is able to degrade oil significantly more effectively than ATCC 17908. In addition, WCO-9 possesses a specific lipase that is not found in homologous strains. In summary, A. junii WCO-9, with a complete triglyceride degradation pathway and the specific lipase gene, has great potential in environmental remediation and lipase for industry.

CMIP interacts with WT1 and targets it on the proteasome degradation pathway.

BACKGROUND: The Wilms tumor 1 suppressor gene, WT1, is expressed throughout life in podocytes and is essential for their function. Downregulation of WT1 has been reported in podocyte diseases but the underlying mechanisms remain unclear. Podocyte injury is the hallmark of idiopathic nephrotic syndrome (INS), the most frequent glomerular disease in children and young adults. An increase in the abundance of Cmaf-inducing protein (CMIP) has been found to alter podocyte function, but it is not known whether CMIP affects WT1 expression. METHODS: Transcriptional and post-transcriptional regulation of WT1in the presence of CMIP was studied using transient transfection, mouse models, and siRNA handling. RESULTS: We showed that overproduction of CMIP in the podocyte was consistently associated with a downregulation of WT1 according to two mechanisms. We found that CMIP prevented the NF-kB-mediated transcriptional activation of WT1. We demonstrated that CMIP interacts directly with WT1 through its leucine-rich repeat domain. Overexpression of CMIP in the M15 cell line induced a downregulation of WT1, which was prevented by lactacystin, a potent proteasome inhibitor. We showed that CMIP exhibits an E3 ligase activity and targets WT1 to proteasome degradation. Intravenous injection of Cmip-siRNA specifically prevented the repression of Wt1 in lipopolysaccharides-induced proteinuria in mice. CONCLUSIONS: These data suggest that CMIP is a repressor of WT1 and might be a critical player in the pathophysiology of some podocyte diseases. Because WT1 is required for podocyte integrity, CMIP could be considered a therapeutic target in podocyte diseases.

Induction of C-Mip by IL-17 Plays an Important Role in Adriamycin-Induced Podocyte Damage.

BACKGROUND/AIMS: Although the disturbance of T lymphocyte and glomerular podocyte exerts a crucial function in the pathogenesis of proteinuria, the potential link is still unclear. METHODS: The balance of Treg and Th17 cells, and the expression of IL-17/IL-17R and c-mip were investigated in adrimycin-induced nephropathy (AN) mice. The effect and mechanism of IL-17 on podocyte were explored in cultured podocytes. RESULTS: The proportion of Th17 cells in peripheral blood mononuclear cells, the amount of IL-17 in serum and kidney cortical homogenates, and the expression of IL-17R and c-mip in glomerular podocyte were increased obviously in AN mice. In cultured podocytes, recombinant IL-17 led to an induction of apoptosis and cytoskeletal disorganization, an overproduction of c-mip while down-regulation of phosphor-nephrin, and an increased binding of c-mip to NF-κB/RelA. Silence of c-mip prevented podocyte apoptosis and reduction of phosphor-nephrin by prompting nuclear translocation of NF-κB/RelA in IL-17 treated cells. Persistent activation of NF-κB up-regulated pro-survival protein Bcl-2 and decreased podocyte apoptosis, but had no effect on phosphor-nephrin level. CONCLUSION: These findings demonstrated that induction of IL-17 released by Th17 cells plays a key role in podocytopathy most likely through down-regulation of phosphor-nephrin and Bcl-2 level via overproduction of c-mip.

Protective role of cyclosporine A and minocycline on mitochondrial disequilibrium-related podocyte injury and proteinuria occurrence induced by adriamycin.

BACKGROUND: Dysfunction of mitochondria is involved in podocyte injury in some kidney diseases, but the relationship between abnormal mitochondrial morphology and podocyte injury as well as the underlying mechanism is still unclear. This study aims to investigate dynamic changes of mitochondrial morphology and the potential molecular events in an adriamycin (ADR)-induced podocyte injury model. METHODS: Podocyte apoptosis was evaluated by annexin V assay. Podocyte mitochondrial membrane potential (MMP) was measured with MitoCapture kit. Double staining was used to show the distribution changes of mitochondria and actin filament as well as mitofusin proteins and podocin. Mitochondrial shape descriptors were obtained using analySIS Image system. Effects of cyclosporine A (CsA) or minocycline (Mcy) on mitochondrial morphology were explored in ADR-induced nephropathy rats. RESULTS: ADR caused podocyte damage displaying as induction of cellular apoptosis and increase of activated caspase 3 and cytochrome c. The MMP level was decreased remarkably in ADR-treated podocytes. Mitochondrial morphological changes induced by ADR occurred rapidly from large and ellipsoid shape to the small, long and irregular. ADR significantly decreased surface area, perimeter and circularity, while increasing aspect ratio of mitochondria. In addition, mitochondria number transiently increased at 6 h following ADR application. Mitochondria intensity was increased along with punctate mitochondria formation, which co-localized with polymerized actin cytoskeleton in ADR podocytes. In ADR-induced nephropathy rats, 24-h proteinuria was decreased significantly by CsA or Mcy. ADR-induced abnormal changes of mitochondrial morphology were restored by CsA or Mcy. The induction of mitofusin proteins and the reduction of podocin in ADR rat glomeruli were rescued by CsA or Mcy. CONCLUSIONS: Mitochondrial dysfunction may be an early event in ADR-induced podocyte damage, and the protective role of CsA or Mcy may be mediated partially by improving mitochondrial function through inhibiting the induction of mitofusin proteins.

Crk1/2 and CrkL form a hetero-oligomer and functionally complement each other during podocyte morphogenesis.

Activation of the slit diaphragm protein nephrin induces actin cytoskeletal remodeling, resulting in lamellipodia formation in podocytes in vitro in a phosphatidylinositol-3 kinase-, focal adhesion kinase-, Cas-, and Crk1/2-dependent fashion. In mice, podocyte-specific deletion of Crk1/2 prevents or attenuates foot process effacement in two models of podocyte injury. This suggests that cellular mechanisms governing lamellipodial protrusion in vitro are similar to those in vivo during foot process effacement. As Crk1/2-null mice developed and aged normally, we tested whether the Crk1/2 paralog, CrkL, functionally complements Crk1/2 in a podocyte-specific context. Podocyte-specific CrkL-null mice, like podocyte-specific Crk1/2-null mice, developed and aged normally but were protected from protamine sulfate-induced foot process effacement. Simultaneous podocyte-specific deletion of Crk1/2 and CrkL resulted in albuminuria detected by 6 weeks postpartum and associated with altered podocyte process architecture. Nephrin-induced lamellipodia formation in podocytes in vitro was CrkL-dependent. CrkL formed a hetero-oligomer with Crk2 and, like Crk2, was recruited to tyrosine phosphorylated nephrin. Thus, Crk1/2 and CrkL are physically linked, functionally complement each other during podocyte foot process spreading, and together are required for developing typical foot process architecture.

c-mip down-regulates NF-κB activity and promotes apoptosis in podocytes.

The mechanisms of podocyte disorders in cases of idiopathic nephrotic syndrome (INS) are complex and remain incompletely elucidated. The abnormal regulation of NF-κB may play a key role in the pathophysiology of these podocyte diseases, but at present, NF-κB has not been thoroughly investigated. In this study, we report that induction of c-mip in podocytes of patients with INS is associated with a down-regulation of RelA, a potent antiapoptotic factor that belongs to the NF-κB family. Overexpression of c-mip in differentiated podocytes promotes apoptosis by inducing caspase-3 activity and up-regulating the proapoptotic protein Bax, whereas the overall levels of the antiapoptotic protein Bcl-2 was concomitantly decreased. The associated overexpression of RelA prevented the proapoptotic effects of c-mip. In addition, the targeted induction of c-mip in podocytes in vivo inhibited the expression of the RelA protein and increased the Bax/Bcl-2 ratio. The expression of both c-mip and active caspase-3 increased in focal and segmental glomerulosclerosis biopsies, and both proteins displayed a close spatial relationship. These results suggest that alterations in NF-κB activity might result from the up-regulation of c-mip and are likely to contribute to podocyte disorders in cases of INS.

Reduction in VEGF protein and phosphorylated nephrin associated with proteinuria in adriamycin nephropathy rats.

BACKGROUND/AIMS: The relationship between vascular endothelial growth factor (VEGF) and the phosphorylated critical podocyte slit diaphragm molecule nephrin is not fully clarified. This study investigated the dynamic changes in VEGF expression and nephrin phosphorylation, and the effects of the antiproteinuric drugs prednisone and lisinopril on them in Adriamycin nephropathy rats. METHODS: Renal tissues from Adriamycin rats were collected at days 3, 7, 14, and 28. Distribution and expression of VEGF was revealed by immunohistochemistry, real-time PCR and Western blot. Phosphorylated nephrin was evaluated by immunoprecipitation. RESULTS: A discontinuous redistribution of VEGF was displayed at day 3, followed by significant protein reduction at day 7 with persistent downregulation to day 28. Phosphorylated nephrin decreased evidently at day 14 and persisted to day 28. The reduction in VEGF and phosphorylated nephrin was not a result of podocyte loss. The intervention of prednisone and lisinopril evidently reduced proteinuria, effectively attenuated the severe lesions of podocyte foot processes, and restored the reduction in VEGF and nephrin phosphorylation. At day 28, the reduction in VEGF and phosphorylated nephrin was negatively correlated with proteinuria, whereas the phosphorylated nephrin was positively correlated with VEGF protein from day 7 to day 28. CONCLUSION: The reduction in VEGF protein and nephrin phosphorylation was possibly involved in the proteinuria in Adriamycin rats, and there might be some relationship between VEGF and nephrin phosphorylation. The antiproteinuric effects of lisinopril and prednisone were achieved at least partially by restoring VEGF protein and nephrin phosphorylation.

WT1 mutation and podocyte molecular expression in a Chinese Frasier syndrome patient.

We report on a Chinese girl with Frasier syndrome (FS). She presented with steroid-resistant focal segmental glomerulosclerosis (FSGS) and male pseudohermaphroditism. The WT1 IVS 9 + 5 G>A mutation was detected in one allele in the proband. The ratio of +KTS/-KTS was 0.67 in the proband's cDNA. The expression of podocyte molecules (WT1, nephrin, podocin, alpha-actinin 4 and CD2AP) were also investigated in a renal specimen of this FS patient. WT1 expression showed diffuse nuclear staining, with less obvious speckles in the patient's glomeruli than in those of controls. The distribution and intensity of podocyte molecules were altered both in normal- and abnormal-appearing glomeruli. In conclusion, the study presented a case of FS by clinical manifestation, renal pathology, karyotype analysis and genetic testing. A lower ratio of +KTS/-KTS and an abnormal distribution of WT1, as well as abnormal expressions of other podocyte molecules, were also revealed. The mechanisms of WT1 mutation causing FS still need to be investigated.

Diversities of podocyte molecular changes induced by different antiproteinuria drugs.

Nephrin, podocin, CD2AP, and alpha-actinin-4 are important podocyte proteins that help maintain the integrity of the slit diaphragm and prevent proteinuria. Studies have shown that angiotensin-converting enzyme inhibitors, glucocorticoids, and all-trans retinoic acid (ATRA) have antiproteinuric effects. However, it is still unclear whether these drugs, with different pharmacological mechanisms, lead to a reduction in proteinuria by changing the expression and distribution of these important podocyte proteins. In this study, changes in the expression and distribution of nephrin, podocin, CD2AP, and alpha-actinin-4 were dynamically detected in Adriamycin-induced nephrotic (ADR) rats treated with three different drugs: lisinopril, prednisone, and ATRA. Nephropathy was induced by an intravenous injection of Adriamycin. After Adriamycin injection, rats received lisinopril, prednisone, and ATRA treatment, respectively. Renal tissues were collected at Days 3, 7, 14, and 28. The distribution and the expression of messenger RNA and protein of nephrin, podocin, CD2AP, and alpha-actinin-4 were detected by indirect immunofluorescence, real-time polymerase chain reaction, and Western blotting, respectively. With the intervention of lisinopril, prednisone, and ATRA, changes in the expression of nephrin, podocin, and CD2AP were diverse, which was different from that detected in ADR rats. After lisinopril and prednisone intervention, podocin exhibited prominent earlier changes compared with those of nephrin and CD2AP, whereas CD2AP showed more prominent changes after ATRA intervention. There was no change in the expression of alpha-actinin-4 molecule. In summary, we conclude that the antiproteinuric effects of lisinopril, prednisone, and ATRA were achieved by changes in the expression and distribution of the important podocyte molecules nephrin, podocin, CD2AP, and alpha-actinin-4. The pattern in the change of podocyte molecules after lisinopril and prednisone intervention was similar, but the pattern in the change of podocyte molecules after ATRA intervention was different from that of lisinopril or prednisone intervention.